Bibliothek

Notizen: Abstract: Axonal transport of glycoconjugates was studied in the motoneurons of rat sciatic nerve following injection of [3H]glucosamine into the lumbosacral spinal cord. After varying time intervals, the sciatic nerve was exposed, and two ligatures were tied for collection of materials undergoing anterograde and retrograde transport. Gangliosides and glycoproteins were found to undergo fast anterograde transport, estimated at 284–446 mm/day. Both classes underwent retrograde transport as well, with labeled glycoproteins returning slightly ahead of labeled gangliosides. Only minor quantities of labeled proteoglycans were detected. Purified gangliosides extracted from nerve segments were fractionated according to sialic acid number on diethylaminoethyl-Sephadex; the distributional pattern tended to resemble that of brain gangliosides. The similarity between anterograde and retrograde patterns suggested absence of metabolic changes in gangliosides entering and leaving the axon-nerve terminal structures.

Notizen: Abstract: Catecholamine secretory organelles were partially purified from PC12 cells. Measurement of the sedimentation coefficient (540S in 0.32 M sucrose), density in an isoosmotic gradient (1.139 g/cm), and density in an isoosmotic gradient using D2O as a solvent (1.205 g/cm3) have allowed us to calculate the molecular weight (1.17 × 109 daltons), radius (74 nm), and water content (62% vol/vol) of the secretory vesicle. The vesicle appears to contain ATP, but the molar ratio of 3,4-dihydroxyphenylethylamine (dopamine) to ATP in the particles is high (16.5) and the ATP was frequently asymmetrically distributed in the vesicle fraction. The particle behaves like a true secretory particle in that the dopamine content of the particle is increased by pargyline, diminished by depolarization, and abolished by reserpine. Sequential purification of PC12 lysates on controlled pore glass columns and isoosmotic Ficoll gradients produced a 20–30-fold purification, but this enrichment is not sufficient to produce a homogeneous population of vesicles. An 82,000-dalton protein copurifies with secretory granules and appears to be the major secreted protein. At this stage of purification this single protein makes up about 30% of the protein in the vesicle-containing fractions and so the vesicles must be approaching homogeneity.

Notizen: Abstract: Neurons do not divide during adult life and thus they provide a unique system to study the effects of age-accumulated damage to DNA in the absence of DNA replication. We have analyzed DNA polymerase activity in neurons isolated from young adult and very aged mice. The predominant catalytic activity is DNA polymerase-β and it is present in similar amounts in neurons from young and old mice. This polymerase is highly errorprone in copying φX174 DNA, the error frequency being about 1/7,000 and not significantly different when obtained from young and old animals. This high infidelity is considered with respect to DNA repair and the protein synthesis error catastrophe theory of aging.

Notizen: Abstract: Previous work from this laboratory has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by nerve growth factor treatment. The protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. In the present work, the tissue distribution of Nsp100 phosphorylation in 5-day-old-and adult rats was studied. In adult rats, phosphorylation of an Nsp100-like protein was observed in brain, adrenal gland, testis, and muscle, but not in liver or kidney. In 5-day-old rats, a similar phosphorylation was observed in brain, adrenal gland, superior cervical ganglia, liver, spleen, kidney, and muscle. In PC12 cells, Nsp100 phosphorylation is completely inhibited by 5 × 10−5M Zn2+ and is completely inactivated by treatment at 50°C for 2 min. The phosphorylation of the Nsp100-like protein in both adult and 5-day-old rats showed the same characteristics. Partial purification of Nsp100 and Nsp100 kinase from the brains of 5-day-old rats was carried out using the procedures developed for PC12 cells. Nsp100 and Nsp100 kinase were separated on diethylaminoethyl-Sephacel, and the kinase was eluted with 0.3 M NaCl; the same results have previously been obtained with PC12 cells. Phosphorylated Nsp100 from brain and from PC12 cells was compared by proteolysis on sodium dodecyl sulfate gels; similar peptide patterns were generated from the two samples. The phosphorylation of the Nsp100-like protein by extracts of superior cervical ganglia was decreased by prior in vitro treatment of the ganglia with nerve growth factor, showing that the effect of nerve growth factor on Nsp100 phosphorylation observed in PC12 cells also occurs in normal target tissues.

Notizen: Abstract: Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.

Notizen: Abstract: We recently characterized two developmentally regulated myelin-associated glycoprotein (MAG) polypeptides synthesized by mouse brain mRNA in vitro. We now extended these studies to include the peripheral nervous system (PNS). Total cytoplasmic RNA was isolated from the sciatic nerves of 7-, 12-, and 17-day-old and adult rats and translated in vitro in a rabbit reticulocyte lysate system. In contrast to results in the CNS, it appears that only one MAG polypeptide, p67MAG, is synthesized by PNS mRNA at all ages. The implications of these findings are discussed with respect to recent observations concerning both the localization of MAG and the synthesis of MAG in the PNS of dysmyelinating mutant mice.

Notizen: Abstract: Rats received intraventricular (i.v.t.) injections of 5,7-dihydroxytryptamine (5,7-DHT) (100–600 μg). Some animals also received intraperitoneal injections of the 5-hydroxytryptamine uptake blocker fluoxetine (FX) (20 mg/kg) or the norepinephrine uptake blocker desmethylimipramine (DMI) (48 mg/kg) 30–90 min prior to i.v.t. 5,7-DHT. Rats were killed between 2 and 35 days following i.v.t. 5,7-DHT, brains were dissected, and regions were assayed for thyrotropin-releasing hormone (TRH) by radioimmunoassay. Dose-dependent increases in TRH content following i.v.t. 5,7-DHT were noted in the brainstem and hippocampus. DMI pretreatment blocked the increase in hippocampal TRH, but not in brainstem TRH. FX pretreatment was ineffective in blocking any increases in TRH content. These results suggest differential regulation of regional TRH content by interactions with specific neurotransmitter systems.

Notizen: Abstract: The effect of muscle extract on cell survival and choline acetyltransferase (ChAT) activity in cultures of enriched cholinergic neurones from 7-day chick embryo spinal cord was examined. When neurones were grown on hydrated collagen gels, considerable cell survival and ChAT activity were obtained even in the absence of tissue extract. These parameters were stimulated twofold in the presence of skeletal muscle extract but not liver or skin extracts. The cholinergic neurotrophic activity was found to be heat- and trypsin-sensitive, non-dialysable, and to act in the virtual absence of glial cells. These data are consistent with a retrogradely acting motor neurone trophic activity.

Notizen: Abstract: This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl-β-carboline-3-carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (∼ 16-fold) and dipyridamole (∼sevenfold) for the [3H]Ro 15–1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.

Notizen: Abstract: When grown in primary cell culture in the absence of neurons, muscle cells from a variety of species synthesize several forms of acetylcholinesterase (AChE), including the collagen-tailed A12 form. A12 AChE has been the subject of much study because it is thought to be a major functional enzyme form normally found in the basal lamina at the neuromuscular junction. In this paper, we show that muscle fibers derived from mouse embryos and neonates are also able to synthesize substantial percentages of their AChE as the A12 form when grown in vitro. This synthesis is modulated by a process associated with spontaneous muscle contractile activity since both total enzyme levels and the proportion of A12 AChE expressed on the cell surface are decreased when the cells are grown in the sodium channel blocker tetrodotoxin, which blocks muscle contraction. On the other hand, when the cells are treated with veratridine, which opens sodium channels, thereby mimicking one aspect of muscle contraction, their AChE levels are comparable to those of untreated cells. Although smaller in magnitude, these changes are similar to those seen in rat muscle cultures. A novel feature of mouse muscle cultures. not seen in those from rat and chick, is the presence of a secreted enzyme form that sediments in the same position as the cellular A12 form (when separated on sucrose density gradients containing high salt) and is also collagenase sensitive.

Notizen: Abstract: The ontogeny of muscarinic receptors was studied in human fetal striatum, brainstem, and cerebellum to investigate general principles of synaptogenesis as well as the physiological balance between various chemical synapses during development in a given region of the brain. [3H]Quinuclidinyl benzilate ([3H]QNB) binding was assayed in total particulate fraction (TPF) from various parts of brain. In the corpus striatum, QNB binding sites are present at 16 weeks of gestation (average concentration 180 fmol/mg protein of TPF), slowly increase up to 24 weeks (average concentration 217 fmol/mg protein), and rapidly increase during the third trimester to 480 fmol/mg protein of TPF. In contrast, dopaminergic receptors exist as two subpopulations, one with low affinity and the other with high affinity up to the 24th week of gestation; all of them acquire the high-affinity characteristic during the third trimester. In brainstem, the muscarinic receptors show maximum concentration by 16 weeks of age (360 fmol/mg protein of TPF). Subsequently the muscarinic receptor concentration shows a gradual decline in the brainstem. In cerebellum, except for a slight increase at 24 weeks (average concentration 90 fmol/mg protein of TPF), the receptor concentration remained nearly constant at about 60–70 fmol/mg protein of TPF throughout fetal life. This study demonstrates that the ontogeny of muscarinic receptors varies among the different regions, and the patterns observed suggest that receptor formation occurs principally in the third trimester. Also noteworthy is the finding that the QNB binding sites decreased in all regions of the human brain during adult life.

Notizen: Book Reviewed in this article: Medicinal Chemistry—A Biochemical Approach by T. Nogrady. Ethopharmacological Aggression Research (Progress in Clinical and Biological Research, Vol. 167) edited by K. Miczek, M. Kruk, and B. Olivier.

Notizen: Abstract: Extracellular levels of amino acids were estimated in dialysates of the rat striatum that were collected 1,2, and/or more than 5 days after surgery, before (resting release) and during exposure to high K concentrations (50 mM) or electroconvulsive shocks. The resting release of several amino acids (Glu, Asn, Thr, Tau, Tyr, Gly, and Ala) was higher 9 days as compared to 1 day after surgery. In the 1-day preparation the resting release correlated highly with that observed with push-pull cannulas. The correlation with the tissue content of the amino acids was high only when they were divided into two groups (putative transmitters and metabolic intermediates). High K exposure produced increased output of Ala, ethanolamine (Eam). Asp, Glu. Tau, and Gly and a decrease in the egress of Gln 1 or 2 days after surgery. The effects on Asp and Glu had disappeared, and that on Gln reversed after 4–9 days. Electrically induced convulsions produced increased output of Ala, Gln, and Eam 1 or 2 days and 2 weeks after implantation of the probe. Changes were seen not only during but also (and some cases even more prominent) after the seizure. This study shows the usefulness of dialysis to monitor extracellular transmitter amino acids in the striatum of conscious rats (also bilateral dialysis was possible) for only a limited time after implantation of the probe. The dialysis method is suitable for longer time, when metabolic changes in amino acids are to be followed. In addition to transmitter release, glycolysis can be monitored by the measurement of Ala in the dialysate.

Notizen: Abstract: Cholesterol ester hydrolase activities previously have been identified in brain and linked to the production of myelin, which has very low levels of esterified cholesterol. We have studied two cholesterol ester hydrolase activities (termed the pH 6.0 and pH 7.2 activities) in cultures derived from 19- to 21-day-old dissociated fetal rat brains and in developing rat brain. In vivo the levels of both the pH 6.0 and pH 7.2 activities began to increase by about 10 postnatal days, reached maximal levels at 20 days (20 and 1.5 nmol/h/mg protein, respectively), and thereafter remained nearly constant (pH 6.0) or decreased somewhat before becoming constant (pH 7.2). In contrast, in the cultures the pH 6.0 cholesterol ester hydrolase activity was low until 21 days in culture (DIC; 20 nmol/h/mg protein), increased to a peak activity at 31 DIC (60 nmol/h/mg protein), remained high for 24 days, and finally decreased (18 nmol/h/mg protein at 63 DIC); the pH 7.2 cholesterol ester hydrolase activity was very low until 20 DIC, increased to a peak activity at 31 days (3 nmol/h/mg protein), and thereafter decreased to a lower level (2 nmol/h/mg protein) that was maintained for about 24 days before decreasing (0.7 nmol/h/mg protein at 63 DIC). Therefore, (a) the time courses of appearance of both cholesterol ester hydrolase activities were delayed by 10–14 days relative to that seen in vivo, and (b) the specific activities observed in the cultures were transiently two- to three-fold higher than in rat brain, but then declined to levels characteristic of whole brain homogenates. Subcellular fractionation of the cultures demonstrated that the pH 7.2 cholesterol ester hydrolase activity, along with myelin basic protein and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity, was enriched in a membrane fraction collected at an interface between 0.32 M and 0.9 M sucrose; the pH 6.0 cholesterol ester hydrolase activity, in contrast, was enriched in the microsomal fraction.

Notizen: Abstract: Aging was associated with an increase in the density of specific binding sites for [3H]imipramine in postmortem specimens of human hypothalamus, frontal cortex, and parietal cortex. In general, [3H]imipramine binding was not affected by factors considered difficult to control in postmortem studies, i.e., time from death to autopsy and cause of death. The in vitro regulation of [3H]imipramine binding by sodium was impaired with age in hypothalamic homogenates. In vitro regulation of [3H]imipramine binding by chloride was intact. Determination of the concentrations of 5-hydroxytryptamine (serotonin) and 5-hydroxyindoleacetic acid in hypothalamus and frontal cortex indicated no apparent age-related changes in indole metabolism. The age-related increase in brain [3H]imipramine binding and impairment in the in vitro regulation of binding by ions are similar to changes observed previously in aged mouse brain. The increase in brain antidepressant binding sites is discussed in relationship to other indices of brain serotonergic function in aging and to the relationship of [3H]imipramine binding and depression.

Notizen: Abstract: The mechanisms by which uridine enters and leaves brain, choroid plexus, and cerebrospinal fluid (CSF) were investigated in the isolated choroid plexus in vitro and by injection [3H]uridine intravenously and intraventricularly. Consistent with its postulated role in transporting uridine from blood into CSF, the isolated rabbit choroid plexus concentrated uridine with 10 μM uridine in the medium. [3H]Uridine, with and without unlabeled uridine, was infused at a constant rate into conscious adult rabbits. At 180 min, [3H]uridine entered CSF, choroid plexus, and brain more rapidly than mannitol. In brain, approximately 60–80% of the nonvolatile radioactivity was [3H]uridine phosphates. The addition of 2.1 mmol/kg of unlabeled uridine to the infusion syringe decreased the relative entry of [3H]uridine into brain by approximately 75% due mainly to the decreased formation of [3H]uridine phosphates and increased formation of [3H]uracil. Two hours after the intraventricular injection of [3H]uridine. [3H]uridine was cleared from CSF more rapidly than mannitol, in part to brain, where approximately 75% of the [3H]uridine was converted to [3H]uridine phosphates. The intraventricular injection of 42μmol unlabeled uridine with the [3H]uridine decreased the phosphorylation of [3H]uridine in brain significantly and also decreased the clearance of [3H]uridine from the CSF. From blood, uridine enters CSF and the extracellular space of brain. Uridine then can enter brain cells, be phosphorylated to uridine phosphates, and subsequently incorporated into RNA or be catabolized to uracil.

Notizen: Abstract: The molecular forms of somatostatin contained in the rat striatum were separated by size-exclusion HPLC. Three major peaks of somatostatin-like immunoreactivity (SLI) were resolved. Two peaks cochromatographed with synthetic somatostatin-14 (SS-14) and somatostatin-28 (SS-28), respectively. One peak exhibited a higher molecular weight (about 10,000) and may contain a proform of somatostatin. Local injection of the neurotoxin kainic acid (1 μg) into the left striatum resulted in a persistent decrease (65–85%) of all three forms of somatostatin. In the contralateral—not injected—striatum a decrease of SLI was also observed which was maximal (45%) after 2 days and was largely abolished after 7 days. This decrease of SLI in the contralateral striatum, however, was due mainly to a decrease of SS-14 and SS-28 but not of the putative proform. Our data suggest that kainic acid causes a destruction of somatostatin-containing perikarya in the injected striatum, whereas in the contralateral striatum increased release with subsequent inactivation of SS-14 and SS-28 takes place. The putative somatostatin proform may serve as neurochemical marker for somatostatin-containing perikarya in the striatum.

Notizen: Abstract: A technique for studying the binding of La3+ to synaptosomes in a double-beam spectrophotometer, using murexide as indicator, is described. The binding of La3+ was very rapid and Scatchard plots revealed two components, with KD values of 0.6 and 27 μM in a Na+-free medium (sucrose medium) and 2.3 and 63 μM in an ionic medium containing 135 mM Na+. The binding of the cationic dye ruthenium red (RuR) showed only one site, with a KD of 3.7 μM. La3+ binding was partially inhibited by RuR and vice versa, and La3+ was also capable of partially displacing RuR previously bound to the synaptosomes, particularly in the sucrose medium. The release of labeled γ-aminobutyric acid (GABA) stimulated by K+ depolarization was inhibited by La3+ concentrations at or above 1 μM. in the ionic medium, whereas in the sucrose medium 2.5 μM or higher La3+ concentrations notably stimulated the spontaneous release of both GABA and glutamic acid. It is concluded that La3+ and RuR share at least one type of binding site, which is probably the high-affinity La3+ site. Since both La3+ and RuR at low concentrations have been shown to block the depolarization-induced Ca2+ entry in synaptosomes, this site might be related to the voltage-dependent Ca2+ entry involved in neurotransmitter release.

Notizen: Abstract: Phosphatidylinositol-specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.OS upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably of soubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a ∼ 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.

Notizen: Abstract: We have recently isolated from bovine adrenal medulla a novel C-terminally amidated opioid peptide, amidorphin, which derives from proenkephalin A. Amidorphin revealed a widespread distribution in bovine, ovine, and porcine tissue. Particularly high concentrations of amidorphin immunoreactivity were detected in adrenal medulla, posterior pituitary, and striatum, similar to the major gene products of proenkephalin A. In the adrenal medulla of each species. authentic amidorphin was the predominant immunoreactive form. Pituitary and brain, however, contained predominantly putative N-terminally shortened fragments of amidorphin of a slightly lower molecular weight and shorter retention times on HPLC. In addition, in ovine adrenal medulla, a putative high-molecular-weight form of amidorphin was detected. These findings are indicative of a tissue-specific processing of the proenkephalin A precursor, leading predominantly to authentic amidorphin in the adrenal medulla and further processing to smaller C-terminal fragments in the brain and pituitary.

Notizen: Abstract: The accumulation of labelled inositol mono-, bis-, and trisphosphate in rat cerebral cortex slices was examined following preincubation with [3H]inositol. The muscarinic receptor agonist carbachol produced a rapid and sustained increased accumulation of each labelled inositol phosphate both in the presence and absence of 5 mM lithium. Lithium potentiated carbachol-stimulated accumulation of inositol monophosphate (EC50 0.5 mM) and inositol bisphosphate (EC50 4 mM) in a concentration-dependent manner. However, exposure to lithium in the presence of the muscarinic agonist produced a concentration- and time-dependent inhibition of inositol trisphosphate accumulation that was not related to receptor desensitisation. Although the present data do suggest that polyphosphoinositides are substrates for agonist-stimulated phospholipase C in brain, these results may not be entirely consistent with the production of inositol mono- and bisphosphate through inositol trisphosphate dephosphorylation. Furthermore, these data suggest site(s) additional to inositol monophosphatase that are affected by lithium.

Notizen: Abstract: Conditioned medium by a variety of rat nonneuronal cells contains a protein involved in the differentiation of cholinergic neurons in cultures prepared from newborn rat superior cervical ganglion, from nodose ganglion, and from embryonic spinal cord. We have determined some hydrodynamic properties of this factor using as a bioassay the increase in choline acetyltransferase activity in sympathetic neurons grown for 12–15 days in the presence of the factor. The Stokes' radius, measured by molecular sieving chromatography on an Ultrogel AcA 44 column, was similar to that of ovalbumin (27.6 Å). By analysis on 5–20% linear sucrose gradients made in H2O and D2O, we determined the partial specific volume (0.68 ml × g−1) and the sedimentation coefficient (2.1S). These data allowed the calculation of the molecular weight (21,000) and the frictional ratio f/f0 (1.56). The elution pattern of the factor from a SynChropak CM 300 HPLC cation exchange column suggested that it was a basic protein. The activity of this factor was unaffected by heat treatment at 100°C for 10 min.

Notizen: Abstract: The effect of a single oral 750 mg/kg dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain and spinal cord proteins was assessed in hens during the development of and recovery from delayed neurotoxicity. Crude membrane and cytosolic fractions were prepared from the brains and spinal cords of control and TOCP-treated hens at 1, 7, 14, 21, 35, and 55 days after treatment. Brain and spinal cord protein phosphorylation with [γ-32P]ATP was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and microdensitometry. TOCP administration conferred calcium and calmodulin dependence on the phosphorylation of a few brain cytosolic proteins and caused an increase in the phosphorylation of a number of other cytosolic and membrane proteins. This effect of TOCP was large in magnitude, and its time course reflected the onset of and recovery from the signs of ataxia and paralysis associated with delayed neurotoxicity in the hen. The molecular weights (Mr) and maximal phosphorylation (percent of control) for the most prominently affected bands were as follows: brain cytosol—50K (183%), 55K (575%), 60K (529%), 65K (273%), and 70K (548%); brain membranes—50K (622%) and 60K (697%); and spinal cord cytosol—20K (182%). The role of endogenous phosphorylation reactions in and their potential usefulness as biochemical indicators of delayed neurotoxicity are being explored further.

Notizen: Abstract: Male, Fischer strain 344 adult rats were given various doses (25–100 mg/kg) of p,p′-DDT by oral gavage, and levels of biogenic amines, their metabolites, and amino acid neurotransmitters, tremor activity, and rectal temperature were measured at several intervals (2, 5, 12, and 24 h) after dosing. Dose-related increases in rectal temperature and in tremor activity were observed at 50–100 mg/kg 12 h after dosing. Tremorigenic doses of DDT increased the 5-hydroxyindoleacetic acid (5-HIAA) level in hypothalamus, brainstem, and striatum, whereas doses of 75 and 100 mg/kg increased the 3-methoxy-4-hydroxyphenylglycol (MHPG) level in hypothalamus and brainstem and the 3,4-dihydroxyphenylacetic acid level in striatum. Six amino acids were assayed in the brainstem, hypothalamus, and striatum; aspartate and glutamate levels were increased only in brainstem at 25–100 mg/kg. No consistent changes in concentrations of taurine, glutamine, glycine, or γ-aminobutyric acid were observed in any of the regions assayed. Time-related increases in rectal temperature were seen 2–12 h after dosing, and the presence of tremor was observed 5–12 h after dosing; for both the time of peak effect was at 12 h. The DDT-induced hyperthermia and tremor were associated with dose- and time-related increases in levels of 5-HIAA, MHPG, aspartate, and glutamate. It is suggested that an increase in the turnover rate of 5-hydroxy-tryptamine (5-HT) may be responsible for the DDT-induced hyperthermia, whereas increases in the metabolism of 5-HT and norepinephrine may be involved in the tremor.

Notizen: Abstract: Galactolipid metabolism was investigated as a function of development in primary cultures initiated from 19–21-day-old dissociated fetal rat brain. Significant amounts of galactocerebrosides, sulfatides, and monogalactosylglycerides were synthesized and accumulated by 8 days in culture. Thereafter the synthetic rates and levels of these galactolipids increased rapidly, reaching maximal values ∼22–29 days in culture. Galactolipids containing nonhydroxy or 2-hydroxy fatty acid were both synthesized at approximately equal rates. The initial rates of synthesis, investigated at 15, 29, and 50 days in culture, were three- to fivefold higher for galactocerebrosides than for sulfatides and two- to threefold higher than for monogalactosylglycerides. The total number of cells staining with antisera against galactocerebroside of sulfatide also increased very rapidly between 8 and 22 days in culture, reaching levels of 4–5 million cells per seeded fetal brain. The amount of galactocerebroside or sulfatide per cell stained with the corresponding antiserum increased severalfold from 10 to 27 days in culture and remained high until at least 36 days in culture (the latest time point examined). Thus, the temporal expression of galactolipid accumulation in the cell cultures was comparable to that occurring in rat brain, but some important quantitative reductions in the levels of accumulation per cell in culture were noted. In addition, in contrast to normal brain in which galactolipid synthetic rates are reduced after the period of most active myelination, in culture both synthesis and turnover of these galactolipids remained high, suggestive of a partial arrest in myelin maturation.

Notizen: Abstract: Quantitative autoradiography was used to ascertain alterations in [3H]muscimol, [3H]flunitrazepam (FLU), [3H]naloxone, [3H]d-alanine-d-leucine-enkephalin (DADL), and [3H]spiroperidol binding in basal ganglia 1 week, 4 weeks, and 5 months after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle (MFB) in the rat. At 1 and 4 weeks following lesions, [3H]spiroperidol binding increased 33% in striatum. At 5 months, [3H]spiroperidol was only nonsignificantly increased above control. At 1 week, [3H]muscimol binding decreased 39% in ipsilateral globus pallidus (GP), but increased 41% and 11% in entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr), respectively. At 4 weeks, [3H]muscimol binding was reduced 19% in striatum and 44% in GP and remained enhanced by 32% in both EPN and SNr. These changes in [3H]muscimol binding persisted at 5 months. [3H]FLU binding was altered in the same direction as [3H]muscimol binding; however, changes were slower in onset and became significant (and remained so) only at 4 weeks after lesions. Decreases in [3H]naloxone and [3H]DADL binding were seen in striatum, GP, EPN, and SNr. Scatchard analyses revealed that only receptor numbers were altered. This study provides biochemical evidence for differential regulation of striatal GABAergic output to GP and EPN/SNr.

Notizen: Abstract: We attempted to define whether thyroid hormone can ameliorate the cerebral hypomyelination present in the congenitally hypothyroid (hyt) neonatal mouse, and to define the critical time period during early postnatal life when thyroxine (T4) is essential for myelin formation. We administered T4) to the hyt mouse by breast milk during the first 20 days of postnatal life, and through the diet during the second 20 days of postnatal life. Positive results were obtained only when hormone was given during the first 20 days of postnatal life. A distinct increase in cerebral 2′,3′-cyclic nucleotide 3′-phosphohy drolase activity was noted, and brain sections stained for myelin basic protein correlated with the biochemical findings. The later administration of hormone through diet was ineffective.

Notizen: Abstract: Detergent extraction of brain slices and mouse fibroblast 3T3 cells was performed to determine rates and relative amounts of extraction of inositol versus the glycolytic enzymes. The two detergents, Triton X-100 and Brij 58, led to similar results for extraction of myo-inositol. The extraction of enzymes from brain slices or cells varied with the detergent. In brain slices, a buffered solution containing 0.2% of the detergent Brij 58 led to the extraction of 85% of the inositol before 3% of the aldolase or before 37% of either lactate dehydrogenase or triose phosphate isomerase was extracted. In contrast, with 0.1% Triton X-100 in isotonic phosphate-buffered saline, when 70% of the inositol was extracted, 33% of the aldolase and 48% of the triose phosphate isomerase were extracted. Lesser amounts of aldolase and glyceraldehyde phosphate dehydrogenase were extracted than most of the other glycolytic enzymes under all conditions, implying that these enzymes may be interacting with nonextractable subcellular components. In 3T3 cells, both detergents were of similar effectiveness for inositol extraction. Triton X-100 caused 89% of the inositol to be released and Brij 58 caused 84% to be released. With the enzymes, Brij 58 caused between 15 and 38% extraction and Triton X-100 caused between 61 and 85% extraction of the different glycolytic enzymes. Thus Brij 58 was as effective as Triton X-100 in inositol extraction but not nearly as effective in glycolytic enzyme extraction. The results demonstrate that inositol leakage from tissues or cells is a better indicator of detergent-mediated alterations in membrane porosity than glycolytic enzyme leakage. In addition, it may be suggested that Brij 58 caused plasma membrane perforations prior to destruction of the cytomatrix, whereas Triton X-100 appeared to affect both the membrane integrity and the cytomatrix as indicated by dramatic losses of both inositol and glycolytic enzymes. This distinction between detergents should be considered and used to advantage in the design of histochemical, immunocytochemical, or further biochemical studies.

Notizen: Abstract: The conversion of succinic semialdehyde into γ-aminobutyric acid (GABA) by GABA-transaminase was measured in rat brain homogenate in the presence of different concentrations of the cosubstrate glutamate. The calculated kinetic parameters of succinic semialdehyde for GABA-transaminase were a limiting Km value of 168 μM and a limiting Vmax value of 38 μmol g−1 h−1. Combination with previously obtained data for the conversion of GABA into succinic semialdehyde revealed a kEq value of 0.04, indicating that equilibrium of GABAtransaminase is biased toward the formation of GABA. The increased formation of GABA in the presence of succinic semialdehyde was not due to an increased conversion of glutamate into GABA by glutamic acid decarboxylase. Therefore these results indicate that succinic semialdehyde can act as a precursor for GABA synthesis.

Notizen: Abstract: One of the primary inactivating cleavages of neurotensin (NT) by rat brain synaptic membranes occurs at the Arg8-Arg9 peptide bond, leading to the formation of NT1-8 and NT9-13. The involvement at this site of a recently purified metalloendopeptidase was demonstrated by the use of its specific inhibitor, N-[1(R,S)-carboxy-2-phenylethyl]-alanylalanylphenylalanine-p-aminobenzoate, which exerts an inhibition on NT1-8 formation with an IC50 (0.6 μM) close to its Ki for the purified metalloendopeptidase (1.94 μM). Furthermore, we established the role of a postproline dipeptidyl-aminopeptidase in the secondary processing of NT9-13 formation.

Notizen: Abstract: Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific α1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+ -free medium, but was abolished when EGTA was also present in the Ca2+ -free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.

Notizen: Abstract: The in vivo formation of [1-14C]acetyl-coenzyme A from D-[3-14C]3-hydroxybutyrate in the brain of the suckling rat was not affected by postnatal exposure to phenyl acetate. However, utilization of the generated acetyl-coenzyme A was significantly inhibited in certain metabolic reactions, namely synthesis of fatty acids and of sterols, but not in others as the Krebs cycle reactions that lead to the production of dicarboxylic amino acids. The incorporation of D-[U-14C]glucosamine into N-acetylneuraminic acid bound to glycoproteins was appreciably diminished in the rat pup previously exposed to maternal phenylketonuria induced by phenyl acetate. During the period of very rapid development of the brain, interference by phenyl acetate and/or its metabolites with certain critical biosynthetic pathways that require acetylcoenzyme A would significantly contribute to retarded maturation of the brain that occurs in phenylketonuria.

Notizen: Abstract: Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the α-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an “adipocyte′’ form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.

Notizen: Abstract: A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.

Notizen: Abstract: The concentration dependence of regional isoleucine transport across the blood–brain barrier was determined in anesthetized rats with the in situ brain perfusion technique of Takasato et al. [Am. J. Physiol.247, H484–493 (1984)]. This technique allows, for the first time, accurate measurements of cerebrovascular amino acid transport in the absence of competing amino acids using saline perfusate, and in the presence of physiological concentrations of amino acids using plasma perfusate. Cerebrovascular isoleucine transport from saline perfusate followed Michaelis-Menten saturation kinetics where Vmax= 9–11 × 10−4μmol · s−1· g−1 and Km= 0.054–0.068 μmol · ml−1 in six brain regions. A component of nonsaturable transport was not detected in any brain region even though perfusate isoleucine concentration was increased to · 150 times the normal plasma concentration. Isoleucine influx during plasma perfusion was only 8% of that predicted from the saline perfusion data due to transport inhibition by competing amino acids in plasma. Competitive inhibition increased the apparent Km for isoleucine transport from plasma by ≥24-fold to 1.5–1.7 μmol · ml−1. These data provide accurate new estimates of the kinetic constants that describe amino acid transport across the blood–brain barrier. In addition, they indicate that the cerebrovascular transfer-site affinity (1/Km) for isoleucine is ∼fivefold greater than previously reported with the brain uptake index technique.

Notizen: Abstract: Previous studies have shown that Ro 5–4864 is a potent convulsant and increases the firing rate of substantia nigra zona reticulata neurons. The pharmacologic profile of compounds that antagonize these actions suggested that the effects of Ro 5–4864 were not mediated by “brain-type”benzodiazepine receptors. We examined a number of compounds that are structurally related to Ro 5–4864 for their capacities to displace [3H]Ro 5–4864 from “peripheral-type”binding sites and their potencies as convulsants (or as antagonists of Ro 5-4864-induced convulsions). It was observed that compounds such as KW 3600 (the N-desmethyl analog of Ro 5–4864), which have very low affinities for “peripheral-type”sites, are convulsants with a potency nearly equal to that of Ro 5–4864. In contrast, compounds such as Ro 5–6900 and PK 11195, which bind with very high affinities to “peripheral-type”binding sites, are neither convulsants nor do they antagonize the convulsant actions of Ro 5–4864. Within a series of compounds that are structurally related to Ro 5–4864 there is a good correlation (r = 0.93; p 〈 0.01) between their potencies as convulsants and their capacities to displace [35S]t-butylbicyclophosphorothionate from sites that may be associated with the chloride ionophore. Thus, it appears that occupation of “peripheral-type”binding sites by high-affinity ligands may not be directly involved in the convulsant actions of Ro 5–4864 and related compounds.

Notizen: Abstract: Distinct regional differences in dolichol content were defined in human brain from 15 to 76 years of age. Concerning the regional distribution of dolichol, levels were: (1) higher in cortical gray matter than in subcortical white matter, (2) highest among cortical regions in temporal gray matter, (3) highest among all brain regions in thalamus, and (4) lowest among all brain regions in lower brain stem and spinal cord. The developmental changes in the contents of dolichol were found to be different among brain regions. For example, among regions with the highest levels of dolichol, in thalamus there was a six to sevenfold increase, but in parietal gray matter, only a 2.5-fold increase. Regional and developmental changes in the proportions of the individual molecular species (isoprenologues) of dolichol were also observed. The findings indicate that the metabolism of dolichol is not uniform among regions of developing and aging human brain and may have implications for the role of dolichol in normal and diseased human brain.

Notizen: Abstract: Published experiments both support and contradict the hypothesis that nerve growth factor (NGF) can regulate adenylate cyclase activity. Using a sensitive assay that measures the conversion of [2-3H]adenine to [3H]cyclic AMP, we have shown that NGF alone cannot measurably stimulate cyclic AMP production, whereas the adenosine analog phenylisopropyladenosine (PIA) stimulates adenylate cyclase 20-fold over basal activity. NGF potentiates the capacity of both PIA and cholera toxin to stimulate cyclic AMP accumulation at all concentrations tested. This potentiation occurs at the earliest measurable times and does not require RNA synthesis. Therefore, we conclude that cyclase activation alone does not account for the effect of NGF on cyclic AMP accumulation and we discuss possible mechanisms.

Notizen: Abstract: Ascorbic acid in fetal rat brain increases from 374 mg/g on the 15th day of gestation to 710 mg/g by the 20th day and remains at that level until birth. There is an 18% drop from this plateau after birth.

Notizen: Abstract: The neurochemical profile of a new compound, Lu 19–005 [(±)trans-3-(3,4-dichlorophenyl)-N-methyl-1-indanamine hydrochloride], has been investigated. Lu 19–005 is a potent inhibitor of the synaptosomal uptake of 3,4-dihydroxyphenylethylamine (dopamine, DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT, serotonin). In this respect it resembles diclofensine, whereas compounds such as GBR 13.069 and bupropion are more selective DA-uptake inhibitors. Although Lu 19–005 releases DA in in higher concentrations it must be considered as an uptake inhibitor, as the accumulation of DA is inhibited in much lower concentrations. Lu 19–005 attenuates the DA and NA depletion caused by 6-hydroxydopamine in mouse brain. These properties confirm the DA- and NA-uptake-inhibitory properties of the compound. In receptor-binding models and functional in vitro tests Lu 19–005 is devoid of dopaminergic-, serotonergic-, noradrenergic-, histaminergic-, and cholinergic-inhibiting properties. Since DA, NA, and 5-HT seem to be involved in depression, the profile of Lu 19-005–with equally potent activity on the three neuronal systems–makes it an interesting experimental tool and a potential new antidepressant agent.

Notizen: Abstract: Basal activity of adenylate cyclase from the amygdala of sheep brain and the neostriatum of turkey brain decays in two phases at 37°C. The first phase is rapid (t1/2= 2.3 ± 0.3 min) and results in the loss of 60–70% of basal activity. The second phase is slow (t1/2± 100 min) during which time the catalytic units denature irreversibly. The GTP analogue guanosine-5’(β-γ-imino) triphosphate (p[NH]ppG) prevents the rapid decay by stabilizing the enzyme at its initial level of activity and also reactivates the enzyme to initial levels during or immediately following the early phase, indicating that denaturation of neither the guanylnucleotide units nor the catalytic units causes the rapid decline in basal activity. Activation by p[NH]ppG is rapid at 37°C, but the binding of p[NH]ppG to the guanylnucleotide subunit also occurs at nonactivatory temperatures. This is determined by the protection of catalytic units from thermal or N-ethylmaleimide inactivation after extensive washing. Thus, at 25°C all of the catalytic units can be stabilized by saturating p[NH]ppG concentrations. At 0°C, 35% of the catalytic units can be stabilized by saturating p[NH]ppG concentrations within 30 s. The half-saturation constant for the binding of p[NH]ppG at 0°C is identical to that derived in an assay at 37°C, or after an incubation of the membranes for 10 min at 45°C, when the process of thermal denaturation is 80% complete (K1/2∼ 3 ± 2 μM). Taken together these results suggest that the catalytic units of adenylate cyclase in brain membranes are coupled to a freely accessible, preactive state of guanylnucleotide units when the membranes are prepared, leading to an initial state of high activity.

Notizen: Abstract: Chemical changes in central glutamate neurotransmission were assessed by measuring synaptic membrane receptor binding, the uptake and release by synaptosomes of glutamate in rats treated acutely with tetraethyl lead and chronically with lead acetate. The activity of Na+, K+-ATPase in synaptosomes was measured to correlate with the changes in uptake/release studies. The affinity of receptor binding and uptake systems was significantly reduced although the number of receptor sites and the capacity of uptake systems were increased. The activity of Na+, K+-ATPase was also found to be increased in synaptosomes. The changes were more marked in inorganic lead toxicity, and all three regions studied—cerebral cortex, cerebellum, and brainstem—showed significant alterations.

Notizen: Abstract: Calmodulin activity in 68 discrete areas of rat brain, obtained by micropunch technique, was assessed by its capacity to activate a calmodulin-sensitive form of phosphodiesterase. In general, the activity of calmodulin was higher in the telencephalon, limbic system, and hypothalamus than in the mesencephalon, pons, cerebellum, and medulla. However, there were substantial differences in calmodulin activity in discrete nuclei of each region. The regional distribution of calmodulin activity in rat brain does not appear to correlate with that of any of the known putative neurotransmitters or peptides.

Notizen: Abstract: Slices from the forebrains of day-old chicks represent a highly active in vitro protein-synthesising system. The in vitro incorporation of l[14C]leucine into protein of slices was estimated to be 2.5 mmol/mg protein/h. Incorporation was linear over 90 min of incubation and was suppressed by 92% by 1 mM cycloheximide. The highest incorporation was into microsomal and cell-soluble fractions. Under the electron microscope, slices appeared vacuolated near the cut surfaces, but well preserved internally (〉40 μm from the edge). Autoradiography showed that radioactivity was incorporated evenly across the slice with no decrease in label in the central part of the tissue. The rate of incorporation was only weakly dependent on leucine concentration in the medium (0.04–1 mM). Addition of a mixture of unlabelled amino acids (1 mM) produced a 20–50% inhibition of incorporation of radioactive l-leucine depending on the amino acids involved. In slices prepared from chicks 1 h after training on a one-trial passive avoidance paradigm, l-[14C]leucine incorporation was 23% higher (p 〈 0.01) in the forebrain roof than in slices from control chicks. This figure is comparable to the one previously reported in vivo. Subcellular fractionation of incubated slices from the forebrain roof of trained and control birds revealed that the increased protein synthesis was due mainly to an elevated leucine incorporation into the soluble fraction.

Notizen: Abstract: Transport of polyamines across the blood-brain barrier of adult rats was examined by measuring the amount of radioactivity that reached the forebrain 5 s after a “bolus’ intracarotid injection. The values were expressed by the brain uptake index (BUI), which is the percentage of material transported in relation to freely diffusible water in a single passage through the brain. Transport was restricted as indicated by the respective BUI values, presented as means ± SD (number of animals): putrescine, 5.3 ± 0.8 (11); spermidine, 6.1 ± 1.3 (7); and spermine, 5.8 ± 0.5 (4). A kinetic study of the transport of [14C]putrescine showed that transport due to passive diffusion accounted for the majority of the observed influx (66% at 1 mM putrescine). However, a small saturable component exists with a Km value of 4–5 mM and a Vmax of 30 nmol ± min−1± g−1. This Km value is considerably higher than the circulating levels of the polyamine in the normal mature animal, and thus is unlikely to be of physiological significance. Competition studies indicated that putrescine does not interact with carriers for adenosine, arginine, choline, or leucine.

Notizen: Abstract: Phosphoprotein B-50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat-bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5–5.0). As visualized by two-dimensional gel electrophoresis, B-50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B-50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B-50 was in part due to a glycoprotein nature of B-50 was studied extensively. However, none of the six different methods used revealed the presence of glyco-moieties in B-50.

Notizen: Abstract: Male ICR mice, young (25-days old), mature (3-months old), and old (22 months), were injected with morphine sulfate (10 mg/kg, s.c.) or were implanted with morphine pellets (75 mg). Controls received saline injections or placebo pellets. One hour after injections and 72 h after pellet implantations, the mice were decapitated and striatal regions were removed for the following analyses: calmodulin (CaM) levels via radioimmunoassay and activities of cyclic nucleotide phosphodiesterases, adenylate and guanylate cyclases, and Ca2+, Mg2+-ATPase. Acute morphine treatment produced the following: (1) increases in calmodulin levels in the young and old mice while having no effect on mature levels; (2) increases in activities of guanylate cyclase of mature mice while decreasing those of the old mice; (3) no effects on activity of adenylate cyclase; (4) decreased activity of cyclic AMP-phosphodiesterase in young mice only; (5) decreased activity of Ca2+, Mg2+-ATPase in the old mice only. The only changes found in striata from morphinetolerant mice when compared with age-matched controls were elevations in cyclic GMP-phosphodiesterase activities in all three age groups. Differences in control values of the three age groups were as follows: CaM levels, mature 〉 old 〉 young; Ca2+, Mg2+-ATPase activity, old 〉 mature-young. The results indicate age-induced changes in cellular regulation and biochemical responses to morphine.

Notizen: Abstract: DL-Allylglycine causes a marked increase in mouse brain ornithine decarboxylase (ODC) activity. The amount of immunoreactive enzyme protein increases concomitantly with the activity, but the enzyme protein decreases more slowly than that of the activity. The amount of immunoreactive ODC in brain is many hundred times that of the catalytically active enzyme. The fact that mouse brain cytosol contains high amounts of dissociable antizyme (an inactivating protein) indicates the existence of an inactive, immunoreactive ODC-antizyme pool. The total antizyme content does not change markedly, but instead there are significant changes in different antizyme pools. Putrescine concentrations start to increase 8 h after treatment with allylglycine and concomitantly with this increase, antizyme is released to inhibit enzyme activity. These results indicate the involvement of antizyme in the inactivation process of ODC.

Notizen: Abstract: Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to membranes from cerebral hemispheres of adult rat and chicken was determined over a range of radioligand concentrations from 0.25 to 500 nM. Scatchard plots of these data were curvilinear and non-linear regression analysis indicated binding to two sites that differ in affinity. For rat cerebrum, KD(1)= 1.15 nM, Bmax(1) 0.085 pmol/mg; KD(2)= 232 nM, Bmax(2)= 16.9 pmol/mg. For chicken cerebrum, KD(1)= 1.39 nM, Bmax(1)= 0.111 pmol/mg; KD(2)= 166 nM, Bmax(2)= 17.6 pmol/mg. This multiplicity of [35S]TBPS binding was further confirmed when unlabeled TBPS or picrotoxinin displaced radioligand. The displacement curves were biphasic and yielded Hill coefficients from 0.65 to 0.70. These displacement curves were also resolved into two components with distinct IC50 values for unlabeled TBPS (rat, 1.55 and 271 nM; chicken, 2.40 and 224 nM). The IC50 values were similar to the dissociation constants obtained from equilibrium binding measurements.

Notizen: Abstract: The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5-and for Leu5-enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5- and Leu5-enkephalin and with substance P. The ontogeny of Met5-enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5-enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.

Notizen: Abstract: Mn2+ greatly increases the incorporation of myo-[3H]inositol into phosphatidylinositol (PI) of brain and other tissues by stimulating the activity of a PI-myoinositol exchange enzyme. This study examined the ability of norepinephrine (NE) and carbachol to stimulate the hydrolysis of [3H]PI formed in the absence and presence of Mn2+-stimulated [3H]inositol exchange. Rat cerebral cortical slices were incubated with myo-[3H]inositol for 60 min in an N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES) buffer without or with MnCl2 (1 mM). The tissue was washed and further incubated with unlabeled myo-inositol and LiCl (10 mM). Prelabeled slices were then incubated with NE (0.1 mM) or carbachol (1 mM) to induce agonist-stimulated [3H]PI hydrolysis. Mn2+ treatment resulted in eight- and sixfold increases in control levels of [3H]PI and [3H]inositol monophosphate ([3H]IP), respectively. Both NE and carbachol stimulated [3H]IP formation in tissue prelabeled without or with manganese. However, the degree of stimulation (percentage of control values) was greatly attenuated in the presence of Mn2+. In the absence of Mn2+ treatment, NE decreased [3H]PI radioactivity in the tissue to 80% of control values. However, NE did not decrease [3H]PI radioactivity in the Mn2+ -treated tissue. These data demonstrate that Mn2+ stimulates incorporation of myo-[3H]inositol into a pool of PI in brain that has a rapid turnover but is not coupled to agonist-induced hydrolysis.

Notizen: Abstract: The activity of transglutaminase was characterized in the rat brain. In adults, comparable levels of transglutaminase activity are present in all brain regions examined. The activity is present in all subcellular fractions, as studied by differential centrifugation, but the soluble fraction contains the highest specific activity. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate casein) is very low in all subcellular fractions, except in the synaptosomal fraction where its highest levels are about 40–60% of the activity assayed in the presence of casein. Furthermore, enzyme activity is present on the external surface of synaptosomes. In the soluble fraction, maximal activity can be detected between pH values of 9 and 10 when assayed in the presence of 5 mM CaCl2 (with half-maximal activity requiring 0.75 mM CaCl2) and 0.4 mM putrescine (with an apparent Km for putrescine of 0.1 mM). The activity can be partially inhibited by ZnCl2 (with an IC50 of 4.5 mM) and by AlCl3 (with an IC50 of 5.1 mM). In the cerebellum, where the full span of neuronal development can be studied after birth, the highest specific activity is observed just after birth, thereafter the activity starts to decline and by 14 days, after a reduction of about 65%, it reaches levels observed throughout life.

Notizen: Abstract: Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 μM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 μM reserpine, 30 mM NH4+, or 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules within digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 μM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-β-hydroxylase. The studies demonstrate that 20 μM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.

Notizen: Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (Ni) and stimulatory (Ns) components. Inactivation of Ni by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of Ns, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E1, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through Ni resulting in the unimpeded expression of Ns activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which Ni regulates adenylate cyclase in this cell line.

Notizen: Abstract: A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional γ-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GAGA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.

Notizen: Abstract: Brainstem slices prepared from 22-day-old rats, were employed to study the intracellular translocation of radioactively labeled myelin proteolipid protein (PLP). Double-isotope and short pulse-chase procedures allowed us to demonstrate the flux of PLP through nine different subcellular membrane fractions that were isolated on the basis of their particle size and buoyant density. Tagged PLP was rapidly depleted from microsomes, showed transient passage through a number of presumably intermediate membranous pools, and accumulated in myelin. On the basis of the kinetics of PLP labeling and isotope ratios, the membranes can be arranged as they participate in the intracellular translocation of PLP and consistently show a pattern indicating possible precursor-product relationships.

Notizen: Abstract: Valproic acid distribution in brain is less than that of other anticonvulsants such as phenytoin or phenobarbital. Possible mechanisms for this decreased distribution space in brain include (a) increased plasma protein binding of valproate relative to the other anticonvulsants and (b) asymmetric blood-brain barrier (BBB) transport of valproate such that the brain-to-blood flux exceeds the blood-to-brain flux. These mechanisms are investigated in the present studies using the intracarotid injection technique in rats and rabbits. In the rat, the brain uptake index (BUI) of [14C]valproate relative to [3H]water is 51 ± 6%, indicating the blood-to-brain transport of water is twofold greater than that of valproate. However, the BUI of [14C]valproate relative to [3H]water decreased with time after carotid injection during a 4-min washout period, which indicates that brain-to-ßlood transport of valproate is greater than that of water. This suggests that the permeability of the BBB to valproate is polarized, with antiluminal permeability being much greater than luminal permeability. In rabbits, the BUI of [14C]valproate is 47 ± 7% in newborns and 17 ± 6% in adult animals. However, the high drug extraction in newborns may be attributed to decreased cerebral blood flow in the neonate as the BBB permeability-surface area (PS) products are unchanged (e.g., PS= 0.13 and 0.11 ml min−1, g−1 in the newborn and adult rabbit, respectively). With regard to plasma protein binding effects on valproate transport, brain valproate uptake was also measured in the presence of human, lamb, pig, rat, horse, goat, hamster, dog, and mouse sera. Higher brain uptakes were observed when the unbound fraction of drug increased. However, our data indicate that a fraction of the valproic acid entering the capillaries bound to plasma proteins had the capacity to equilibrate with brain because of enhanced drug dissociation from albumin in the brain microcirculation. Since plasma protein-bound valproate is available for uptake by brain, the major factor underlying the diminished distribution of the drug in brain appears to be the asymmetric transport properties of the BBB to valproic acid.

Notizen: Abstract: Calmodulin-dependent kinase activity was investigated in cold-stable microtubule fractions. Calmodulin-dependent kinase activity was enriched approximately 20-fold over cytosol in cold-stable microtubule preparations. Calmodulin-dependent kinase activity in cold-stable microtubule preparations phosphorylated microtubule-associated protein-2, α- and β-tubulin, an 80,000-dalton doublet, and several minor phosphoproteins. The endogenous calmodulin-dependent kinase in cold-stable microtubule fractions was identical to a previously purified calmodulin-dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin-binding properties, (4) subunit autophosphorylation, (5) calmodulin-binding subunit composition on high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on β-tubulin, and (9) phosphopeptide mapping. Endogenous cold-stable calmodulin-dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin-binding, autophosphorylating p and σ subunits of the previously purified kinase. The p and σ subunits of this kinase represented the major calmodulin-binding proteins in the cold-stable microtubule fractions as assessed by denaturing and nondenaturing procedures. These results indicate that calmodulin-dependent kinase is a major calmodulin-binding enzyme system in cold-stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.

Notizen: Abstract: The binding of [3H]indalpine {4-[2-(3-indolyl)]ethyl piperidine} to slide-mounted sections of rat brain has been characterized. This 5-hydroxytryptamine (5-HT) uptake blocker binds to sections with high affinity (KD∼ 1 nM). The binding is saturable, and can be displaced by the addition of clomipramine (1 μM). Other drugs inhibiting the uptake of 5-HT also have the capacity to inhibit the binding of [3H]indalpine. A significant correlation (r = 0.86) was found between the capacity of these compounds to inhibit the uptake of 5-HT and their potencies as inhibitors of [3H]indalpine binding. Binding was Na+- and Cl−dependent and was inhibited competitively by 5-HT. Furthermore, electrolytic lesions of the dorsal raphe or medial forebrain bundle, which cause a degeneration of 5-HT cell bodies and fibers, respectively, resulted in a 30–40% reduction in the binding of [3H]indalpine. [3H]Indalpine binds to the 5-HT uptake recognition sites in a different manner from imipraminelike compounds.

Notizen: Abstract: The localization of binding sites for [3H]indalpine to sections of rat brain was studied by a quantitative autoradiographic technique. Binding sites for this specific neuronal 5-hydroxytryptamine (5-HT) uptake inhibitor are concentrated in areas rich in 5-HT neuronal cell bodies, fibers, and synaptic terminals. One of the most interesting features of this regional distribution is the very high density of these sites found in the dorsal raphe, substantia nigra, ventral tegmental area, and locus ceruleus. Components of the visual system also show pronounced labelling with [3H]indalpine. The finding that limbic structures are strongly labelled by this drug may be related to the antidepressant activity of indalpine. The anatomical distribution of binding sites demonstrated is Consistent with the specific labelling of 5-HT neurons by [3H]indalpine and confirms previous studies carried out with another 5-HT uptake inhibitor, [3H]imipramine.

Notizen: Abstract: We report a method for the isolation of enriched fractions of intact Golgi apparatus from neurons of 10- to 12-day-old rat brains. Neurons were prepared according to a modified method of Farooq and Norton [J. Neurochem.31, 887–894 (1978)]. Golgi-enriched fractions were obtained after centrifugation of postmitochondrial supernatants in a discontinuous sucrose gradient. Golgi fractions 1 and 2, recovered at the interfaces of 28–34% and 34–36% sucrose densities, respectively, were examined with morphometric and enzymatic methods. Morphometric analyses showed that 21–34% of fraction 1 and 11–29% of fraction 2 consisted of intact Golgi apparatus. Lysosomes, mitochondria, ribosomes, and rough endoplasmic reticulum contaminated fraction 1 (6–10%) and fraction 2 (14–26%). Golgi fraction 1 showed a 25- to 65-fold enrichment over neurons of UDP Gal:GlcNAc galactosyltransferase, CMP-sialic acid:lactosylceramide sialyltransferase, and PAPS:cerebroside sulfotransferase activities. Golgi fraction 2 showed a 8-to 23-fold enrichment over neurons of the activities of the above glycolipid- and glycoprotein-synthesizing enzymes. The activities of the possible marker enzymes rotenone-insensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and arylsulfatase were low or minimally elevated in the Golgi fractions. A sevenfold enrichment of Na+,K+-ATPase activities was found in the Golgi fractions. This is consistent either with significant plasma membrane contamination or with the presence of this enzyme in the neuronal Golgi apparatus.

Notizen: Abstract: The total amount of gangliosides per cerebellum of a wild-type mouse increased 126-fold during postnatal development. Although all major gangliosides were synthesized, the relative amount of individual ganglioside species changed during this period. In the developing wild-type cerebellum a transient accumulation of GD3 occurred between birth and postnatal day 20 (P20) with the largest portion (23%) of the total ganglioside content at postnatal day 7 (P7). In the adult cerebellum GD3 was only a minor component (3.2%) of the ganglioside pattern. As demonstrated by immunofluorescence the accumulation of GD3 was predominantly associated with premigratory and early postmigratory granule cells. The ganglioside GD3 was found in two alkali-stable forms in the young cerebellum, whereas the ganglioside species with the higher Rf value (migrating in the same position as the upper GD3 band) in the adult cerebellum was alkali labile. The cerebellum of the neurological mutant staggerer (sg/sg) was characterized by a low amount of GD1a in adult animals, due to the massive death of neurons in the postnatal cerebellar cortex. The neonatal loss of sialic acid residues from cerebellar cell surfaces in wild-type mice and the maintenance of embryonic sialoglycoconjugates in the staggerer cerebellum cannot be explained by the alterations of ganglioside patterns observed during postnatal development.

Notizen: Abstract: The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific sites in CNS membranes was investigated using cortical tissue from a variety of mammalian species. Mass law analysis of the site-specific binding of NBMPR data revealed that rat, mouse, guinea pig, and dog cortical membranes each contained an apparent single class of high-affinity (KD 0.11–4.9 nM) binding sites for NBMPR; rabbit cortical membranes, however, exhibited two distinct classes of NBMPR binding sites with KD values of 0.4 nM and 13.8 nM. Dipyridamole, a potent inhibitor of nucleoside transport, produced a biphasic profile of inhibition of the binding of NBMPR to guinea pig, rabbit, and dog membranes (IC50 〈 20 nM and IC50 〉 6 μM for NBMPR binding sites displaying high and low affinity for dipyridamole, respectively). These results are indicative of heterogeneity of NBMPR binding sites in mammalian cortical membranes. Rat and mouse cortical membranes appear to possess only one type of NBMPR binding site, which has low affinity for dipyridamole. Detailed analysis of inhibitorinduced dissociation of NBMPR from its sites in each species led to the conclusion that these multiple forms of NBMPR binding sites are different conformations of a single site associated with the CNS nucleoside transport system, rather than two distinct sites. It is also suggested that the affinity of dipyridamole for each conformation of NBMPR site indicates the susceptibility of that conformation of the nucleoside transport system to inhibition by dipyridamole.

Notizen: Abstract: Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15–16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2′:3′-cyclic nucleotide 3′-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.

Notizen: Abstract: Two neuroblastoma cell lines were cultured in control (euthyroid) and hypothyroid media and examined for protein, RNA and DNA content, activity of the catecholaminergic enzymes tyrosine hydroxylase (TH, EC 1.14. 16.2) and monoamine oxidase-A (MAO-A, EC 1.4.3.4), and for L-triiodothyronine (T3) nuclear receptors. In the hypothyroid condition, the rate of cell division and the levels of RNA and protein as well as the activities of TH and MAO were lower than in the euthyroid condition, the reduction being more marked in the Ē than in the A2(1) cell line. T3 nuclear receptors, unaltered in affinity, were increased in number in the hypothyroid medium, possibly as a regulatory response to hormonal deficiency. Examination of a possible relationship between T3 occupancy and TH activity in the Ē cells, most sensitive to thyroid hormone deficiency, revealed that induction of TH activity by T3 is dose-dependent and correlates with the number of nuclear sites occupied by the hormone. When neuroblastoma cells were induced to differentiate by the addition of sodium butyrate to the medium, parameters of cell growth (protein, RNA) and enzyme activity (TH and MAO-A) increased in both cell lines irrespective of the presence of thyroid hormones. These data indicate that thyroid hormones, through their nuclear receptors, directly affect the activity of catecholaminergic enzymes in cultured, immature (undifferentiated) neurons.

Notizen: Abstract: The kinetic constants for large neutral amino acid (LNAA) transport across the blood–brain barrier (BBB) of conscious rats were determined in four brain regions: cortex, caudate-putamen, hippocampus, and thalamus-hypothalamus. Indwelling external carotid artery catheters allowed for single-bolus (200 μ1) injections directly into the arterial system of unanesthetized and lightly restrained animals. Our results showed lower brain uptake index values for conscious rats compared to previous reports for anesthetized animals which are consistent with higher rates of cerebral blood flow in the conscious animals. Km values were lower in the conscious animals and ranged from 29% to 87% of the Km values in pentobarbital-anesthetized animals whereas the KD values were about twofold higher in the conscious animals. No apparent regional differences were observed. Influx rates were determined which take into consideration flow rates and plasma amino acid concentrations. Our results showed an average amino acid influx value of 5.2 nmol/min/g, which is 53% higher than the average influx in pentobarbital-anesthetized animals. The present results in conscious animals regarding the low Km of LNAA transport across the BBB lend further support to the importance of fluctuations in plasma amino acid concentrations and LNAA transport competitive effects on brain amino acid availability.

Notizen: Abstract: Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.

Notizen: Abstract: The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse-labeled with [35S]sulfate. The radioactively labeled products were characterized by one- and two-dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein. much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000 100,000; pI 4.3–5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue-stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.

Notizen: Abstract: Influences of α2-adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet-activating protein and NAD resulted in a significant increase in basal activity as well as in GTP-or forskolin/GTP-induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet-activating protein and NAD. It is suggested that α-receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex.

Notizen: Abstract: Adrenal chromaffin cells normally synthesize and release catecholamines. In the present study, [3H]acetylcholine synthesis and another characteristic of cholinergic neurons, [3H]choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells. Chromaffin cell cultures took up [3H]choline from the medium and acetylated the [3H]choline to form [3H]acetylcholine. The rate of [3H]acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture. [3H]Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+. The ability for K+ to stimulate synthesis of [3H]acetylcholine developed only after 28 days in culture. [3H]Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline. [3H]Choline uptake was enhanced by Na+ omission in day-14 cultures, but was at least partially Na+-dependent in day-29 cultures. Hemicholinium-3 (IC50〈 10 μM) inhibited [3H]choline uptake into chromaffin cell cultures. It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.

Notizen: Abstract: An oligodendroglial specific property, glucocorticoid regulation of glycerol-3-phosphate dehydrogenase (GPDH) levels, was inhibited in C6 rat glioma cells when 4β-phorbol 12-myristate 13-acetate (PMA) was added to the cultures. PMA inhibited GPDH induction in both logarithmic- and stationary-phase cells. These events are most likely mediated through the phorbol ester receptor since the ability of various phorbol ester analogs to compete with the ligand [3H]4β-phorbol 12,13-dibutyrate for binding to the receptor correlates with the ability the particular analog has to inhibit GPDH induction. Additionally, like tumor promotion in vivo, the inhibition of GPDH induction is reversible. The PMA effect is not restricted to the C6 cell line since PMA also inhibits GPDH inducibility in another rat glioma cell line. This PMA-mediated event has been partially characterized. PMA did not affect the overall rate of protein or RNA synthesis. It was ineffective in altering both glucocorticoid accumulation to the nucleus and the rate of GPDH degradation. It appears likely that PMA's inhibitory action occurs at the transcriptional or translational level.

Notizen: Abstract: Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal γ-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 μM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.

Notizen: Abstract: The ATP-stimulated accumulation of L-[3H]glutamate by whole brain synaptic vesicle preparations from long-sleep and short-sleep mice, lines selectively bred for difference in sleep time response to acute ethanol administration, was examined. L-[3H]Glutamate accumulation in vesicles from short-sleep mice was approximately twice that observed in vesicles from longsleep mice at three glutamate loading concentrations. The vesicular content of endogenous L-glutamate in crude and enriched vesicle preparations from short-sleep mice was approximately 1.5-fold higher than in vesicles from long-sleep mice. The data suggest that L-glutamate associated with synaptic vesicles may serve a role in glutamate neurosecretion.

Notizen: Abstract: The enzyme UDP-N-acetylglucosamine: dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase initiates the synthesis of the oligosaccharide chain of complex-type glycoproteins. In view of the high content of glycoprotein in peripheral nerve myelin, the properties of this enzyme, its changes with age, and the effect of the specific inhibitor tunicamycin were investigated. The enzyme activity in rat peripheral nerve homogenate was completely dependent on the presence of exogenous dolichyl phosphate as well as Mg2+ and a detergent (Triton X-100) and was also greatly stimulated by a high salt concentration (0.4 M KCl) and AMP. The highest specific activity was present in the postmitochondrial membranes. The specific activity in postmitochondrial membranes in the presence of exogenous dolichyl phosphate reached a maximum at 17 days and remained relatively high throughout development, up to 2 years of age, but the activity was much lower when dolichyl phosphate was not added. This indicates that the enzyme level does not decrease with age, but that the content of the lipid cofactor may limit glycoprotein synthesis in vivo. Tunicamycin (5 μg) was injected intraneurally into 24-day-old rat sciatic nerve, and the enzyme was assayed from 1 to 24 days after injection. The specific activity of the transferase remained at low levels (5–40% of the level in control nerve) in most injected nerves assayed throughout this postinjection period. A protein previously identified as the unglycosylated P0 protein was synthesized in vitro by the tunicamycin-injected nerve and could be demonstrated to be incorporated into myelin in large amounts at 2 days and in small amounts at 6 days after injection. At 11 days, the protein was synthesized but not incorporated into myelin, and morphological examination showed evidence of myelin disintegration at 14 and 30 days after tunicamycin injection. These results indicate an important role for the enzyme UDP-N-acetylglucosamine:dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase in the synthesis of P0 in peripheral nerve myelin. The activity of this enzyme may be a limiting factor in the maintenance of the nerve.

Notizen: Abstract: Myelin proteolipid protein is known to contain covalently bound fatty acid. To determine the contribution of the fatty acid to the multiple bands observed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the electrophoretic parameters of the proteolipid protein were compared with those of the deacylated form. The relative mobility and proportion of each band, as well as the retardation coefficient and free electrophoretic mobility, were not altered by removal of the fatty acid moiety. Furthermore, the acylated and deacylated forms bound the same amounts of sodium dodecyl sulfate. These data demonstrate that the presence of covalently bound fatty acids does not account for the electrophoretic heterogeneity of the proteolipid.

Notizen: Abstract: The effect of acute ethanol administration on histamine (HA) dynamics was examined in the mouse hypothalamus. The steady-state level of HA did not change after intraperitoneal administration of ethanol (0.5–5 g/kg), whereas the level of tele-methylhistamine (t-MH), a predominant metabolite of brain HA, increased when 3 and 5 g/kg of ethanol was given. Pargyline hydrochloride (80 mg/kg, i.p.) increased the level of t-MH by 72.2% 90 min after the treatment. Ethanol at any dose given did not significantly affect the t-MH level in the pargyline-pretreated mice. Decrease in the t-MH level induced by metoprine (10 mg/kg, i.p.), an inhibitor of HA-N-methyltransferase, was suppressed by ethanol (5 g/kg), thereby suggesting inhibition of the elimination of brain t-MH. Ethanol (5 g/kg) significantly delayed the depletion of HA induced by (S)-α-fluoromethylhistidine (50 mg/kg, i.v.), a specific inhibitor of histidine decarboxylase. Therefore, a large dose of ethanol apparently decreases HA turnover in the mouse hypothalamus.

Notizen: Abstract: A superfusion system employed to measure the K+ -stimulated release of [3H]5-hydroxytryptamine ([3H]5-HT,[3H]serotonin) from a synaptosomal-rich spinal cord tissue preparation was carefully characterized, then used to examine the regulation of spinal 5-HT release. Spinal 5-HT release is apparently modulated by an autoreceptor. Exogenous 5-HT depressed, in a concentration-dependent manner, the K+-stimulated release of [3H]5-HT. Similarly, lysergic acid diethylamide (LSD) produced a concentration-dependent decrease in [3H]5-HT release. Methiothepin and quipazine blocked the inhibition of release induced by exogenous 5-HT. The 5-HT2 receptor antagonists spiperone and ketanserin failed to alter the action of 5-HT at the spinal 5-HT autoreceptor. Spiperone and ketanserin were shown, however, to alter the storage of [3H]5-HT. When used in concentrations greater than 10 nM, the drugs evoked increases in basal [3H]5-HT and [3H]5-hydroxyindoleacetic acid ([3H]5-HIAA) effluxes which were independent of the presence of calcium ions. A good agreement existed between the potencies of drugs for modifying autoreceptor function and their abilities to compete for high-affinity [3H]5-HT binding in the spinal cord (designated 5-HT1). Furthermore quipazine, in concentrations that preferentially interact with the 5-HT1B subtype, antagonized the actions of exogenous 5-HT on K+ -stimulated release. Spiperone, in a concentration that approximated the affinity constant of 5-HT1A sites for the drug, was ineffective in altering the ability of exogenous 5-HT to modulate K+-stimulated [3H]5-HT release. These results suggest that 5-HT1B sites are associated with serotonergic autoreceptor function in the spinal cord.

Notizen: Abstract: A saturable, specific, high-affinity binding site for [3H] 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine was found in rat brain homogenates. The CNS regional distribution, the subcellular fractionation, and the displacement by pargyline, clorgyline, and deprenyl suggest that this binding site may correspond to monoamine oxidase. I -Methyl-4-phenyl-1,2,3,6-tetrahydropyridine inhibited the oxidative deamination of dopamine, both in vivo and in vitro. Striatal levels of 3,4-dihydroxyphenylacetic acid were significantly reduced shortly after intravenous administration, and returned to normal values after a few hours. The in vitro formation of 3,4-dihydroxyphenylacetic acid from dopamine was inhibited by concentrations of 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine comparable to those of pargyline.

Notizen: Abstract: In this study we examined the interaction of opiates with K binding sites in the bovine adrenal medulla. [3H]Ethylketocyclazocine (EKC), [3H]etorphine, and [3H]bremazocine stereoselective bindings were used to assay these interactions. The K sites were found to be heterogeneous: [3H]bremazocine identified with high affinity all subtypes of these sites. [3H]EKC, in the presence of saturating concentrations of [D-Ala2, D-Leut]-enkephalin (DADLE) (5μM), was used to identify K1 sites, on which dynorphin A (1–13) bound with high affinity. Either [3H]EKC or [3H]etorphine in the presence of 5μM DADLE identified the K2 subtype. This subtype was found to interact with β-endorphin and especially with the octapeptide Met5-enkephalyl-Arg6-Gly7-Leu8. Furthermore, [3H]etorphine identified in the bovine adrenal medulla a third high-affinity component, in the presence of 5 μM DADLE. This residual interaction was found to be equally stereoselective and presenting K selectivity. Met5-enkephalyl-Arg6-Phe7 interacted preferentially with this site. The three K subtypes interacted differentially with monovalent (Na+, K+, and Li+) and divalent (Ca2+, Mg2+, and Mn2+) ions by modification of the apparent concentration of the accessible sites and/or by changes of the apparent KD for radioligands. Modifying agents (proteolytic enzymes, thiol-modifying reagents, and A2-phospholipase) produced different effects on each subtype of the K site, suggesting a different protein (or protein-lipid?) composition.

Notizen: Abstract: Two major components of human brain S100 fraction were purified by HPLC and an amino acid sequence was elucidated for the S100β component. Human S100 proteins showed absorption spectra and amino acid compositions similar to S100α and S100β from bovine brain. However, the relative amounts of the human proteins were 4% S100α and 96% S100β by weight, while the bovine protein distribution was 47% S100α and 53% S100β by weight. An amino acid sequence of human S100β was established by analysis of overlapping fragments generated by cyanogen bromide and trypsin cleavage. Three amino acid sequence differences between the human and bovine S100β were found at residues 7, 62, and 80. These differences were chemically conservative and compatible with minimum single base changes in the codon structures. These results document that S100β is a conserved protein among mammals and provide the necessary foundation for current clinical studies.

Notizen: Abstract: We examined the soluble fraction from homogenates of 12-day embryonic chick heart for the presence of an endogenous modulator of muscarinic acetylcholine receptors (mAChR). Homogenates were separated into 100,000 g soluble and crude membrane fractions by differential centrifugation. Aliquots of membranes were incubated in the presence or absence of the soluble fraction and the muscarinic antagonist, [3H]quinuclidinyl benzilate ([3H]QNB), and the data subjected to Scatchard analysis. In the presence of the soluble fraction, mAChR number decreased up to 70% and the affinity for [3H]QNB decreased six- to eightfold. These results suggested that an endogenous soluble factor (ESF) affected cholinergic ligand binding to the receptor. The amount of ESF extracted from 〈 10 mg of brain was sufficient to reduce by 50% [3H]QNB binding to 50 fmol mAChR. ESF activity was partially purified by heat and acid treatment. The loss of receptors was dependent upon the amount of ESF added and was time dependent. QNB protected some receptors from loss due to ESF. The change in mAChR affinity for [3H]QNB was observed only if ESF was present continuously during the [3H]QNB binding assay. Ultrafiltration and gel filtration showed that ESF was 〈 10,000 daltons and probably 〈700 daltons. ESF activity was blocked by EDTA. However, ESF was not a divalent cation since it was base labile, and removal of divalent cations with Chelex-100 did not inhibit ESF activity. ESF activity was also blocked by catechol, catecholamines, ascorbate, and dithiothreitol. ESF was present in embryonic but not in adult heart.

Notizen: Abstract: The complex pharmacological profile (excitation/inhibition) of ibotenic acid on single neurons in the mammalian CNS prompted studies on the stability of ibotenic acid and a number of structurally related excitatory amino acids under different in vitro conditions in the presence or absence of enzymes. Ibotenic acid, (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA), (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and (RS)-α-amino-3-hydroxy-4-bromo-5-isoxazolepropionic acid (4-Br-homoibotenic acid) were all inhibitors of (S)-glutamic acid decarboxylase (GAD) in mouse brain homogenates, but only ibotenic acid was shown to undergo decarboxylation during incubation with brain homogenates. The formation of the decarboxylated product, muscimol, which primarily occurred in a synaptosomal fraction, was dependent on the presence of pyridoxal-5-phosphate (PALP) and was inhibited by (S)-glutamic acid, 3-mercaptopropionic acid (3MPA), aminooxyacetic acid (AOAA), and allylglycine, suggesting that ibotenic acid is a substrate for GAD. The overall decomposition rate for ibotenic acid (8.7 nmol min−1 mg−1 of protein), which apparently embraces other reactions in addition to decarboxylation to muscimol, was higher than the rate of decarboxylation of (S)-glutamic acid (3.2 nmol min−1 mg−1 of protein). At pH 7.4 and 37°C, but in the absence of enzymes, none of the excitatory amino acids under study underwent any detectable decomposition, whereas ibotenic acid and 7-HPCA, but not AMPA and 4-Br-homoibotenic acid, decomposed, partially by decarboxylation, at 100°C in a pH-dependent manner. In the presence of liver homogenates, ibotenic acid was also shown to decompose. Although muscimol was the only detectable reaction product, mechanisms other than decarboxylation may be involved. Under these conditions, the degradation reaction or reactions were partially dependent on PALP and were inhibited by AOAA and 3MPA but not by allylglycine. The present in vitro studies indicate that ibotenic acid is likely to undergo enzyme-catalyzed decomposition to give muscimol in brain tissue and after systemic administration to animals. These aspects must be taken into consideration in the interpretation of the pharmacological or neurotoxic effects of ibotenic acid after direct application near central neurons, after local injections into animal brains, or after systemic administration to animals.

Notizen: Abstract: The synthesis of a series of γ-glutamyl amines (γ-Glu-amines), including γ-Glu-dopamine, γ-Glu-5-hydroxytryptamine, γ-Glu-octopamine, γ-Glu-tryptamine, γ-Glu-tyramine, and γ-Glu-phenylethylamine, by nervous tissue of the marine mollusc Aplysia californica is described. After ganglia were incubated in vitro with 14C-amines, the unchanged amine and a new 14C-labeled product, identified as the γ-Glu conjugate of the amine, were isolated from the tissue extracts. Identification was made by comparing the chromatographic properties (HPLC, TLC, and LC) of the isolated conjugates with chemically synthesized γ-Glu-amines before and after acid hydrolysis.

Notizen: Abstract: At D2 3,4-dihydroxyphenylethylamine (dopamine) receptors in anterior pituitary tissue, magnesium ions shifted receptors to agonist high-affinity states, but decreased the affinity of the antagonist [3H]spiperone. Conversely, sodium ions shifted the receptors to agonist low-affinity states, but increased the affinity of [3H]spiperone. Magnesium is proposed to stabilize the hormone–receptor–guanine nucleotide regulatory protein complex, whereas sodium appears to destabilize this ternary complex. Thus, magnesium and sodium appear to mediate their regulatory effects via a common component at the D2 dopamine-receptor ternary complex.

Notizen: Abstract: As part of a systematic study of the evolution of the nervous system, the lipid composition of the ventral nerves of earthworms was examined. The nerve axons are wrapped in copious layers of loosely bound membrane, superficially resembling the myelin sheath of vertebrates. However, neither galactocerebroside nor sulfatide, both of which are considered to be markers for myelin, was present, and only traces of glucocerebroside, which is abundant in shrimp nerve, were detected. The lipids were rich in cholesterol (15.3 μmol/g of fresh tissue) and phospholipids (21.7 μmol/g of fresh tissue). The phospholipids were composed of phosphatidylethanolamine, -choline, -serine, and -inositol in the ratio of 41:44:8:7. Most of the ethanolamine-containing phospholipids were in the form of plasmalogens. The fatty acid moieties of these phospholipids were predominantly 18:1, 18:0, and 20:1, whereas the aldehyde moieties of plasmalogen were mostly 18:0. Sphingomyelin, which is considered a ubiquitous component of animal membranes, was not detected. How the unique structure of the membranes of earthworm nerves may be related to the function of the nervous system in this organism is discussed.

Notizen: Abstract: The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC;EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [I-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.

Notizen: Abstract: Substance P is known to modulate acetylcholine-induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol-induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol-induced 45Ca2+ uptake in both Na+-containing and Na+-free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage-sensitive or acetylcholine receptor-linked channels. However, substance P produced only a small inhibition of K+-induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage-sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol-induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, and increasing concentrations of extracellular Ca2+ were unable to overcome substance P's inhibition of [3H]-norepinephrine ([3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.

Notizen: Abstract: Three monoamine oxidase (MAO) inhibitors—pargyline, clorgyline and deprenyl—as well as the serotonin (5-HT, agent hydroxytryptamine) releasing agent fenfluramine were administered to developing chick embryos and the effects on [3H]5-HT binding parameters and endogenous 5-HT levels were assessed. Multiple, but not acute, pretreatments with any of the three MAO inhibitors significantly increased 5-HT levels (p 〈 0.01) and decreased receptor number (Bmax) to a maximum of 20% (p 〈 0.01) without affecting the affinity (KD). When d,l-5-hydroxytryptophan (d,l-5-HTP) was similarly administered there were large increases in 5-HT levels (p 〈 0.01), but no significant effects on either Bmax or KD. However, if d,l-5-HTP was co-administered with any of the MAO inhibitors there was a significant (p 〈 0.01) enhancement of the MAO inhibitor-induced down-regulation to a maximum of about 40%. Multiple pretreatments with fenfluramine resulted in dose-related decreases in 5-HT levels (p 〈 0.01) and Bmax (p 〈 0.01) without affecting KD. The largest decrease in [3H]5-HT binding sites inducible by fenfluramine treatment alone was also about 40%. When given in combination with d,l-5-HTP, there was a potentiation of the down-regulation capabilities of fenfluramine at several different dosage levels; however, maximal down-regulation was also limited to 40%. Evidence was presented suggesting that these effects were not due to endogenous 5-HT or drugs remaining in the tissue preparation. The overall evidence implies that merely increasing endogenous 5-HT levels, as by precursor administration, does not necessarily induce down-regulation unless the 5-HT is also made available as functional 5-HT.

Notizen: Abstract: The blood–brain barrier (BBB) transport of a highly lipid-soluble peptide, [3H]cyclosporin, was studied in ketamine-anesthetized rats using the carotid artery injection technique. For comparison, peptide transport into rat liver was also assessed with the portal vein injection technique. Despite the high lipid solubility of this peptide (1-octanol/Ringer's partition coefficient = 991 ± 55), the extraction by rat brain was only 2.9 ± 0.5% in the presence of 80% human serum, and this value approximated the extraction for a poorly diffusible substance such as [3H]inulin, 2.0 ± 0.1%. In contrast, the hepatic extraction of [3H]cyclosporin was high, 84 ± 2%, in the presence of 80% human serum. The BBB transport of cyclosporin is markedly restricted owing to the combined effects of binding by serum proteins and a paradoxically low permeability of the BBB to the peptide.

Notizen: Abstract: 1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by mono-amine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxy-phenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 μM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 μM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.

Notizen: Abstract: The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces neuropathology and clinical symptoms that resemble Parkinsonism in primates and humans. In mice it induces a long-lasting depletion of neostriatal 3,4-dihydroxyphenylethylamine (dopamine) content. Using the mouse, we found that MPTP induces a fall of dopamine and a rise of acetylcholine in the neostriatum. Both responses to MPTP can be blocked by prior treatment with atropine or trihexyphenidyl.

Notizen: Abstract: The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and olfactory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25–30°C and pH 7.8–8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD= 0.7 ± 0.1 nM; Bmax= 347 ± 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.

Notizen: Abstract: Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6–15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 μg CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.

Notizen: Abstract: L-Aspartate N-acetyltransferase, a nervous system enzyme that mediates the synthesis of N-acetyl-L-aspartic acid, has been characterized. In the presence of acetyl-CoA, L-aspartate was acetylated 10-fold more efficiently than L-glutamate, and the acetylation of aspartylglutamate was not detectable. Within the nervous system, a 10-fold variation in the enzyme activity was observed, with the brainstem and spinal cord exhibiting the highest activity (10–15 pmol/min/mg tissue) and retina the lowest detectable activity (1–1.5 pmol/min/mg). No enzyme activity was detected in pituitary, heart, liver, or kidney. The enzyme activity was found to be membrane-associated and was solubilized by treatment with Triton X-100.