Library

Notes: Abstract: Specific binding characteristics of acetylcholine receptors at the diaphragm neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months were assayed by measuring 125I-α-bungarotoxin binding. Maximal binding to intact tissue samples was greater in the older rats; this could be attributed to an age-related increase in terminal branching. The toxin concentration at which half-maximal binding occurred increased in the older rats. Binding kinetics were assayed in finely minced tissue samples, and the association rate constant was observed to decrease in the 28-month animals. Retardation of the initial rate of toxin binding by d-tubocurarine (dTC) in minced tissue was described by a two-component nonlinear Hofstee plot; IC50 values (7.1–7.2 μM and 39.0–46.5 nM) were about the same for both age groups, but there was a significant shift toward the low-affinity values in the aged rats. Rhodamine-conju-gated α-bungarotoxin was used to visualize receptor localization. There were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. The data indicate a shift toward lower binding affinity during aging, which may involve changes either in one of the two toxin-binding sites on individual receptors, in dTC blocking of the channel moiety, or in receptor types.

Notes: Abstract: The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) on activities of enzyme complexes in the electron transport system were studied using isolated mitochondrial preparations from C57BL/6J mouse brains. Both MPTP and MPP+ dose-dependently inhibited activity of NADH-ubiquinone oxidoreductase (EC 1.6.5.3). The inhibition was reversible. Preincubation of freeze-thawed mitochondria with MPTP or MPP+ had no effect on the inhibition; however, when nonfrozen mitochondria were used, NADH-ubiquinone oxidoreductase activity was reduced to 46% of that in the nonincubated sample after a 5-min preincubation with MPTP and to 77% of that in the nonincubated sample after a 5-min preincubation with MPP+. Kinetic analyses revealed that inhibition of MPTP was non-competitive and that of MPP+ uncompetitive with respect to NADH. On the other hand, inhibition of MPTP was uncompetitive and that of MPP+ noncompetitive with respect to ubiquinone. Succinate – ubiquinone oxidoreductase (complex II), dihydroubiquinone-cytochrome c oxidoreductase (complex III), and ferrocytochrome c-oxygen oxidoreductase (EC 1.9.3.1) activities were either slightly inhibited or not inhibited by MPTP or MPP+. The significance of these findings is discussed in relation to the mechanism of MPTP-induced neuronal degeneration.

Notes: Abstract: Substance P (SP), the widely distributed undeca-peptide, is synthesized in cell bodies of vagal sensory ganglia and transported bidirectionally toward the CNS and thoracic and abdominal viscera. In explants of the guinea pig inferior (nodose) vagal sensory ganglion and attached 2 cm of distal vagus nerve, SP is synthesized within the ganglion and transported predominantly distally. The quantity of distal transport is similar to that observed in vivo and provides an index of ongoing synthesis within the ganglion. In this report, the model is further characterized. Double ligation of the explant distal to the ganglion demonstrates that all the transported peptide is derived from the ganglion; there is no evidence of intraaxonal processing of peptide precursor. Approximately 50% of the peptide is in a rapid transport vs. an apparent stationary compartment. Not only transport, but also synthesis, of SP was blocked by 20 mM colchicine. Ongoing SP biosynthesis is dependent on a nutrient medium [medium 199 (M-199)] and is partially inhibited with added fetal bovine serum (FBS; 10%): total ex-plant content in M-199/FBS vs. M-199, 1, 785 ± 101 (n = 8) vs. 2, 254 ± 123 pg (n = 9); p 〈 0.02. Addition of 2-deoxyglucose (2-DG) decreased both total SP synthesis and transport (total explant content for 2-DG vs. control, 986 ± 94 vs. 1, 391 ± 111; p 〈 0.05). Medium supplemented with glucose to a final concentration of 600 mg/100 ml or with glucose (300 mg/100 ml) with or without insulin (50 ng/ml) did not alter explant SP content or transport. Veratridine (5 ± 10−−6M) inhibited both SP synthesis and transport; ouabain (10−−4M) also inhibited synthesis, but less so transport. Tetrodo-toxin reversed the effects of veratridine. These studies demonstrate the usefulness of this model, which can examine factors regulating both synthesis and transport of sensory neuropeptides in vitro. The results suggest that SP synthesis/transport may be under tonic inhibition, perhaps by both neural and humoral mechanisms.

Notes: Abstract: Antisera were raised against L-3,4-dihydroxyphe-nylalanine (L-DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme-linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catechol-amine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the non-conjugated compound was poorly recognized. The nonre-duced conjugates of L-DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross-reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L-DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti-NA and anti-OA antibodies, the lateral chain is important.

Notes: Abstract: The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120–500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF—both lumbar and ventricular—was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.

Notes: Abstract: Goldfish CNS was fractionated by differential and density gradient centrifugation. The fractions obtained were characterized by marker enzymes typical of various subcellular organelles. They were further analyzed by radioimmunoassay for their contents of ependymins, two CNS glyco-proteins known to participate in biochemical reactions after learning events. Ependymins were shown to be major constituents of the soluble cytoplasm (5.6% of the total protein content). The nuclear fraction was virtually devoid of ependymins (0.6% of protein). Small amounts were observed in the crude synaptosomal and microsomal fractions (1.0 and 3.5%, respectively). The highest steady-state concentration of ependymins, however, was measured in the brain extracellular fluid (15.6% of the protein), including the CSF. The specificity of the distribution was examined by intracerebro-ventricular injection of 125I-labeled ependymins as exogenous marker substances. No indication of an artificial redistribution of the radiolabel during homogenization and frac-tionation was obtained. The exogenous analogues of ependymins were, however, incorporated in vivo into organelles recovered in the nuclear and crude synaptosomal fractions. Our results suggest that ependymins may interact with synaptic membranes from the extracellular fluid, although so far no evidence for a specific receptor-type binding site could be obtained in vitro.

Notes: Abstract: Binding of the selective dopamine (DA) uptake inhibitor [3H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [3H]-GBR 12935 binding at 0°C was reversible and saturable and Scatchard analysis indicated a single binding site with a KD of 5.5 nM and a Bmax of 760 pmol/mg tissue. [3H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19–005 potently inhibited [3H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC50 values for inhibition of [3H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1-methyl-4-phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [3H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these “weak” inhibitors affected the binding by alloste-ric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [3H]GBR 12935 was potently displaced from it by disubstituted piper-azine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [3H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6-OH-DA. Thus, [3H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [3H]GBR 12935 binding with low potency, but did not alter the rate constants for [3H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.

Notes: Abstract: The effects of the neurotoxin N-methyl-4-phenylpyridinium ion (MPP+) on the enzymes involved in synthesis and catabolism of catecholamines were examined using a clonal rat pheochromocytoma cell line, PC12h, as a model of dopaminergic neurons. MPP+ added in the culture medium was found to be accumulated in PC12h cells after 30-min incubation. Monoamine oxidase (MAO) activity in PC12h cells was inhibited by MPP+ in a dose-dependent way from 10 nM to 10 μM, but concentrations of MPP+ higher than 100 μM were found to increase the MAO activity. At the lower concentrations MPP+ inhibited MAO non-competitively with respect to the substrate, kynuramine, and at the higher concentrations it increased both the Km and the Vmax values of MAO toward the substrate. On the other hand, tyrosine hydroxylase activity and the dopamine concentrations in PC 12 cells were not changed by incubation with MPP+ for 30 min, 60 min, or 24 h.

Notes: Abstract: In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the —C(O)NH2 of NAD+ was replaced with C(S)NH2, −C(O)CH3, and −C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki∼ 1 nM for the A form). Even at high concentrations (20 μM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, α-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.

Notes: Books review in this article: Nutrition and Neurobiology edited by J. C. Somogyi and D. Hötzel Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties edited by R. W. Olsen and J. C. Venter Progress in Retinal Research, Volume 5 edited by N. N. Osborne and G. J. Chader

Notes: Abstract: Glucocorticoid hormones are known to inhibit the production of prostaglandins in many cell types and tissues. The effect of these hormones on the biosynthesis of brain tissue is not yet clear. In the present study we investigated the effect of dexamethasone on the release of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-PGF1α from various brain areas of male rats. Slices from cortex, hippocampus, hypothalamus, and striatum taken from rats pretreated with dexamethasone 4 mg/kg body weight or vehicle, 18 h and 2 h prior to killing, were incubated in Krebs-Ringer-bicarbonate for 1 h. The accumulation of PGs in the medium was determined by radioimmu-noassay. Pretreatment with dexamethasone significantly reduced the release of all PGs from the cortex by 40–50%. In the striatum and hippocampus only TXB2 was reduced by ∼40%. In the hypothalamus the effect of dexamethasone was not significant. When slices of the same brain areas from intactrats were incubated for 1 h in the presence of 40 μM dexamethasone, only the release of PGE2 from the cortex was reduced (by 30%). These results suggest that glucocorticoids can inhibit PG synthesis in brain tissue, and that the cortex is the most sensitive area to the inhibitory effect of the hormone.

Notes: Abstract: The effect of intrastriatal injection of fluorocitrate on amino acid pattern, cell enzyme markers, and ultrastruc-tural appearance was investigated. A dose of 1 nmol of fluorocitrate resulted in temporarily decreased levels of glutamine, glutamate, and aspartate, whereas the level of alanine was increased. The glutamine level was severely reduced after 4 h but was reversed after 24 h. The activity of different cellular enzyme markers did not change markedly after this dose. Ultrastructural changes in glial cells were observed, concomitant with the biochemical changes. A dose of ≥2 nmol of fluorocitrate resulted in more marked and irreversible changes in amino acid levels. By 24–72 h after the injection of this dose, several marker enzyme activities decreased markedly. The ultrastructural changes affected the neurons as well as the glial cells and were not reversible. The use of microinjection of 1 nmol of fluorocitrate into the neostria-tum of the rat to provide a model for studying transmitter amino acid metabolism in brain devoid of glial cell activity is discussed.

Notes: Abstract: The content of polyunsaturated fatty acids, the activities of superoxide dismutase (SOD), glutathione per-oxidase, glutathione reductase, and catalase, and the concentration of reduced glutathione were measured in cerebral microvessels isolated from rat brain. Polyunsaturated fatty acids, mainly arachidonic, linoleic, and docosahexaenoic acids, accounted for 32% of total fatty acids in cerebral microvessels. Whereas total SOD activity in the microvessels was slightly lower than that found in cerebrum and cerebellum, glutathione peroxidase and glutathione reductase activities were twice as high and catalase activity was four times higher. Glutathione peroxidase in microvessels is active on both hydrogen peroxide and cumen hydroperoxide, and it is strongly inhibited by mercaptosuccinate. After several hours of preparation, the concentration of reduced glutathione in isolated microvessels was 0.7 μmol/mg of protein, which corresponds to a concentration of ∼3.5 mM. Our results indicate that the blood-brain barrier contains large amounts of peroxide-detoxifying enzymes, which may act, in vivo, to protect its highly polyunsaturated membranes against oxidative alterations.

Notes: Abstract: Structural analogues of the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (3′,5′-PAPS) were examined for their ability to inhibit dopamine and phenol sulfation by the M and P forms of phenol sulfotransferase (PST), respectively. The Ki values for each of the adenosine derivatives were calculated from the rate equation for PST. For both M and P PST, the naturally occurring product 3′-phosphoadenosine-5′-phosphate, (3′,5′-PAP), was shown to be the most effective inhibitor. The weakest inhibitors of the two sulfotransferases were 5′-adenosine phosphosulfate and the three AMP derivatives, which were 〈1,000 times as effective as 3′,5′-PAP. 5′-ATP, 2′,5′-PAPS, 2′,5′-PAP, and 5′-ADP were similar in their inhibition of M and P PST and were all ∼ 100 times less effective than the natural end product. These data reveal that there is a rigid structural requirement for binding of the ribose portion of adenosine to both M and P PST that involves the groups on both the 3′ and 5′ positions. The effectiveness of binding to the two enzymes may depend on both steric factors as well as the distribution of negative charges on the ribose ring.

Notes: Rabbit anti-bovine myo-inositol-1-phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion-fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo-inositol-1-phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo-inosose reductase and myo-inositol-1-phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo-inositol-1-phosphate synthase in brain raises new questions about the mechanism by which myo-inositol is concentrated to such high cellular levels in the principal substance of that organ.

Notes: Abstract: It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: (a) nominally calcium-free perfusion solution (no calcium added), (b) EGTA-containing calcium-free perfusion solution, and (c) perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.

Notes: Abstract: We sought to establish whether the endogenous opiate-receptor agonist Met-enkephalin (m-ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m-ENK (1-10 μM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylal-anine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), tau-rine (Tau), and glycine (Gly) were similarly increased by 50–150% with m-ENK in slices of CS, but not Cx. The enhanced release of AAs by m-ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5–55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as γ-aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m-ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8–51%) compared to that of GABA, Gly, Glu, and Tau (49–123%). Nonetheless, m-ENK (10 μM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction. Other neuropeptides including cholecystokinin octapeptide (CCK-8), thyrotropin-releasing hormone (TRH), and vaso-active intestinal peptide (VIP) facilitated the release of Tyr from brain slices in a regionally specific pattern. We conclude that: (1) Tyr is released from nerve terminal-enriched preparations of CS, but not Cx, by m-ENK via an opiate receptor-mediated, Ca2+-dependent process with regional selectivity; (2) neuronal depolarization alone, however, does not affect the release of Tyr; (3) CCK-8, TRH, and VIP also increase Tyr release with regional specificity, suggesting that receptors for other neuropeptides may also modulate Tyr release. The specific neuronal source and functional role of Tyr released from elements of CS via activation of opiate receptors remain to be elucidated.

Notes: Abstract: Antisera were raised in rabbits against the α sub-unit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the α subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Goα-coupled Sepharose column. Purified antibodies reacted only with Goα and did not cross-react with the Giα subunit or βγ subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Goα was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab′)2 fragments and the same antibody Fab’ fragments labeled with β-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Goa, and it did not cross-react with Giα or βγ. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Goα were determined. Goα was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neu-ropil was rich in Goα.

Notes: Abstract: Chronic ingestion of ethanol, which produced tolerance and physical dependence, resulted in altered function of the cerebral cortical β-adrenergic receptor-coupled adenylate cyclase system in mice. Although there was no change in basal adenylate cyclase activity, or in the activity of the digitonin-solubilized catalytic unit, stimulation of adenylate cyclase activity by the nonhydrolyzable guanine nu-cleotide analog guanylylimidodiphosphate [Gpp(NH)p] was reduced in brains of ethanol-fed animals. Ethanol added in vitro increased adenylate cyclase activity, and this enhancement, in the presence of Gpp(NH)p, was also reduced in cortical membranes of ethanol-fed mice. Furthermore, the maximal response to isoproterenol was decreased, and the EC50 for isoproterenol stimulation of adenylate cyclase activity was increased in ethanol-fed animals. The results are consistent with a qualitative or quantitative defect in the function of the stimulatory guanine nucleotide-bind-ing protein (Ns), as well as in the β-adrenergic receptor, after chronic ethanol exposure. In part, these changes appear to be similar to those that occur during heterologous desensiti-zation of various receptor systems, and may be associated with dependence on or tolerance to ethanol.

Notes: Abstract: A mass fragmentographic method for the simultaneous quantification of γ-aminobutyric acid (GABA) and glutamic acid is described. In a convenient one-step reaction, the two ammo acids were derivatized with pentafluor-opropionic anhydride and pentafluoropropanol. The deri-vatization products were stable for several days. The technique has been applied to the assay of GABA and Glu in five amygdaloid nuclei of the rat brain. The GABA level was high in the central and medial nuclei, whereas the Glu level was high in the lateral and basal nuclei. The regional distribution of GABA was different from that of Glu within the amygdaloid nuclei.

Notes: Abstract: The origin of afferent somatostatin-containing fibers terminating in medial and ventral parts of the striatum has been investigated by performing various neuro-chemical and surgical lesions in the rat. Lesions of the anterior hypothalamus, amygdala, and the hippocampal commissure as well as lesions with 6-hydroxydopamine and 5,7-dihydroxytryptamine failed to decrease striatal soma-tostatin levels. However, thermal coagulation of the globus pallidus or knife-cut lesions performed ventrally to the striatum resulted in significant decreases in striatal somatostatin content. Analysis of the topographical distribution of somatostatin within the striatum after thermal lesions of the globus pallidus as well as after kainic acid-induced seizures revealed a preferential loss of the peptide in medial and ventral portions of the striatum, the site of terminating afferent somatostatin nerve fibers. The data suggest that the striatal afferent somatostatin-containing neurons may originate in the area of the globus pallidus.

Notes: Abstract: Goldfish were trained for 4 h to swim with an attached polystyrene foam float and tested for retention 3 days later. Intracerebroventricular injection of anti-ependymin antisera was shown to prevent long-term memory formation of this vestibulomotor learning task, as reported previously. In further experiments, fish were killed 4–14 h after the start of training. The brains were dissected, incubated in an isoosmolar solution for collection of proteins of the brain extracellular fluid (ECF), homogenized, and fractionated by differential centrifugation. The ECF, a supernatant fraction enriched in cytoplasmic constituents (S3), and various par-ticulate subcellular fractions were analyzed for their epen-dymin contents by radioimmunoassay. No statistically significant changes that might be induced by the learning were revealed in any of the participate fractions. Steady-state concentrations of ependymins in the cytoplasm, however, increased temporarily by 39% in fish that had mastered the training task as compared with nonlearning animals (passive and active controls). In the ECF, the specific concentration of ependymins first decreased to 88% of control levels (4–5 h after the start of training), but later on, it increased to 138% (8–14 h). Apparently, ependymins present in the ECF are used during biochemical reactions of memory consolidation. The resulting decrease in extracellular epen-dymin concentrations might trigger their resynthesis in the cytoplasm and lead to an increased release of these glyco-proteins into the ECF.

Notes: Abstract: The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified γ-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydro-chloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cys-tine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but SH and S-S-groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glyco-peptidase A, β-galactosidase, and α-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with β-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with β-galactosidase and glycopeptidase A were much higher than that with α-mannosidase may also indicate a special importance of the β-galactosyl residue in the inhibition of GABA receptor binding activity. Furthermore, the observation that the activation of high-affinity [3H]muscimol binding by benzodiazepines disappeared following β-galactosidase treatment suggests that the carbohydrate chain in the receptor complex may also be involved in the functional coupling between the GABA receptor and the benzodiazepine receptor.

Notes: Abstract: Hippocampal brain slices were incubated with depolarizing agents or excitatory amino acids either alone or in the presence of excitatory amino acid antagonists [ω-phosphonic α-aminocarboxylic acids—2-amino-4-phosphonobutyric acid (AP4), 2-amino-5-phosphonovaleric acid (AP5), or 2-amino-7-phosphonoheptanoic acid (AP7)—or γ-D-glutamylaminomethylsulphonic acid (GAMS)] or a calcium-channel blocker, (S)-1-(3-methoxyphenyl)-3-methylaza-7-cyano-7- (3,4-dimethoxyphenyl)-8-methyl-nonane hydrochloride [(-)-D888]. The uptake of 45Ca2+ and the efflux of glutamate or aspartate induced by veratrine or high K+ was blocked (54–76%) by AP7 (IC50 46–250 μM). AP5 and AP4 were less effective. (-)-D888 (10 μM) caused 100% block of evoked 45Ca2+ uptake. Uptake of 45Ca2+ induced by exogenous glutamate, aspartate, and N-methyl-D-aspartate (NMDA) was also inhibited by AP7, whereas GAMS completely blocked the action of kainate and partially blocked that of glutamate. The action of NMDA in stimulating 45Ca2+ uptake was Mg2+-sensitive, low Mg2+ levels in the incubation medium selectively enhancing the response. It is concluded that Ca2+ uptake evoked by excitatory amino acids is receptor-mediated, and that released excitatory amino acids are responsible for a large part of the action of veratrine and high K+ in stimulating 45Ca2+ uptake.

Notes: Abstract: The human gene coding for vasoactive intestinal peptide was recently isolated and shown to contain seven exons. We now demonstrate that an intron-containing precursor RNA can be the major vasoactive intestinal peptide-related RNA in the cell, which is in contrast to most known genes. By RNA blot analysis using a variety of genomic and cDNA-related probes we show that in a human tumor producing vasoactive intestinal peptide, most of the RNA encoding the peptide is of the precursor type. Similar precursor transcripts were found in total rat brain RNA as well. As proof of the identity of the intron-containing RNA, cDNA clones corresponding to this RNA sequence have been isolated.

Notes: Abstract: Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both ara-chidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 μM and 5.8 μM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol min−1 mg protein−1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 μM unlabelled arachidonic acid, with a Ki of 7.1 μM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 μM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 μM unlabelled palmitic acid, with a Ki of 5.0 μM and to a much lesser extent by arachidonic acid (Ki, 23 μM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40°C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis. These data together strongly suggest that rat brain microvessels have the capacity to catalyze specifically the formation of acyl-CoA derivatives from several polyunsaturated long-chain fatty acids, including arachidonic acid in the first place. Besides this particular arachidonoyl-CoA synthetase, palmitic acid could be activated with the aid of a second acyl-CoA synthetase.

Notes: Abstract: The nitrendipine receptor associated with the voltage-dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3-[(3-cholamidopropyl)dimethylammonio] - 1 - propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S-500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is ∼10 times that of the membrane-bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 ∼ 10−7M, and they caused maximal inhibitions of 70 and 100%, respectively.

Notes: Abstract: A S-adenosylmethionineiprotein-lysine N-methyltransferase (EC 2.1.1.43) has been purified from rat brain cytosol 7,080-fold with a yield of 8%, using octopus calmodulin as a substrate. It contains a lysine residue that is not fully methylated. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and phosphocellulose and octopus calmodulin-Sepharose affinity chromatographies. Among protein substrates, it was highly specific toward octupus calmodulin. The Km values for octopus calmodulin and S-adenosyl-L-methionine were found to be 2.2 × 10−8M and 0.8 × 10−6M, respectively. The molecular weight was estimated to be 57,000 by gel filtration and the pH optimum was between 7.5 and 8.5. The enzyme was stimulated in the presence of 10−7M Mn2+ and 10−4M Ca2+. HPLC of the acid hydrolysate of methyl-3H-labeled calmodulin showed the formation of ε-N-mono-, ε-N-di-, and ε-N-trimethyllysine. Reverse-phase HPLC of tryptic peptides of the methyl-3H-labeled calmodulin demonstrated that the labeled N-methyllysine lies in the 107–126 peptide. These findings suggest that this enzyme methylated a specific lysine residue of octopus calmodulin.

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.