Library

Notes: Abstract: The binding of [3H]dipyridamole ([3H]DPR) to guinea pig brain membranes is described and compared to that of [3H]nitrobenzylthioinosine ([3H]NBI). The binding of [3H]DPR is saturable, reversible, and specific with phar-macologic evidence indicating that this ligand is binding to the adenosine uptake site. Compared to [3H]NBI the binding of [3H]DPR is of higher capacity (Bmax= 208 ±16 fmol/ mg protein for [3H]NBI and 530 ± 40 fmol/mg protein for [3H]DPR) and lower affinity (KD= 0.35 ± 0.02 nM for [3H]NBI and 7.6 ± 0.7 nM for [3H]DPR). The adenosine uptake inhibitors are the most potent inhibitors of binding (Ki of 10−8-10−7M) whereas adenosine receptor ligands such as cyclohexyladenosine, 2-chloroadenosine, and various methylxanthines are several orders of magnitude less potent (Ki 10−5-10−2). The inhibition of [3H]DPR binding by NBI is biphasic, with only 40% of binding being susceptible to inhibition by NBI concentrations 〈 10−5M. The tissue distribution of [3H]DPR binding parallels that of [3H]NBI although in most cases significantly more sites are observed with [3H]DPR. Calcium channel blocking agents such as nifedipine, nimodipine, and verapamil are also inhibitors of [3H]DPR binding with potencies in the micromolar range. The data are consistent with [3H]DPR being a useful additional ligand for the adenosine uptake site and provide evidence that multiple uptake binding sites exist of which only about 40% are NBI-sensitive.

Notes: Abstract: Polyclonal antibodies to ganglioside Gmi have been prepared and characterised by direct and competitive enzyme-linked immunoassay. An immunoglobulin fraction was prepared from a rabbit antisera showing high specificity and antibody titre for GMI relative to the other major brain gangliosides. The anti-GMI immunoglobulin fraction and B-cholera toxin specifically labelled neurons in primary cultures of embryonic chick dorsal root ganglia and there was a good correlation between the relative increase in binding of anti-GMI immunoglobulin and B-cholera toxin following neuraminidase treatment of a variety of cell types. At antibody concentrations that show saturable binding to endogenous ganglioside in the neuronal membrane, the anti-GM1 immunoglobulin fraction did not interfere with the nerve growth factor (NGF)-mediated fibre outgrowth and neuronal survival as indexed by measurement of neu-rofilament protein levels. Similarly, at levels in excess of those shown to stimulate thymocyte proliferation, B-cholera toxin was also without effect. These data are not consistent with GMI in the neuronal membrane functioning as a receptor molecule for NGF and/or other differentiation factors present in the tissue culture media.

Notes: Abstract: Delta sleep-inducing peptide (DSIP) has been isolated and characterized by its capacity to enhance delta sleep in rabbits. Up to now, sleep was the main target of DSIP research, but different extra-sleep effects of the peptide have been reported as well. Several mechanisms of action have been proposed, though no convincing evidence for any of them has been obtained so far. We recently detected that DSIP reduced the nocturnal increase of N-acetyltransferase (NAT) activity in rat pineal in a dose-dependent manner. The activity of this enzyme is known to be induced by adrenergic agonists and several studies have suggested that stimulation of α1-adrenergic receptors potentiates the “basic” effect of β-receptors. DSIP in the range between 20 and 300 nM significantly enhanced NAT activity induced by 10-6M norepinephrine in vitro, and a similar effect was observed with 2 nM P-DSIP, a phosphorylated analog. Incubation with prazosin eliminated the enhancement, whereas propranolol reduced norepinephrine stimulation that was still increased by P-DSIP and probably DSIP. It was concluded that the sleep-peptide and its analog modulate the α1-adrenergic receptor of rat pineal in its response to adrenergic agonists. The same mechanism may also be responsible for other biological activities of DSIP such as sleep-induction and stress-tolerance.

Notes: Abstract: When rat brain membranes were incubated with the benzodiazepine agonist [3H]flunitrazepam or the partial inverse benzodiazepine agonist [3H]Ro 15–4513 in the presence of ultraviolet light one protein (P51) was specifically and irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. After digestion of the membranes with trypsin, protein P51 was degraded into several peptides. When P51 was photolabeled with [3H]Ro 15–4513, four peptides with apparent molecular weights of 39,000, 29,000, 21,000, and 17,000 were observed. When P51 was labeled with [3H]flunitrazepam, only two peptides with apparent molecular weights of 39,000 and 25,000 were obtained. Protein P55 was only partially degraded by trypsin, and whether it was labeled with [3H]-flunitrazepam or [3H]Ro 15–4513 it yielded the same two proteolytic peptides with apparent molecular weights of 42,000 and 45,000. These results support the existence of at least two different benzodiazepine receptor subtypes associated with proteins P51 and P55. The different receptors seem to be differentially protected against treatment with trypsin. In addition, these results indicate that in the benzodiazepine receptor subtype associated with P51 benzodiazepine agonists and partial inverse benzodiazepine agonists irreversibly bind to different parts of the molecule.

Notes: Abstract: In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10–8M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. α-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating eifects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.

Notes: Résumé: La biosynthèse de la carcinine est réalisée in vitro à partir de ses deux constituants: la β-alanine et l'histamine. La réaction est catalysée par des extraits de muscles, de coeur, et de CNS de Carcinus maenas. L'activité spécifique de l'enzyme, la carcinine synthétase, est 15 fois plus élevée dans le CNS que dans les autres organes. Seul le CNS réalise la biosynthèse de la carcinine à partir de l'histidine et uniquement en présence de phosphate-5’ de pyridoxal. Le siège de la biosynthèse de la carcinine serait done le CNS. Dans cet organe, l'histidine serait transformée en histamine qui serait ensuite catabolisée sous forme de carcinine. Celle-ci serait alors transportée jusque dans le tissu cardiaque où elle s'accumulerait. L'histamine dont le métabolisme s'effectue en totalité dans le CNS serait done impliquée dans l'activité neuronale du Crustacé. La carcinine synthétase est une enzyme soluble qui requiert la présence d'ATP, de β-alanine, et d'histamine. Mg2+ et le dithiothréitol sont aussi essentiels pour son activité. Son pH optimum se situe à∼7,6. La carcinine synthétase se distingue de la carnosine synthétase et de la γ-glutamylhistamine synthétase car elle ne catalyse ni la synthèse de la β-alanylhistidine, ni celie de la γ-glutamylhistamine. Mots Clés: Métabolisme de l'histamine—β-Alanylhistamine—Carcinine—Carcinus maenas. Arnould J.-M. Mise en evidence de la carcinine synthétase, une nouvelle enzyme catalysant le métabolisme de l'histamine dans le systeme nerveux central de Carcinus maenas. J. Neurochem. 48, 1316–1324 (1987).

Notes: Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [14C]sialic acid was found to be associated with the high-molecular-weight [〉200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.

Notes: Abstract: Stimulation of glutamate binding by the dipeptide l-phenylalanyl-l-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.

Notes: Abstract: Using ligand binding techniques, we studied β-adrenergic receptor subtypes in brains obtained at autopsy from seven histologically normal controls and seven histopathologically verified cases with Alzheimer-type dementia (ATD). Inhibition of [3H]dihydroalprenolol ([3H]DHA) binding by the selective β, antagonist, metoprolol, results in nonlinear Hofstee plots, suggesting the presence of the two receptor subtypes in the human brain. The calculated ratios of β1/β2-adrenergic receptors in control brains are as follows: frontal cortex, 49:51; temporal cortex, 31:69; hippocampus, 66:34; thalamus, 23:77; putamen, 70:30; caudate, 48:52; nucleus basalis of Meynert (NbM), 43:57; cerebellar hemisphere, 25:75. Compared with the controls, total concentrations of β-adrenergic receptors were significantly reduced only in the thalamus of the ATD brains. β1-Adrener gic receptor concentrations were significantly reduced in the hippocampus and increased in the NbM and cerebellar hemisphere, whereas β2-adrenergic receptor concentrations were significantly reduced in the thalamus, NbM, and cerebellar hemisphere and increased in the hippocampus and putamen of the ATD brains. These results suggest that β1-and β2-adrenergic receptors are present in the human brain and that there are significant changes in both receptor subtypes in selected brain regions in patients with ATD.

Notes: Abstract: Leukotriene B4 [5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.

Notes: Abstract: Stressors such as tissue slicing, toxic chemicals, and heat shock applied to cultured cells, organ tissues, or whole animals in vivo induce the synthesis of a 71,000-kilodalton stress protein (SP71) that is not normally present in most organ tissues. In the present experiment, an attempt was made to inhibit selectively the synthesis of SP71 in rat brain tissue slices. Of several manipulations to the brain slice incubation medium that were examined, only addition of very high concentrations of certain polyhydroxyl alcohols, i.e., 1.0 M glycerol, selectively inhibited SP71 synthesis. Glycerol also selectively inhibited SP71 synthesis in heat-shocked cerebral microvascular cells in culture but failed to inhibit SP71 synthesis in anesthetized rats in vivo in response to heat shock. The effects of glycerol on SP71 synthesis are discussed in relationship to current hypotheses concerning the function of SP71.

Notes: Abstract: Phosphatidylethanol (Peth) formation catalyzed by the transphosphatidylation activity of phospholipase D was demonstrated to occur in a rat brain synaptosomal enriched preparation. The optimal pH was determined to be 6.5, and the optimal ethanol concentration was determined to be 0.3–0.4 M with an apparent Km of 0.2 M. Peth formation was barely detectable in the absence of an appropriate activator and several unsaturated fatty acids were found to be effective activators. The concentrations of oleic acid required for maximum activation varied with the concentration of exogenous phosphatidylcholine present in the incubation mixtures. All detergents tested were significantly less active than the unsaturated fatty acids and divalent ions were not required for Peth formation. Phosphatidylcholine was the most effective phosphatidyl donor of the phospho-lipids tested. Peth forming activity was greatest in the synap-tic membrane fraction of the various brain subfractions examined. The 12,000 g-100,000 g paniculate fraction of lung, heart, and adipose tissue had activities similar to that of brain.

Notes: Abstract: Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-celluiose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.

Notes: Abstract: HPLC with electrochemical detection was used to determine the levels of p-hydroxyphenylethanolamine (octopamine), 3,4-dihydroxyphenylethylamine (dopamine), and 5-hydroxytryptamine (5-HT) in the brains of control, reserpine, and d-amphetamine-treated blow flies, Phormia regina Meigen. Parallel studies were carried out to assess the effects of the two drugs on fly feeding behavior, measured as mean acceptance threshold: the minimum sucrose concentration to which the average fly in a population will respond by proboscis extension when its tarsi contact the solution. In saline-injected control flies, all three amines were found at levels of approximately 2 pmol/brain. Thirty minutes after injection with d-amphetamine (12 μg/fly), brain octopamine was depleted by 85%, whereas dopamine and 5-HT were depleted by 70%. Reserpine (5 μg/fly) caused 70% depletion of dopamine and 〉90% depletion of both octopamine and 5-HT 24 h after injection. However, the effect of reserpine was much slower in onset (hours versus minutes) and more persistent (days versus hours) than was the effect of d-amphetamine. With either drug, the time course of amine depletion closely matched the time course of the increase in feeding threshold observed in drug-treated flies. These results suggest that CNS pools of the biogenic amines, octopamine, dopamine, and 5-HT are important in governing blow fly responsiveness to food stimuli.

Notes: Abstract: Rubrophilin, a unique brain specific polypeptide, was purified to apparent homogeneity from microsomal fractions of bovine brains. The peptide stains pink with Coomassie Brilliant Blue R-250 (C.I. No. 42660) under specific conditions, has an apparent Mr of 53,000, and is acidic with an apparent pI of 4.9. The purification involves initial solubilization of delipidated microsomes in sodium dodecyl sulfate, followed by ammonium sulfate fractionation, reversed ammonium sulfate gradient elution from diatoma-ceous earth, gel filtration on polyacrylamide (Biogel P-200). gradient elution chromatography from hydroxylapatite, and reverse-phase chromatography from phenyl-Sepharose. A yield of about 5 mg of rubrophilin was obtained from 9 g of microsomal proteins. Amino acid analysis shows that rubrophilin contains only nine amino acids with residues/ mol as follows: alanine (102), glutamic acid (97), lysine (65), proline (55), aspartic acid (48), glycine (44), serine (37), threonine (35), and valine (10). Cysteine, methionine, tryp-tophan, tyrosine, isoleucine, phenylalanine, histidine, and arginine could not be detected. Relative rubrophilin content of vertebrate brains was as follows: mammals 〉 birds 〉 reptiles 〉 fishes. It is present in mouse retina and human neu-roblastoma cell cultures but could not be detected in octopus optic lobe or in cultured C-6 rat glioma cells.

Notes: Abstract: To probe the activities of various pathways of lipid metabolism in peripheral nerve, six phospholipid-directed precursors were individually injected into the exposed sciatic nerves of adult mice, and their incorporation into phos-pholipids and proteins was studied over a 2-week period. Tritiated choline, inositol, ethanolamine, serine, and glyc-erol were mainly used in phospholipid synthesis; in contrast, methyl-labeled methionine was primarily incorporated into protein. Phosphatidylcholine was the main lipid formed from tritiated choline, glycerol, and methionine precursors. Phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol were the main lipids formed from serine, ethanolamine, and inositol, respectively. With time there was a shift in label among phospholipids, with higher proportions of choline appearing in sphingomyelin, glycerol in phosphatidylserine, ethanolamine in phosphatidylethanolamine (plasmalogen), and inositol in polyphosphoinosi-tides, especially phosphatidylinositol 4,5-bisphosphate. We suggest that the delay in formation of these phospholipids, which are concentrated in peripheral nerve myelin, may, at least in part, be due to their formation at a site(s) distant from the sites where the bulk of Schwann cell lipids are made. We propose that separating the synthesis of these my-elin-destined lipids to near the Schwann cell's plasma membrane would facilitate their concentration in peripheral nerve myelin sheaths. At earlier labeling times, ethanolamine and glycerol were more actively incorporated into phosphatidylcholine and phosphatidylinositol, respectively, than later. The transient labeling of these phospholipids may reflect some unique role in peripheral nerve function.

Notes: Abstract: The veratridine/tetrodotoxin-sensitive sodium influx was measured in membrane fractions isolated from the electric organ of Electrophorus electricus. The fractions were characterized, and the main biochemical markers and their acetylcholine receptor content were determined. The innervated and noninnervated faces of the electroplax were separated. The different biochemical criteria used indicate that the pre- and Postsynaptic. membranes of the innervated face were isolated. Sodium influx increased by veratridine and blocked by tetrodotoxin was found in fractions from the presynaptic membrane. Because some of the vesicles in this fraction are in the inside-out conformation, tetrodotoxin had to be applied to both faces of the vesicles so that sodium influx was blocked completely. The fractions from the innervated face of the electroplax contained sodium channels with sensitivities to tetrodotoxin and veratridine similar to those of fractions from other nerve membrane preparations.

Notes: Abstract: HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.

Notes: Abstract: The activities of tyrosine hydroxylase and tryptophan hydroxylase, and the concentrations of the biopterin cofactor and the precursor neopterin were measured in 14 regions of postmortem brains from four histologically verified patients of senile dementia of the Alzheimer type (SDAT) and eight histologically normal controls. Neopterin concentrations were measured in the human brain for the first time. The activities of tyrosine hydroxylase and tryptophan hydroxylase in the brains of patients with SDAT were significantly reduced in the substantia nigra and in the lateral segment of the globus pallidus, locus ceruleus, and substantia nigra, respectively. The concentrations of total biopterin in the brains of patients with SDAT were signifycantly reduced in the putamen and substantia nigra, but the total neopterin concentrations did not change significantly. These results suggest that the reduction in biogenic amines in SDAT might be related to reductions in biosynthetic enzymes associated with biogenic amines, due to destruction of monoaminergic neurons.

Notes: Abstract: Modulation of [3H]dopamine release by cholinergic agents (acetylcholine, atropine, d-tubocurarine, oxo-tremorine, and nicotine) was studied in primary cell cultures derived from whole brains of foetal rats (17 days of gestation). Monolayer and aggregated neuron-enriched cultures were maintained for 17 days in vitro. [3H]Dopamine basal outflow was enhanced by acetylcholine, nicotine, and atropine and was unaffected by oxotremorine, hexametho-nium, and d-tubocurarine. The action of nicotine was antagonized by d-tubocurarine, and that of atropine was partially blocked by oxotremorine. A similar picture was seen when the influence of cholinergic agents was studied under depolarizing conditions. The action of oxotremorine was dependent on nerve activity. The presence of both musca-rinic and nicotinic antagonists was necessary for abolishing the effect of acetylcholine on the dopamine outflow. These results show that dopamine release in both types of neuron-enriched cultures can be influenced by cholinergic agents and that both muscarinic and nicotinic receptors are involved in regulation of the amine's outflow.

Notes: Abstract: Tyrosine hydroxylase purified from rat pheochro-mocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichio-metric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.

Notes: Abstract: The biochemical basis of a case of GM2 gangliosidosis in a Japanese Spaniel was studied. This dog had a massive accumulation of GM2 ganglioside in the brain. The β-hexosaminidase activity in this affected dog brain was ∼ 12 times higher than that of normal brain. However, the activity toward p-nitrophenyl-6-sulfo-2-acetamido-2-de-oxyglucopyranoside was only four times higher in the affected brain than in normal brain. The GM2 activator preparation obtained from the normal dog brain could stimulate the hydrolysis of GM2 ganglioside by β-hexosaminidase isolated from the affected dog. However, the corresponding activator fraction from the affected dog could not stimulate such a reaction. It was concluded that the biochemical basis of the Gm2 gangliosidosis in this Japanese Spaniel was due to the attenuation in the stimulatory activity of GM2 activator. This case represents the first animal form similar to the activator deficiency (or defect) of Type AB GM2 gangliosidosis in humans.

Notes: Abstract: Triethyllead (TEL) is a CNS neurotoxin producing bizarre neurobehavioral changes. The principal objective of this study was to determine if TEL-induced defects in energy metabolism were responsible for the inhibition of synaptosomal Na+-dependent high-affinity uptake of γ-aminobutyric acid (GABA). A dose-dependent inhibition of GAB A uptake (ID50= 10 μMTEL) was found during 30-s incubations. Uptake of glutamate was more resistant to the inhibitory effects of TEL. A TEL-induced Cl− -dependent synaptosomal deficit of ATP was observed. Such deficit in high-energy phosphate was time-dependent and did not occur in the absence of Cl− or as early as 30 s. Inhibition of GABA uptake, on the other hand, was a Cl−-independent phenomenon and was observed at as early as 30 s. TEL was not competitive with Na+ or GABA itself, as the effects of TEL were not overcome with high [Na+] or [GABA]. These results indicate that the locus of TEL inhibition of GABA uptake is not a Cl−-dependent event and does not involve a perturbed transmembrane electrochemical gradient, due to either an observed mitochondrial defect or an inhibition of Na+, K+-ATPase directly.

Notes: Abstract: A study of the onset of cation and guanine nucleotide regulation of δ, μ, and k rat brain opioid receptors during postnatal development was undertaken. Site-specific binding assays were utilized for each receptor type and the effects of 0.5 mM MnCl2, 100 mM NaCl, and/or 50 μM guanosine-5′-(β,γ-imido) triphosphate [GPP(NH)P] were assessed. The most pronounced changes of opioid binding were seen in the presence of Mn2+. In adults, agonist binding to δ sites was stimulated by Mn2+, whereas that to μ. sites was not affected and k binding was inhibited. The postnatal development of Mn2+ regulation for the three receptor subtypes was distinctly different. The largest effects were seen on δ sites detected in the early neonatal period, Mn2+ eliciting a 68% stimulation of binding over controls at day 1. Significant inhibition of k site binding by Mn2+ was detected only after the third postnatal week. Mn2+ caused a significant reversal of Gpp(NH)p inhibition of δ binding in the early neonatal period, exceeding that in the absence of regulators. Inhibition of μ and δ receptor binding by Na+ was greater, and the Mn2+ reversal of this effect was smaller, in the first 2 postnatal weeks than in adults. Gpp(NH)p + Na+ regulation did not change appreciably during the postnatal period. However, Mn2+ reversal of the considerable inhibition elicited by the combination of Na+ and Gpp(HN)p was developmental time-dependent. The data are discussed in terms of multiple sites of interaction for guanine nucleotides and cations. Our results demonstrate that the characteristics and postnatal development of guanine nucleotide and cat-ionic regulation of μ, δ, and k binding are distinctly different. Furthermore, neonates may serve as a model for the examination of individual regulatory effects on opioid receptors.

Notes: Abstract: The main objective of this study was to determine whether the excitotoxic cholinesterase inhibitor soman increases the catabolism of phospholipids in rat brain. Injections of soman (70 μg/kg, s.c), at a dose that produced toxic effects, increased the levels of both free fatty acids (175–250% of control) and free choline (250% of control) in rat cerebrum 1 h after administration. All fatty acids contained in brain phosphatidylcholine were elevated significantly including palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4), and docosahexaenoic (22:6) acids. The changes observed were consistent with those reported to occur following ischemia and the administration of other convulsants. Pretreatment of rats with the anticonvulsant diazepam (4 mg/kg, i.p.) prevented both the signs of soman toxicity and the soman-induced increase of choline and free fatty acids. Diazepam alone did not affect the levels of choline or free fatty acids, cholinesterase activity, or soman-induced cholinesterase inhibition, suggesting that soman toxicity involves a convulsant-mediated increase in phosphatidylcholine catabolism. In addition, administration of the convulsant bicuculline, at a dose that produces seizures and increases the levels of free fatty acids in brain, significantly increased the levels of choline. Results suggest that excitotoxic events enhance the hydrolysis of phosphatidylcholine in brain as evidenced by a concomitant increase in the levels of choline and free fatty acids.

Notes: Abstract: 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+), have been shown to cause a number of lesions in dopaminergic pathways of the nigro-striatal region of the brain. However, data on the effects of these neurotoxins on other aspects of brain metabolism are scarce. The data presented here show that MPTP and MPP+ inhibit glucose oxidation via the tricarboxylic acid cycle, and acetylcholine synthesis in synaptosomal preparations from rat forebrain. Monoamine oxidase B inhibitors (e.g., pargyline, MDL 72145) relieve the inhibition caused by MPTP but not MPP+. The inhibitory effects of MPP+ on glucose oxidation and acetylcholine synthesis are a consequence of the decreased glucose metabolism in synaptosomes and are consistent with its role as an inhibitor of the Complex I (NADH-CoQ reductase) of the mitochondrial respiratory chain.

Notes: Abstract: Reserpine, a competitive inhibitor of catecholamine transport into adrenal medullary chromaffin vesicles, consists of a trimethoxybenzoyl group esterified to an alkaloid ring system. Reserpine inhibits norepinephrine transport with a Ki of ∼ 1 nM and binds to chromaffin-vesicle membranes with a KD of about the same value. Methyl reserpate and reserpinediol, derivatives that incorporate the alkaloid ring system, also competitively inhibit norepinephrine transport into chromaffin vesicles with Ki values of 38 ± 10 nM and 440 ± 240 nM, respectively. Similar concentrations inhibit [3H]reserpine binding to chromaffin-vesicle membranes. 3,4,5-Trimethoxybenzyl alcohol and 3,4,5-trimethoxybenzoic acid, derivatives of the other part of the reserpine molecule, do not inhibit either norepinephrine transport or [3H]reserpine binding at concentrations up to 100 μM. Moreover, trimethoxybenzyl alcohol does not potentiate the inhibitory action of methyl reserpate. Therefore, the amine binding site of the catecholamine transporter appears to bind the alkaloid ring system of reserpine rather than the trimethoxybenzoyl moiety. The more potent inhibitors are more hydrophobic compounds, suggesting that the reserpine binding site is hydrophobic.

Notes: Abstract: Omission of Mg2+ from the incubation buffer results in a six- to eightfold increase in [3H]inositol-1-phosphate ([3H]Ins-1-P) accumulation in primary cultures of cerebellar granule cells at 7–9 days in vitro. This increase is reversed by low concentrations of 2-amino-5-phosphonovalerate (APV), a result indicating that the absence of Mg2+ facilitates the activation of a specific receptor by the endogenous excitatory amino acids (presumably l-glutamate and l-aspartate) released from the granule cells. The absence of Mg2+ also potentiates the action of exogenously applied N-methyl-d-aspartate (NMDA), l-glutamate, l-aspartate, and kainate. In contrast, the action of quisqualate is virtually unaffected by Mg2+ and is resistant to APV inhibition. Addition of the depolarizing agent veratridine enhances the accumulation of [3H]Ins-1-P also in Mg2+-containing buffer. The action of veratridine is antagonized by APV, a result suggesting that, under depolarized conditions, the NMDA receptor can be activated by the endogenously released excitatory amino acids, despite the presence of Mg2+. Accordingly, in the presence of Mg2+, veratridine potentiates the action of exogenously applied NMDA but does not facilitate the action of quisqualate.

Notes: Abstract: Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain mi-crotubule preparations. Both antibodies bind to the 200-ki-lodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and t. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing elec-troblotted NF proteins with Escherichia coli alkaline phos-phatase completely blocks AD 10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with α-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD 10 and AB18 in NF-H is ∼ 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the α-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.

Notes: Abstract: Saturable low-affinity binding sites for [3H]mazin-dol have been demonstrated in crude synaptosomal membranes from rat brain using both a centrifugation and a fil-tion assay. Studies on the regional distribution of these binding sites revealed that the hypothalamus and brainstem had the highest density of sites. Kinetic analysis of the binding of [3H]mazindol to hypothalamic membranes demonstrated a single class of noninteracting binding sites with an apparent affinity constant (KD) of 10.2 ± 0.7 μM and maximal number of binding sites (Bmax) of 786 ± 94 pmol/mg of protein. Specific [3H]mazindol binding was rapidly reversible, temperature sensitive, labile to pretreatment with proteolytic enzymes, and inhibited by physiological concentrations of sodium. In most peripheral tissues, such as the liver and kidney, very low levels of binding were observed; however, the adrenal gland had a relatively high density of sites. The potency of a series of anorectic drugs in inhibiting specific [3H]mazindol binding to hypothalamic membranes was highly correlated with their anorectic potencies in rats, but not with their motor stimulatory effects. These results suggest the presence of a specific drug recognition site in the hypothalamus that may mediate the anorectic activity of mazindol and related phenylethylamines.

Notes: Abstract: The presence of the biologically uncommon D-aspartic acid (D-aspartate) in hu manbrain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to ∼35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartateratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.

Notes: Abstract: The binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS), a γ-aminobutyric acid (GABA)-activated chloride ionophore ligand; [3H]diazepam, a benzodiazepine agonist; and [3H]muscimol, a GABA receptor probe, were used to assess the effects at 100 μM of deltamethrin, dichlorodiphenyltrichloroethane (DDT), and three experimental insecticides—a DDT-pyrethroid hybrid, GH414 (cycloprothrin), and two DDT-analogues, GH266 and GH149 (EDO), on GABA receptor ionophore complexes in a rat brain membrane preparation. GH266 and GH149 were found to inhibit a greater percentage of [35S]TBPS binding than the same concentration of deltamethrin or DDT, although GH414 had little effect. GH266 and GH149 enhanced [3H]diazepam binding by nearly 200%, in contrast to the inhibitory effects of deltamethrin, DDT, and GH414. GH266 and GH149 also caused a dramatic enhancement of [3H]muscimol binding, 367 and 236% of control, respectively, whereas DDT and deltamethrin caused only a moderate enhancement. The effects of the insecticides on binding affinity and density were examined for each of the ligands. The results show a differential interaction of the insecticides on the various ligand binding sites.

Notes: Abstract: The effects of corticostriatal deafferentation (de-cortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra-and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. γ-Aminobutyric acid (GABA), tau-nne (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month postlesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of nonneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain bamer transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine. GABA, Tau, and PEA effluxes were also decreased in kainate-lesioned striata. These findings are consistent with the proposed roles of an acidic amino acid as the corticostriatal transmitter, and of GABA as a transmitter in intrinsic striatal neurons. The existence of releasable pools of Tau and PEA within kainate-sensitive striatal neurons would also appear to be likely.

Notes: Abstract: The effect of severe insulin-induced hypoglycemia on the extracellular levels of endogenous amino acids in the rat striatum was examined using the brain microdialysis technique. A characteristic pattern of alterations consisting of a 9–12-fold increase in aspartate (Asp), and more moderate increases in glutamate (Glu), taurine (Tau), and γ-aminobutyric acid (GABA), was noted following cessation of electroencephalographic activity (isoelectricity). Glutamine (Gln) levels were reduced both during and after the isoelectric period and there was a delayed increase in extracellular phosphoethanolamine (PEA) content. The effects of decortication and excitotoxin lesions on the severe hypoglycemia-evoked efflux of endogenous amino acids in the striatum were also examined. Decortication reduced the release of Glu and Asp both 1 week and 1 month post-lesion. The efflux of other neuroactive amino acids was not affected significantly. In contrast, GABA, Tau, and PEA efflux was attenuated in kainate-lesioned striata. Glu and Asp release was also reduced under these conditions, and a smaller decrease in extracellular Gln was noted. These data suggest that GABA, Glu, and Asp are released primarily from their transmitter pools during severe hypoglycemia. The releasable pools of Tau and PEA appear to be located in kainate-sensitive striatal neurons. The significance of these results is discussed with regard to the excitotoxic theory of hypoglyce-mic cell death.

Notes: Abstract: Concentrations of proenkephalin B (PENK B) mRNA in porcine brain, pituitary, spinal cord, and peripheral tissues were measured using RNA blotting and solution hybridization. A single hybridizing species of ∼2,800 bases in size was present in the CNS, with the highest concentration in the caudate nucleus, followed by hypothalamus and hippocampus. The abundance of PENK B mRNA ranged from 22 pg/μg of poly(A)-rich RNA in caudate nucleus to 〈0.1 pg/μg in cerebellum. Concentrations of immunoreac-tive PENK B-derived peptides showed a similar distribution, with the exception of the hypothalamus, which had lower PENK B mRNA levels than expected from peptide concentrations. PENK B mRNA of the same size as in the brain was also found in the anterior lobe of the pituitary and in the heart ventricle, whereas in intestine, lung, and kidney, smaller mRNA species of 1,800 bases became apparent by RNA blot analysis. An intermediate size of 2,200 bases was found in heart atrium. As revealed by S1 mapping, however, these smaller mRNAs are not completely homologous with PENK B mRNA, but rather may represent closely related mRNAs from a different gene(s).

Notes: Abstract: The relative distribution of type A and type B monoamine oxidase (MAO) inside and outside the monoaminergic synaptosomes in preparations from hypothalamus and striatum of the guinea pig was determined by incubation of synaptosomal preparations of these regions with low concentrations of [14C]5-hydroxytryptamine (5-HT), noradrenaline, and dopamine. The deamination within the monoaminergic synaptosomes was hindered by selective amine uptake inhibitors. In the absence of these inhibitors, both intra- and extraneuronal deamination was measured. The two forms of the enzyme were differentiated with the irreversible and selective MAO-A and MAO-B inhibitors clorgyline and selegiline (1-deprenyl), respectively. [14C]5-HT was deaminated 〉90% by MAO-A both inside and outside the 5-hydroxytryptaminergic synaptosomes prepared from the guinea pig hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes of the hypothalamic preparation was in the ratio 75:25% for MAO-A:MAO-B; the corresponding ratio outside these synaptosomes was 45:55%. The deamination of [14C]dopamine within dopaminergic synaptosomes in the striatal preparation was 65% type A:35% type B, whereas outside these synaptosomes the ratio was 35:65%. Because the relative amounts and the distribution of the two forms of MAO in the guinea pig brain seem to be similar to those previously detected for the human brain, the MAO in the guinea pig brain may be a good model for the MAO in the human brain.

Notes: Book reviews in this article: Epilepsy and GABA Receptor Agonists: Basic and Therapeutic Research edited by G. Bartholini, L. Bossi, K. G. Lloyd, and P. L. Morselli. GABA and Mood Disorders: Experimental and Clinical Research edited by G. bartholini, K. G. Lloyd, and P. L. Morselli.

Notes: Abstract: N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively destroys neuronal cell bodies in the neuromelanin-containing substantia nigra of humans and primates. We show that N-methyl-4-phenylpyridine (MPP+), the active metabolite of MPTP, binds to neuromelanin with high affinity. This binding increases at higher pH and is displaced most potently by divalent cations and antimalarial drugs. MPP+ bound intracellularly to neuromelanin may be stored and released gradually, resulting in subsequent damage to neurons of the substantia nigra.

Notes: Abstract: A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-IIe-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.

Notes: Abstract: Incubated slices and freshly dissociated cells from 8-day-old rat cerebellum were used to try to identify the cells that participate in the large increases in cyclic GMP levels that follow activation of excitatory amino acid receptors in this tissue. In the slices, cyclic GMP responses to L-gluta-mate and related excitants were unaffected by tetrodotoxin and could be replicated by the guanylate cyclase activator nitroprusside. Nitroprusside and the receptor agonists appeared to activate the same pool of the enzyme. Prior destruction of neuroblasts, deep nuclei, or Golgi neurones did not cause loss of responses to L-glutamate. If granule cells were rendered necrotic, however, the cyclic GMP responses to all excitants tested were reduced by ≧ 90%. Substantial losses of responses to veratridine and high K+ levels also occurred, but the nitroprusside-induced elevations were unaffected. In dissociated cell suspensions, the magnitude of responses to receptor agonists, but not those to nitroprusside, was markedly dependent on cell concentration. Responses to L-glutamate were the same in cell suspensions that were Purkinje cell depleted and Purkinje cell enriched. It is concluded that granule cells are primarily involved in the cyclic GMP responses to excitatory amino acids but that the cyclic GMP accumulations occur elsewhere, probably in glial cells.

Notes: Abstract: Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo-inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V1max for inositol uptake was significantly decreased with little change in the K1max. These data suggest that glucose decreases myo-inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo-inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo-inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.

Notes: Abstract: Positron emission tomography (PET) with labeled neuroleptics has made possible the study of neurotransmitter-receptor systems in vivo. In this study we investigate the kinetics of the 3, 4-dihydroxyphenylethylamine (dopamine) receptor-ligand binding using PET data from a series of experiments in the baboon with the 18F-labeled drugs spiperone, haloperidol, and benperidol. Models used to describe these systems are based on first-order kinetics which applies at high specific activity (low receptor occupancy). The parameters governing the uptake and loss of drug from the brain were found by fitting PET data from regions with little or no receptor concentration (cerebellum) and from experiments in which specific binding was blocked by pretreatment with the drug (+)-butaclamol. Receptor constants were determined by fitting data from receptor-containing structures. Correcting the arterial plasma activities (the model driving function) for the presence of drug metabolites was found to be important in the modeling of these systems.

Notes: Abstract: Peripheral nerve transection triggers a series of phenotypic alterations in Schwann cells distal to the site of injury. Mitosis is one of the earliest and best characterized of these responses, although the mechanism by which axonal damage triggers this critical event is unknown. This study examines the appearance and spatio-temporal spread of premitotic activity in distal stumps of transected cat tibial nerves. Premitotic activity was determined by measuring incorporation of [3H]thymidine (a marker of DNA synthesis during the S-phase of the cell cycle) into consecutive segments of desheathed tibial nerve. Incorporation of [3H]thymidine spread proximo-distally within distal nerve stumps between 3 and 4 days posttransection with an apparent velocity of at least 199 ± 67 mm/day. This suggests that anterograde fast axonal transport may directly or indirectly be associated with the Schwann cell mitotic response to axon transection.

Notes: Abstract: We have used biologically active derivatives of β-nerve growth factor (NGF), modified by biotinylation via carboxyl groups, to target the specific binding of liposomes to cultured rat and human tumor cells bearing NGF receptors. Liposomes, to be used for targeting, were prepared by conjugating streptavidin to phospholipid amino groups on liposomes prepared by reverse-phase evaporation. Approximately 2,000 streptavidin molecules were incorporated per liposome. Addition of biotinylated NGF, but not of unmodified NGF, could mediate the subsequent binding of radiola-beled streptavidin-liposomes to rat pheochromocytoma PC 12 cells in suspension at 4°C. In contrast, incubation with biotinylated NGF did not mediate the binding of hemoglobin-conjugated liposomes. Under optimal incubation conditions, approximately 570 streptavidin-liposomes were specifically bound per cell. Biotinylated NGF was also used to obtain specific binding of streptavidin-liposomes containing encapsulated fluorescein isothiocyanate-labeled dextran to PC12 cells or human melanoma HS294 cells. When HS294 cells were incubated at 37°C following targeted liposome binding at 4°C, the cell-associated fluorescence appeared to become internalized, displaying a perinu-clear pattern of fluorescence similar to that observed when lysosomes were stained with acridine orange. Trypsin treatment abolished cell-associated fluorescence when cells were held at 4°C but did not alter the fluorescence pattern in cells following incubation at 37°C. When liposomes containing carboxyfluorescein, a dye capable of diffusing out of acidic compartments, were targeted to HS294 cells, subsequent incubation at 37°C resulted in diffuse cytoplasmic fluorescence, suggesting that internalized liposomes encounter ly-sosomal or prelysosomal organelles.

Notes: Abstract: [3H]Neurokinin B ([3H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [3H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (KD= 4.3 nM; Bmax= 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB 〉 eledoisin (E) 〉 kassinin 〉 physalaemin 〉 neurokinin A (NKA) 〉 substance P (SP), a result suggesting that NKB might be the endogenous ligand for [3H]NKB binding sites. It is of interest that 127I-Bolton Hunter (BH) NKA (127I-BHNKA) was much more potent than NKA in inhibiting the specific binding of [3H]NKB, which raises certain questions concerning the use of 125I-BHNKA as a Iigand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with 125I-BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [3H]NKB or 125I-BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and 127I-BHE. The similarity of the [3H]NKB and 125I-BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [3H]NKB and 125I-BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.

Notes: Abstract: Direct treatment of brain myelin with freezing/thawing in 0.2 M2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-la-beled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M2-mercaptoethanol and with a molecular weight of 350, 000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP. Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.

Notes: Abstract: The effects of γaminobutyric acid (GABA) on the uptake of 36Cl into a membrane microsac preparation from isolated nerve cords of the cockroach Periplaneta americana was studied. On addition of 1 μM GABA (after 4-s incubation, then rapid quenching) the influx of 36Cl- was stimulated to a level 75% above that of the control value. This stimulation was reduced by picrotoxin (100 μM), but was not significantly affected by bicuculline (100 μM)Results of 36Cl-influx experiments are in agreement with data obtained from radiolabelled ligand binding assays and electrophysiological investigations on the same tissue. The method described represents a functional in vitro assay for CNS GABA receptors of insects.

Notes: Abstract: In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70–80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionme in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (〈7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.

Notes: Abstract: The nerve growth factor (NGF) receptor, solubilized with Triton X-100 detergent, has been purified from human melanoma cell line A875. Purification to near-homogeneity was achieved by chromatography on wheat germ agglutinin-agarose, followed by immunoaffinity chromatography on Sepharose columns coupled with anti-NGF receptor monoclonal antibody (MAb). The purified receptor, a 75, 000-dalton protein, retains the capacity to bind NGF as well as anti-receptor MAbs. Final purification was achieved by sodium dodecyl sulfate-polyacryiamide gel electrophoresis. The sequence of amino acid residues at the amino terminus has been determined. Possible sequence homology between the NGF receptor and several other proteins is discussed. Using the purified receptor as immunogen, new MAbs to the NGF receptor have been produced. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of dorsal root ganglia from monkeys.

Notes: Abstract: Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.

Notes: Abstract: 5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 ± 10-7M. The phosphoinositide response was blocked by the 5-HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by ˜30%. The 5-HT1A agonist 8-hydroxy-2 (di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 μM) for 1 h increased the incorporation of [2-3H]myo-inositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-3H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 μM). Radioactivity in IP2 and IP3 was very low, was stimulated ˜50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was ˜40 times that in IP3. The EC50 value of 1.8 ± 10-7M was comparable with that obtained from 32Pi studies measuring labeled PI. Ketanserin and spiperone inhibited 5-HT-stimulated [2-3H]IP release with IC50 values of 3.1 ± 10-9 and 1.8 ± 10, -8M, respectively. This study demonstrates that phosphoinositide hydrolysis is enhanced by 5-HT in C6 glioma cells and that this phenomenon is linked to 5-HT2-like binding sites.

Notes: Abstract: Bovine chromaffin granules from adrenal medulla contain three acidic secretory proteins: chromogranins A, B, and C. For isolation of these proteins, methods based mainly on high performance liquid chromatography were developed. After removal of contaminating glycoproteins by lectin affinity chromatography, chromogranins were separated by high performance anion-exchange, gel-filtration, and reverse phase liquid chromatography. As a final purification step sodium dodecyl sulfate-gel electrophoresis was performed. Amino acid analysis of isolated bovine chromogranins revealed a similar composition of all three proteins, with glutamic acid being the most prominent amino acid. The methods developed for bovine proteins also proved suitable for isolating rat chromogranins A and B from a transplantable pheochromocytoma. Chromogranin C was not present in sufficient amounts to be isolated from this tissue. The chromogranins purified by these methods were used to raise specific antibodies in rabbits. The use of purified chromogranins together with specific antisera may be valuable in understanding the still undiscovered function of these proteins.

Notes: Abstract: Luteinizing hormone-releasing hormone was degraded by cells of the N- I8 line of mouse neuroblastoma and their membrane. Cleavage products were separated by HPLC and identified by amino acid analysis. Fragments (1-3), (4-5), and (6-10) were major cleavage products. All the products increased in level as a function of time except for fragment (1-9, which increased in amount only during a short incubation time and then decreased. The accumulation of fragment (1-5) was increased in the presence of captopnl or EDTA, whereas that of fragments (1-3) and (4-5) decreased inversely. On the other hand, the generation of either fragment (1-3) or (4-5) was stimulated by the presence of C1-. The results suggest that the conversion of fragment (1-5) into fragments (1-3) and (4-5) is catalyzed by angiotensin-converting enzyme. p-Chloromercuribenzoate inhibited the formation of fragment (1-5), a result suggesting the involvement of a thiol protease in this formation. Thus, the degradation of luteinizing hormone-releasing hormone by neuroblastoma cells and their membrane seems to take place mainly through the cleavage of the Tyr5-Gly6 bond by a thiol protease, followed by the release of the dipeptide Ser-Tyr from the liberated fragment (1-5) by angotensin-converting enzyme. It is further suggested that the thiol protease and angiotensin-converting enzyme are also responsible for the initial minor cleavages of the Gly6-Leu7 bond and the Trp3-Ser4 bond, respectively.

Notes: Abstract: Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51, 000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51, 000 (P51) and 55, 000 (P55) in hippocampus. Photolabeling was inhibited by 10 μM diazepam but not by 10 μM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and ß-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15–4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same pro-teins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51, 000 and/or 55, 000. In cerebellum, an additional protein (MW 57, 000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15–4513 in the granular than the molecular layer.

Notes: Abstract: The phosphorylation of microtubule-associated protein 2 (MAP2) by four different kinases was studied in vitro to determine whether MAP2 is phosphorylated in its tubulin binding region or in the microtubule projection portion. Fragments corresponding to both regions of MAP2 were produced not only by chymotrypsin or trypsin digestion, but also using pepsin, a broad chain-specificity protease, a result supporting previous notions of the two-domain structure of MAP2. The position of these two functional domains was determined with respect to the carboxy terminal of the molecule, by labeling MAP2 exclusively at the carboxy terminal and subjecting it to pepsin digestion. The results suggested that the projection region of MAP2 contained the carboxy terminal of the protein. A phosphorylation map was constructed by subjecting phosphorylated MAP2 to enzymatic digestion using Staphylococcus aureus V8 protease or to chemical cleavage using N-chlorosuccinimide. The results indicated that all four kinases phosphorylated MAP2 in a 42-kilodalton peptide that contained the tubulin binding region but differed in the level and localization of the sites at which they phosphorylated the projection of MAP2.

Notes: Abstract: Elevated blood levels of prolactin increase the synthesis, turnover, and release of 3, 4-dihydroxyphenylethylamine (dopamine) from the tuberoinfundibular dopaminergic neurons, which project to the median eminence. The present study examined whether hyperprolactinemia also increases local cerebral glucose utilization, as determined by the 2-deoxy-D-[I-14C]glucose method, in the median eminence and other brain structures. Adult male rats were given ovine prolactin (4 mg/kg) subcutaneously every 8 h for 48 h. This treatment exerted an autoregulatory feedback effect on endogenous rat prolactin secretion, as evidenced by decreased circulating levels of rat prolactin. Ovine prolactin treatment also decreased plasma glucose concentrations. However, in both partially immobilized and free-ranging rats, glucose utilization in brain structures containing tuberoinfundibular dopaminergic cell bodies (the arcuate nucleus) and terminals (the median eminence) was not affected by ovine prolactin treatment. Hyperprolactinemia was, however, associated with decreased glucose utilization in the medial forebrain bundle and the CA subfield of the dorsal hippocampus. The lack of a significant effect of prolactin treatment on glucose utilization in the median eminence indicates (a) that the resolution of the deoxyglucose technique, as used here, is not adequate to detect the ovine prolactin-induced increase in tuberoinfundibular dopaminergic neuronal activity, (b) that the median eminence does not utilize glucose as its primary energy substrate, or (c) that ovine prolactin treatment causes a counterbalancing decrease in the activity of other neurons projecting to the median eminence.

Notes: Abstract: The activity of adenosine deaminase (ADA) was determined in whole brain of rats at the embryonic age of 15 days through to adulthood and in nine brain regions in rats 1 day old through to adulthood. In 1-day-old rats, the highest activity was seen in olfactory bulbs (550 ± 15 nmol/mg protein/30 min) and this was 4.5-fold higher than that in the pons, which was the lowest. In adult animals, olfactory bulb still contained the greatest activity, which was about eightfold higher than hippocampus, which had the lowest. Except for hypothalamus, where ADA activity increased nearly twofold in rats between the ages of 1 and 50 days, significant decreases of as much as fivefold were found in whole brain, superior colliculus, cortex, hippocampus, cerebellum, olfactory bulbs, and olfactory nucleus. In contrast, ADA activity in pons and subcortex remained relatively constant throughout the developmental period. The Km values for ADA in whole brain at 18 days gestation (48 ± 5 μM) were not significantly different from that observed in adult rats (38 ± 7 μM), whereas the Vmax values decreased significantly from 339 ± 9 to 108 ± 8 nmol/mg protein/30 min. Taken together, the developmental patterns observed in the various brain regions appear not to correspond to any one particular process such as periods of rapid cell proliferation, cell death, synaptogenesis, or myelination. Nor do they correspond to known developmental profiles of transmitters, their receptors, or their metabolic enzymes. The complex changes in ADA activity during ontogenesis suggest an important role of ADA at very early stages of development as well as in specific regions of the adult rat brain.

Notes: Abstract: Neurochemical correlates of GABAergic synaptic transmission [binding, uptake, metabolism, and tissue content of γ-aminobutyric acid (GABA)] were investigated in the cortex of rats that had been given 27 mM bromide in drinking water for periods of time ranging from 1 day to 1 month. No effect of bromide on any of the parameters was found and it is concluded that chronic administration of bromide has no profound effect on GABAergic inhibitory system in the rat cortex.

Notes: Abstract: I examined whether the phorbol ester-mediated inhibition of glycerol 3-phosphate dehydrogenase (GPDH) induction could be mimicked by raising the cellular diacylglycerol levels. Phorbol ester tumor promoters and diacylglycerols activate protein kinase C. An increase in radiolabeled diacylglycerol levels in C6 rat glioma cells was observed when cells were prelabeled overnight with [3H]arachidonic acid and treated with either phospholipase C (Clostridium perfringens) or 2-bromooctanoate. The increase was dose dependent. The diacylglycerols competed with [20-3H]phorbol 12, 13-dibutyrate in binding to the phorbol ester receptor. A Scatchard analysis of the binding of cells treated with 0.1 unit/ml of phospholipase C demonstrated that the inhibition was mainly due to a decrease in binding affinity and not in the total number of binding sites. 2-Bromooctanoate and phospholipase C, but not the synthetic diacylglycerol l-oleoyl 2-acetyl glycerol, inhibited the glucocorticoid induction of GPDH levels. Boiled phospholipase C, phospholipase A2, or phospholipase D was ineffective in inhibiting induction, a result suggesting that the inhibition was not due to nonspecific membrane perturbation. Thus, inhibition of the glucocorticoid-mediated increase in GPDH induction is most likely mediated by protein kinase C, and not by an alternate phorbol ester receptor.

Notes: Abstract: The presence of γ-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various γ-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosme-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.

Notes: Abstract: Superior cervical ganglion phospholipase A2 activity was characterized using l-palmitoyl-2-[l-14C]arachidonoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme activity exhibited linearity with interval of incubation and tissue concentration; there appeared to be two pH optima of the enzyme, at pH 6.0 and 9.0. A Lineweaver-Burk plot of the reciprocal of activity versus substrate concentration yielded an apparent Km of 0.53 mM and a Vmax of 5.3 nmol/h/mg of protein. The enzyme exhibited a partial Ca2+ dependence; in the absence of Ca2+ and presence of EGTA, activity was reduced by 40%. The phospholipase A2 activity was heat sensitive and was completely inactivated after treatment at 100°C for 30 min. For determination of whether the enzyme had a preference for hydrolysis of specific fatty acid substituents in the 2 position of phosphatidylcholine, several different substrates were tested. The order of preference for hydrolysis by the ganglionic enzyme was l-palmitoyl-2-[l-14C]arachidonoyl-sn-glycero-3-phosphocholine = l-palmitoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine 〉 l-palmitoyl-2-[l-14C]palmitoyl-sn-gly-cero-3-phosphocholine. For determination of the localization of the phospholipase A2 enzyme in sympathetic ganglia, two approaches were used. Guanethidine, which results in destruction of adrenergic cell bodies in sympathetic ganglia, was administered to rats; an ˜50% decline in phospholipase A2 activity was observed after this treatment. In other experiments, the preganglionic nerve to the ganglion was sectioned in rats; after 2 weeks of denervation, no significant change in ganglionic phospholipase A2 activity was seen, although after 4 weeks of denervation, a small but non-significant decrease in enzyme activity was observed. These results suggest that the enzyme has a predominantly post-synaptic localization. Extrinsic phospholipase A2 enzymes have previously been shown to affect postsynaptic function in rat sympathetic ganglia. The presence of a phospholipase A2 in the tissue itself could suggest that endogenous phospholipase A2 enzymes can regulate postsynaptic neuronal function.

Notes: Abstract: Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 μM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 μM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mMK+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.

Notes: Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13–21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that ∼20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from ∼18,000 to 180,000 and in p1 from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including α-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3–4 weeks after an optic tract lesion, five proteins, including protein 4, s lowed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least bur others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.

Notes: Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys-Arg) typical processing site, would generate two opioid fragments, the amino-terminal fragment BAM 18 and the carboxy-terminal fragment Leu-enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu-enkephalin). Here we present evidence that BAM 18 and Leu-enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys-Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu-enkephalin was found to be present in much greater amounts than was BAM 18-like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu-enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu-enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu-enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.

Notes: The two avian benzodiazepine binding proteins offer an opportunity for further studies concerning their regional variation and their phylo-and ontogenetic development. Accordingly, regional variation of the benzodiazepine binding proteins is investigated further in two reptiles and chicken using photoaffinity labeling with [3H]flunitrazepam followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Whereas regional heterogeneity is pronounced in chicken, it is not readily apparent in the two reptiles. The ontogeny of the benzodiazepine binding proteins in chicken forebrain and cerebellum is remarkably similar to that previously reported in rodents. The results are discussed in light of the possible existence of the γ-aminobutyric acid/benzodiazepine receptor as an isoreceptor complex.

Notes: Abstract: The in vivo labeling of electrocyte lipids is followed after injection of radioactive glycerol and two fatty acids, oleate and arachidonate, into the electric organ of an elasmobranch (Discopyge tschudii). De novo synthesis of lipids and acyl-exchange reactions are operative in the electrocyte. The three precursors are preferentially incorporated into phosphatidylcholine, phosphatidylinositol, and triacylglycerols. The highest specific activities are attained by triacylglycerols and polyphosphoinositides. Electrocyte stacks from electric organ show an efficient and continuous esterification of oleate and arachidonate into lipids after several hours of incubation. Except for an apparently more active labeling of triacylglycerols, which is attributed to the larger availability of free fatty acid precursors under the in vitro experimental conditions, the pattern of lipid labeling is similar to that attained in vivo. 32P-labeled lipids are also steadily produced in electrocyte stacks (24 h of incubation with [32P]phosphate) using glucose as the sole exogenous source of energy. Polyphosphoinositides are the lipids preferentially labeled. The ability to sustain the labeling of lipids under in vitro conditions renders isolated electrocyte stacks an interesting model for future research on lipid involvement in cholinergic function.

Notes: Abstract: The in vivo effects of kainate (1 mM) on fluxes of 45Ca2+, and endogenous amino acids, were examined in the rat striatum using the brain microdialysis technique. Kainate evoked a rapid decrease in dialysate 45Ca2+, and an increase in the concentration of amino acids in dialysates in Ca2+-free dialysates. Taurine was elevated six-to 10-fold, glutamate two-to threefold, and aspartate 1.5-to twofold. There was also a delayed increase in phosphoethanolamine, whereas nonneuroactive amino acids were increased only slightly. The kainic acid-evoked reduction in dialysate 45Ca2+ activity was attenuated in striata lesioned previously with kainate, suggesting the involvement of intrinsic striatal neurons in this response. The increase in taurine concentration induced by kainate was slightly smaller under these conditions. Decortication did not affect the kainate-evoked alterations in either dialysate 45Ca2+ or amino acids. These data suggest that kainate does not release acidic amino acids from their transmitter pools located in corticostriatal terminals.

Notes: Abstract: Mitochondrial respiratory function, assessed from the rate of oxygen uptake by homogenates of rat brain subregions, was examined after 30 min of forebrain ischemia and at recirculation periods of up to 48 h. Ischemia-sensitive regions which develop extensive neuronal loss during the recirculation period (dorsal-lateral striatum, CA1 hippocampus) were compared with ischemia-resistant areas (paramedian neocortex, CA3 plus CA4 hippocampus). All areas showed reductions (to 53–69% of control) during ischemia for oxygen uptake rates determined in the presence of ADP or an uncoupling agent, which then recovered within 1 h of cerebral recirculation. In the ischemia-resistant regions, oxygen uptake rates remained similar to control values for at least 48 h of recirculation. After 3 h of recirculation, a significant decrease in respiratory activity (measured in the presence of ADP or uncoupling agent) was observed in the dorsal-lateral striatum which progressed to reductions of greater than 65% of the initial activity by 24 h. In the CA 1 hippocampus, oxygen uptake rates were unchanged for 24h, but were significantly reduced (by 30% in the presence of uncoupling agent) at 48 h. These alterations parallel the development of histological evidence of ischemic cell change determined previously and apparently precede the appearance of differential changes between sensitive and resistant regions in the content of high-energy phosphate compounds. These results suggest that alterations of mitochondria activity are a relatively early change in the development of ischemic cell death and provide a sensitive biochemical marker for this process.

Notes: Abstract: The binding of [3H]PK 11195 and [3H]Ro 5–4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05–10 nM) bound with high affinity to rat and alf cerebral cortical and kidney membranes. [3H]Ro 5–4864 (0.05–30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5–4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf, tissues. The potency of unlabeled Ro 5–4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig 〈 cat = dog 〈 rabbit 〈 calf. Analysis of these displacement curves revealed that Ro 5–4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat 〈 calf 〈 guinea pig 〈 rabbit 〈 dog 〈 rat, whereas when [3H]Ro 5–4864 was used, the rank order of binding capacity was cat 〈 guinea pig 〈 rat 〈 rabbit 〈 calf 〈 dog. These results further demonstrate species differences of “peripheral-type” benzodiazepine binding sites and also provide evidence of their heterogeneity in the kidney of rat and calf and in the cerebral cortex of rat, guinea pig, rabbit, and calf.

Notes: Abstract: A calcium binding protein that is biochemically similar to vertebrate 28,000-Mr vitamin D-dependent calcium binding protein (calbindin-D28k) has been purified from squid brain. Squid brain calbindin was found to have an isoelectric point of 5.0, was heat stable up to 60°C, and showed increased electrophoretic mobility in the presence of chelator Amino acid analysis revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, and tyrosine, a finding similar but not identical to the composition of vertebrate calbindin-D28k. The molecular weight of the squid protein, determined by Ferguson plot analysis of data obtained from sodium dodecyl sulfategel electrophoresis, was calculated to be 25,700, as compared with 27,800 for rat renal calbindin. Immunocyto-chemical analysis demonstrated immunoreactive protein in a selected population of neurons and fibers in several areas of the molluscan nervous system. This study represents the first purification from an invertebrate of a calcium binding protein that is biochemically similar to vitamin D-dependent calcium binding protein. These results demonstrate that calbindin, although not identical in vertebrates and cephalopods, may be phylogenetically conserved in structure. The restricted distribution of immunoreactive calbindin in both the cephalopod and mammalian brain suggests that the function of neuronal calbindin may also be conserved in evolution.).

Notes: Abstract: The effect of treatment with the γ-aminobutyric acid (GABA) agonist tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) on neural development was monitored in rats by following the expression of the neuron-specific proteins neural cell adhesion molecule (NCAM), D1, and D3 as well as the enzymes glutamate decarboxylase (GAD) and glutamate dehydrogenase (GLDH). As judged from the effect of the treatment on the expression of NCAM and GAD, GABA agonists have the capacity to accelerate and enhance neuronal development during the early postnatal period. However, as judged from the expression of D1-and D3-protein some adverse late effects may result from prolonged treatment with high doses of GABA agonists. The decrease in GLDH specific activity observed in THIP-treated rats during their late postnatal development possibly indicates a repression of glutamatergic neurons.

Notes: Abstract: Acetylcholinesterase (AChE) from housefly heads was purified by affinity chromatography. Three different native forms were separated by electrophoresis on poly-acrylamide gradient gels; Two hydrophilic forms presented apparent molecular weights of 75,000 (AChE1) and 150,000 (AChE2). A third component (AChE3) had a migration that depended on the nature and concentration of detergents. In the presence of sodium deoxycholate in the gel, AChE3 showed an apparent molecular weight very close to that of AChE2. Among the three forms, AChE3 was the only one found in purified membranes. The relationships among the various forms were investigated using reduction with 2-mercaptoethanol or proteolytic treatments. Such digestion converted purified AChE3 into AChE2 and AChE1, and reduction of AChE3 and AChE2 by 2-mercaptoethanol gave AChE1, in both cases with a significant loss of activity. These data indicate that the three forms of purified AChE may be classified as an active hydrophilic monomeric unit (G1) plus hydrophilic and amphiphilic dimers. These two components were termed G2sand G2m, where “s” refers to soluble and “m” to membrane bound.).

Notes: Abstract: The source of norepinephrine (NE) in CSF has been unclear. It has been suggested that CSF NE indicates central neural noradrenergic tone and is determined differently from plasma NE. If CSF NE depended specifically on NE release in the CNS, then interference with ganglionic neurotransmission would be expected to decrease plasma NE but not CSF NE. Hypotension caused by ganglionic blockade might be expected to increase CSF NE reflexively. We infused the ganglion blocker, trimethaphan, intravenously into anesthetized dogs and measured the effects on mean arterial blood pressure (MAP) and on cisterna magna CSF levels of NE. The results were compared with those obtained on administration of saline, clonidine (2 μg/kg), yohimbine (0.25 mg/kg), or nitroprusside and with those obtained when hypotension during ganglion blockade was prevented by concurrent treatment with phenylephrine. Trimethaphan decreased MAP by 40%, arterial NE by 64%, and CSF NE by 61%. Nitroprusside administered intravenously to produce the same 40% depressor response increased arterial NE by 612% and CSF NE by 155%. Prevention of ganglion blockade-induced hypotension using phenylephrine did not prevent the decrease in CSF NE caused by trimethaphan, and when phenylephrine was discontinued, the resulting hypotension was not associated with increases in CSF NE. The similar decreases in plasma NE and CSF NE during ganglionic blockade, and the abolition of reflexive increases in CSF NE during hypotension in ganglion-blocked subjects, cast doubt on the hypothesis that CSF NE indicates central noradrenergic tone and are consistent instead with at least partial derivation of CSF NE from postganglionic sympathetic nerve endings.

Notes: Abstract: Transferrin (Tf), the iron mobilization protein, is synthesized mainly in the liver. Recently, both Tf and a mRNA for Tf have been demonstrated in oligodendrocylcs in the rat brain. The present study used a biochemical assay for determining the levels of Tf in various brain regions of normal rats compared with the level of those obtained from rats with a genetic mutation characterized by an almost complete failure to develop myelin. In myelin-deficient (md) rats, no Tf-positive oligodendrocytes were seen immunohistochemically in the gray or white matter of the CNS. Quantitatively, levels of Tf throughout the CNS of the md rat were decreased to ∼5% of the normal values despite a normal hepatic synthetic rate. In the normal rat brain, the cerebellum contained the highest concentration of Tf, followed by the pons, the cerebral cortex, and the caudate-putamen, with the latter two sites being similar. Regional variation in the amount of Tf was in general agreement with published reports on the variation of iron and Tf receptor levels in the CNS. Immunohistochemical examination with antiserum to galactocerebroside (a myelin-specific lipid) was used for extending biochemical reports that glycoiipid-synthesizing enzymes are deficient in md rats. No immunostaining in the md rat was observed following immunoreaction for galactocerebroside, whereas white matter oligodendrocytes were intensely marked in the normal rat. Robust astrogliosis was present in both the gray and white matter of the md rats. It is not known at present whether the ábility to accumulate Tf is necessary for oligodendrocytic survival or if Tf accumulation is more directly related to myelinogenesis. The lack of Tf in the md rat suggests a critical role for oligodendrocytes in Tf accumulation and, consequently, iron regulation in the CNS.

Notes: Abstract: S-100 protein in clonal GA-1 and C6 rat glioma cell lines was released in serum-free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S-100 protein release by ACTH was dose-dependent, showing a half-maximal release at about 5 μM, and the S-100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S-100 protein release was apparently accompanied by a decrease in the membrane-bound form of S-100 protein in the cell. The S-100 protein release was induced not by the ACTH1–24 fragment, which exhibits the known effects of ACTH, but by the ACTH18–39 fragment, which is designated as corticotropin-like intermediate-lobe peptide (CLIP). These results indicate that the C-terminal half of ACTH is responsible for the S-100 protein release. The enhancement of S-100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S-100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S-100 protein release; however, catecholamines (epinephrine, norepi-nephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release. The S-100 protein release was also stimulated by 5 mM ATP, ADP, and GTP, probably by lowering the concentration of Ca2+ in the medium, because addition of Ca2+ inhibited the release by the nucleotides (but not by the cyclic nucleotides or ACTH), and 1 mM EGTA could induce the S-100 protein release from GA-1 glioma cells.

Notes: Abstract: We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [3H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several-fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO-A and MAO-B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO-B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat microvessels after [3H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of ∼6.5,000 for MAO-A and -60,000 for MAO-B. SDS-PAGE resuits of brain microvessels obtained from 1-, 14-, and 42-day-old rats confirm the differential postnatal development of MAO-B in rat brain microvessels.

Notes: Abstract: We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carbox-ylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein frac-tionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3cells was incubated with S-adenosyl-l-[methyl-3H] methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.

Notes: Abstract: The present investigation examined the effects of neonatal and adult 6-hydroxydopamine (6-OHDA)-induced lesions of dopaminergic neurons on opioid and tachykinin peptides and their gene expression in the rat basal ganglia. This work was undertaken to determine if changes in these neuropeptide systems were contributing to the differing behavioral responses observed between neonatally and adult-lesioned rats after dopamine agonist administration. [Met5]Enkephalin (ME) content was increased in striatal tissue from both 6-OHDA-lesioned groups when compared with unlesioned controls. Dynorphin-A (1–8) content was not altered by the 6-OHDA lesions. The tachykinin peptides substance P and neurokinin A were significantly decreased in level in the striatum and substantia nigra of neonatally lesioned rats, but not in the adult-lesioned rats, when compared with unlesioned controls. Proenkephalin mRNA abundance (quantified by an RNA-cDNA hybridization technique) and precursor level (as reflected by cryptic ME content) were increased in the striatum of both neonatally and adult-lesioned rats. The abundance of preprotachykinin mRNA coding for the tachykinin peptides was markedly decreased in the neonatally lesioned rats, whereas only a small reduction was observed in the adult-lesioned rats. These results suggest that destruction of dopamine-containing terminals with 6-OHDA elevates the level of ME by accelerating transcriptional and/or translational processes; conversely, the reduced content of tachykinins in neonatally lesioned rats may be due to a reduction in such processes. Thus, preproenkephalin-A and preprotachykinin gene expression are differentially regulated after lesioning of catecholamine-containing neurons, an observation suggesting a close functional relationship among these neurotransmitter systems. Furthermore, of the peptides studied, only levels of the tachykinin peptides were differentially altered in the striatum and substantia nigra of the neonatally lesioned rats compared with adult-lesioned rats; therefore, these peptides may be associated with the distinctive behavioral differences between neonatally and adult 6-OHDA-lesioned rats given dopamine agonists.

Notes: Abstract: The association of neurotensin to its receptor in differentiated neuroblastoma N1E115 cells led to a fast and transitory increase of the intracellular concentration in inositol trisphosphate and inositol bisphosphate, followed by a slower and more stable increase in inositol monophosphate. The action of inositol 1,4,5-trisphosphate on digitonin-permeabilized N1E115 cells resulted in a stimulation of cyclic GMP levels that mimicked that induced by neurotensin. Therefore, the cyclic GMP stimulation is probably a consequence of the initial inositol trisphosphate formation triggered by neurotensin. Fluoroaluminate ions and pertussis toxin had the capacity to modulate positively and negatively, respectively, the formation of inositol trisphosphate induced by neurotensin, indicating that GTP-binding proteins are involved in the regulation of inositol phosphate levels by neurotensin receptors.

Notes: Abstract: The distribution of high-affinity binding sites for γ-[3H]hydroxybutyrate in coronal sections of rat brain was studied by quantitative autoradiographic techniques. Binding sites for this naturally occumng substance, which may possibly have a neurotransmitter role, are concentrated in some restricted areas of the brain, particularly in the limbic system. The hippocampus (especially field CAI of Ammon's horn, at 292 fmol/mg of tissue), septum (72 fmol/mg of tissue), and cortex (frontal, 113 fmol/mg of tissue; parietal, 103 fmol/mg of tissue; cingulate, 114 fmol/mg of tissue; and entorhinal, 134 fmol/mg of tissue) show pronounced labeling with γ-[3H]hydroxybutyrate. Binding is much lower in caudatus-putamen (50 fmol/mg of tissue), thalamus, and hypothalamus. Caudal parts of the brain (cerebellum, pons, and medulla) are practically devoid of binding sites. These results strongly support a functional role of endogenous y-hydroxybutyrate in particularly restricted areas of the rat brain.

Notes: Abstract: The fate of tetanus toxin bound to neuronal cells at 0°C was followed using an anti-toxin 125I-protein A assay. About 50%; of surface-bound toxin disappeared within 5 min of warming cells to 37°C. Experiments with 125I-toxin showed that much of this loss was due to dissociation of bound toxin into the medium. Some toxin was however rapidly internalised, and could be detected only by permeabilising cells with Triton X-100 prior to assay. To investigate the mechanism of internalisation, tetanus toxin was adsorbed to colloidal gold. Toxin-gold was shown to be stable, and to recognise the same receptor(s) as free toxin. Quantitation of the distribution of toxin-gold particles bound to the cell body at 4°C showed that it was concentrated in coated pits. After 5 min at 37°C, toxin-gold appeared in coated vesicles, endosomes, and tubules. After 15 min, it was found largely in endosomes, and at 30 min in multivesicular bodies. The involvement of coated pits in internalisation of tetanus toxin, but not cholera toxin, was confirmed using the free toxins, anti-toxins, and protein A-gold. Toxin-gold also entered nerve terminals and axons via coated pits, accumulating in synaptic vesicles and in-traaxonal uncoated vesicles, respectively.

Notes: Abstract: Antisera against 2-aminoimipramine covalently coupled to albumin have been raised in two rabbits. Both antisera bind imipramine and related tricyclic compounds as if to a single class of sites with high affinity and high litres. Displacement/inhibition assays showed that the affinities of various tricyclic compounds for the antisera showed a good correlation with the affinities of these drugs for the tricyclic antidepressant inhibitory sites on plasma-membrane 5-hydroxytryptamine carriers of human platelets and rat brain cortex. 5-Hydroxytryptamine and 5-hy-droxytryptamine-uptake-selective drugs did not inhibit [3H]imipramine binding to antisera. The anti-imipramine antibodies were purified using imipramine-Sepharose affinity chromatography and were shown to be IgG class by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein A-Sepharose precipitation.

Notes: Abstract: This study examined the effects of dopamine D1and D2 receptor agonists and antagonists on the spontaneous and calcium-dependent, K+-induced release of γ-[3H]-aminobutyric acid ([3H]GABA) accumulated by slices of rat substantia nigra. SKF 38393 (D1 agonist) and dopamine (dual D1/D2 agonist) were without effect on [3H]GABA efflux by themselves (1–40 μM), or in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (0.5 μM), but potentiated evoked release in the presence of forskolin (0.5 μM), an adenylate cyclase activator. These increases in release were prevented by the D1 antagonist SCH 23390 (0.5 μM), but not by the D2 antagonist metoclopramide (0.5 μM), Higher concentrations of forskolin (10–40 μM) augmented stimulus-evoked [3H]GABA release directly, whereas dibutyryl cyclic AMP (100–200 μM) depressed it. Apomorphine, noradrenaline, and 5-hy-droxytryptamine (1–40 μM) had no effect. The D2 stimulants lisuride, RU 24213, LY 171555, and bromocriptine dose-dependently inhibited depolarisation-induced but not basal [3H]GABA outflow. These inhibitory responses were not modified by the additional presence of SKF 38393 (10 μM) or SCH 23390 (1 μM), or by injection of 6-hydroxydopamine into the medial forebrain bundle 42 days earlier, but were attenuated by metoclopramide (0.5 μM). Higher amounts (10 μM) of SCH 23390, metoclopramide, or other D2 antagonists (loxapine, haloperidol) reduced evoked GABA release by themselves, probably by nonspecific mechanisms. These results suggest D1 and D2 receptors may have opposing effects on nigral GABA output and could explain the variable effects of mixed D1/D2 dopaminomimetics in earlier release and electrophysiological experiments.

Notes: Abstract: The Wobbler mouse (wr) is a mutant that exhibits loss of anterior horn ceils in the spinal cord and brainstem and subsequent muscle wasting, particularly of the fore-limbs and neck. The wr mice, 2–3 months of age, were found to have increased levels of immunoreactive-thyrotrophin-releasing hormone (ir-TRH) in the spinal cord and pons and medulla, but not in other CNS areas. This increase was observed in dorsal and ventral cord and at cervical, thoracic, and lumbar levels and was confirmed by HPLC to be authentic TRH. The levels of immunoreactive-somatostatin,-neurotensin, and-substance P were not raised in the CNS of wr mice. The activities of two peptidases capable of degrading TRH, pyroglutamylaminopeptidase (PGAP, EC 3.4.11.8) and proline endopeptidase (PEP, EC 3.4.21.26), and the level of 5-hydroxyindoleacctic acid were also raised in the spinal cord of 2–3-month-old wr mice although the activities of alanine aminopeptidase and lactate dehydrogenase and the level of 5-hydroxytrypt-amine were not. Increased spinal cord levels of ir-TRH and PGAP and PEP activities were not observed in the 1-month-old wr mice. In addition, a pilot study using spinal cord obtained at autopsy from three patients with motor neurone disease and 12 control subjects indicated no increase in spinal cord ir-TRH, PGAP, or PEP in human motor neurone disease.

Notes: Kainic acid binding sites were solubilized from rat brain using a combination of Triton X-100 and digitonin. The highest percentage of solubilized binding sites (45%) was obtained by treating brain membranes with 1 % Triton-X-100 and 0.2% digitonin in 0.5 M potassium phosphate containing 20% glycerol. The solubilized binding sites were stable and amenable to analysis by gel nitration and lectin affinity chromatography. Computer assisted analyses demonstrated that the solubilized sites displayed high-and low-affinity binding constants similar to the membrane-bound sites. Competition experiments further supported the pharmacological similarities of the solubilized and membrane-bound sites. Gel nitration chromatography of the solubilized binding site indicated that the detergent-bound complex had a Stokes radius of 82.7 A. The [3H]kainic acid binding site appears to be glycosylated based on its capability to bind to lectins. The lectin, wheat-germ agglutinin, proved to be a potentially useful tool for characterization because the solubilized binding sites were bound and eluted in relatively high yield.

Notes: The neuroexcitotoxin kainate has been used as a selective lesioning agent to model the etiology of a number of neurodegenerative disorders. Although excitotoxins cause susceptible neurons to undergo prolonged or repeated depolarization, the proximate metabolic pathology responsible for neuronal necrosis has remained elusive. We report here that kainate-induced death of cerebellar neurons in culture is prevented by inhibiting the enzyme xanthine ox-idase, a cellular source of cytotoxic superoxide radicals (O2-). Moreover, neurons are also protected from excitotoxin-induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenge superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalls formation of xanthine oxidase. These findings indicate that excitotoxin-induced neuronal degeneration is mediated by superoxide radicals generated by xanthine oxidase, a mechanism partially analogous to that proposed for tissue damage seen upon reperfusion of ischemic tissues.

Notes: The effect of NH4Cl on release of amine and amino acid transmitters from rat brain synaptosomes was investigated. Ammonia (0.1–10 mM) stimulated the secretion of dopamine and 5-hydroxytryptamine in a dose-dependent manner, in a process which was additive with the effect of 40 mM K+, almost unaffected by withdrawal of Ca2+, and markedly decreased by increasing [H+] in the medium. The NH4Cl-induced dopamine efflux, in contrast to that caused by high [K+]e, was inhibited by benztropine. The release of γ-aminobutyric acid, aspartate, and glutamate was unaltered by [NH4Cl] 〈 5 mM, but somewhat stimulated at higher levels. Transmembrane pH gradient, acid inside, was dissipated by NH4Cl in a concentration-dependent manner and the internal alkalinization correlated with the stimulation of the rate of dopamine efflux. Transmembrane electrical potential was unaffected by [ammonia] 〈 5 mM, but a small depolarization was observed at higher levels. It is postulated that ammonia-induced alkalinization of the intrasynaptic storage granules causes extrusion of amines into the cytoplasm and their subsequent leakage into the medium through a reversal of the plasma membrane transporters. A lack of correlation between the release of amino acid neurotransmitters and the dissipation of the ΔpH suggests that in rat brain intrasynaptic vesicles, acidic inside, are unlikely to store substantial amounts of γ-aminobutyric acid, aspartate, or glutamate.

Notes: Abstract: The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+-and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.

Notes: Fast and Slow Chemical Signalling in the Nervous SystemAntidepressants and Receptor Function (Ciba Foundation Symposium, Volume 123), D. L. MurphyCurrent Topics in Research on Synapses, Volume 3 edited by D. G. Jones.Electrophysiological Techniques in Pharmacology Modern Methods in Pharmacology.

Notes: Abstract: The adenosine A1 receptors of sheep brain membranes have been identified by the specific binding of radio-labeled cyclohexyl [3H]adenosine ([3H]CHA). Pretreatment of membranes with periodate-oxidized CHA causes a doseand time-dependent decrease in the number of binding sites. No decrease occurs when membranes are pretreated with CHA. Binding of [3H]CHA to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.

Notes: Abstract: We studied the enzyme monoamine oxidase (MAO) in isolated cerebral microvessels, and in mitochondria-enriched brain and liver preparations from six mammalian species, including human. We also studied MAO distribution in various tissues and in discrete brain regions of the rat. MAO was assessed by measuring the specific binding of [3H]pargyline, an irreversible MAO inhibitor, and the rates of oxidation of known MAO substrates: benzylamine, tyramine, tryptamine, and 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP). Molecular forms of MAO were examined by using specific MAO inhibitors, and by polyacrylamide gel electrophoresis after [3H]pargylihe binding. In general, the liver from all species had higher MAO levels than the brain, with minor variation among species in their brain and liver MAO content. However, there were remarkable species differences in brain microvessel MAO, with rat microvessels having one of the highest MAO activity among all tissues, whereas MAO activities in brain microvessels from humans, mice, and guinea pigs were very low. In most rat tissues, including the brain, there was a preponderance of MAO-B over MAO-A. The only exceptions were the heart and skeletal muscle. Estimates of MAO half-life in rat brain microvessels, rat brain, and rat liver indicated that microvessel MAO had a higher turnover rate. The reasons underlying the remarkable enrichment of rat cerebral microvessels with MAO-B are unknown, but it is evident that there are marked species differences in brain capillary endothelium MAO activity. The biological significance of these findings vis a vis the role of MAO as a “biochemical blood-brain barrier” that protects the brain from circulating neurotoxins and biogenic amines should be investigated.

Notes: Abstract: The regional distribution of [3H]zoIpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD= 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-β-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]- zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H- labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover. [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-β-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.

Notes: Abstract: The distribution of dopamine D-1 receptors has been determined in human prefrontal cortex (Brodmann's area 9) by an in vitro light microscopic autoradiographic method. Dopamine D-l receptors were localized by using [3H]SCH 23390 as a ligand. Our results indicated that [3H]-SCH 23390 binding to slide-mounted tissue sections of human brain is specific, saturable, and of high affinity. Lamina Va contained the highest density of D-l receptors, with a Bmax value of 11.2 × 1.3 fmol/mg tissue. The KD values for [3H]SCH 23390 in all laminae ranged from 2.6 to 3.2 nM. Competition studies performed with [3H]SCH 23390 indicated a pharmacologic profile consistent with labeling of the D-l receptor.

Notes: Abstract: The α-adrenergic agonists phenylephrine and methoxamine, at concentrations that have little effect on pineal N-acetyltransferase activity, markedly enhance stimulation of this enzyme by vasoactive intestinal polypeptide (VIP). This augmentation can be blocked by the α1-adrenergic antagonists phenoxybenzamine and prazosin and, at 10 but not 1 μM, by the α2-antagonist yohimbine. The time course for VIP stimulation is not altered by concomitant α-adrenergic stimulation. Augmented activity does not require concomitant α-adrenergic stimulation, but α-adrenergic agonists must be present for augmentation to be maintained. Phorbol 12, 13-diacetate or -dibutyrate but not 4a-phorbol can substitute for phenylephrine, a finding suggesting that protein kinase C is involved in the augmentation. These results are, in general, analogous to α-adrenergic magnification of N-acetyltransferase induction by β-adrenergic agonists.

Notes: Abstract: The ganglioside composition of the brain from an individual with classical Tay-Sachs disease and from an individual with Sandhoff disease was examined using our new quantitative methods for ganglioside content determination and compared with that of age-matched control brains. The concentration of GM2 was found to be 12.2 and 13.0 μmol/ g of fresh tissue in Tay-Sachs disease and in Sandhoff disease cerebral gray matter, respectively. GM2 was 86 and 87% respectively, of total gangliosides. The concentration of GM1 and, in particular, GM3 ganglioside was also found to be increased, whereas the concentration of the major di- and trisialogangliosides (GD1a, GD1b, and GT1b) had diminished markedly. There was no significant increase in level of any other ganglioside than lyso-GM2. Its concentration was 12 and 16 nmol/g in cerebral gray matter of two Tay-Sachs disease brains and 43 nmol/g in Sandhoff disease brain. The SandhofF disease brain also differed from the classical Tay- Sachs disease brain by having a much higher concentration of gangliotriaosylceramide and globotetraosylceramide. The structures of relevant gangliosides and neutral glycolipids were established by fast atom bombardment-mass spectrometry and permethylation studies.