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Notes: The administration of nicotine activates tyrosine hydroxylase in the rat adrenal gland. This activation is apparently maximal 25 min after a single subcutaneous injection of nicotine at 2.3 mg/kg. Repeated injections of nicotine (seven injections once every 30 min) are associated with a persistent activation of adrenal tyrosine hydroxylase for at least 3 h. The nicotinic receptor antagonist hexamethonium docs not significantly inhibit the nicotine-mediated activation of tyrosine hydroxylase in innervated adrenal glands. However, hexamethonium completely blocks the activation of adrenal tyrosine hydroxylase by nicotine in denervated adrenal glands. Furthermore, even though a single injection of nicotine activates tyrosine hydroxylase in both innervated and denervated adrenal glands, repeated injections of nicotine do not activate tyrosine hydroxylase in denervated adrenal glands. Our results suggest that the systemic administration of nicotine activates adrenal tyrosine hydroxylase by two mechanisms: (1) via direct interaction with adrenal chro-maffin cell nicotinic receptors; and (2) via stimulation of the CNS leading to the release from the splanchnic nerve of substances that interact with adrenal chromaffin cell receptors other than the nicotinic receptor.

Notes: Abstract: Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and γ-aminobutyrate evoked by 50 mMK+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 μM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K+-evoked release of aspartate. Conversely, 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.

Notes: Abstract: There is a considerable amount of conflicting evidence from several studies as to the action of applied N-methyl-D-aspartate (NMDA) on the release of glutamate and aspartate in the brain. In the present study the effect of NMDA on extracellular levels of endogenous amino acids was investigated in conscious, unrestrained rats using intracerebral microdialysis. NMDA caused dose-related increases in extracellular levels of glutamate and aspartate; threonine and glutamine were unaffected. The NMDA-evoked release of glutamate and aspartate was significantly decreased by the specific NMDA receptor antagonist 3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid. In addition, increasing the perfusate concentration (and therefore the extracellular concentration) of Ca2+ significantly enhanced the NMDA-evoked release of glutamate and aspartate, whereas removal of Ca2+ and addition of a high Mg2+ concentration to the perfusate caused a significant reduction in their NMDA-evoked release. Moreover, the NMDA-evoked release of glutamate and aspartate was reduced in decorticate animals. These results demonstrate that, in the striatum in vivo, NMDA causes selective release of endogenous glutamate and aspartate from neurone terminals and that this action occurs through an NMDA receptor-mediated mechanism. The ability of NMDA receptor activation to induce release of glutamate and aspartate, perhaps by a positive feedback mechanism, may be relevant to the pathologies underlying epilepsy and ischaemic and hypoglycaemic brain damage.

Notes: In an attempt to examine some functional characteristics of the N-methyl-D-aspartate (NMDA) receptor complex, the NMDA-evoked effluxes of endogenous dopamine (DA) and [3H]acetylcholine ([3H]ACh) were simultaneously examined in a rat Striatal slice preparation. NMDA induced release of both DA and ACh in a concentration-dependent, Ca2+-, Mg2+-, and tetrodotoxin-sensitive manner. These release responses were remarkably reduced by long-term pre-treatment with a low concentration of NMDA. an indication of the desensitization of the NMDA receptor. Glycine was potent in reversing the desensitization-related reduction of DA release but failed to reverse the diminution of ACh release in the same slices. Our results indicate that the NMDA receptors regulating the release of DA and ACh are different with respect to their glycine modulatory site. This finding is consistent with a functional heterogeneity of the NMDA receptor complex in the rat striatum.

Notes: : The effect of angiotensin II on catecholamine release from bovine adrenal medulla has been investigated. In retrogradely perfused, isolated bovine adrenal glands, angiotensin II increased basal efflux of catecholamines, but the presence of angiotensin II did not increase the release of catecholamines evoked either by bolus injections of the secretogogue carbachol or by depolarization with a perfusing solution containing a raised concentration of K+. In chromaffin cells maintained in primary tissue culture, angiotensin II increased 3H release from cells preloaded with [3H]-noradrenaline but did not enhance the release evoked by carbachol or by depolarization with K+. The increase in 3H release evoked by angiotensin II from chromaffin cells in tissue culture was inhibited by its analogue antagonist Sar1,Ala8-angiotensin II (saralasin) and was entirely dependent on the presence of Ca2+ in the experimental medium. These findings suggest that, in the chromaffin cells of the bovine adrenal medulla, angiotensin II acts on specific receptors to cause a calcium-dependent catecholamine release but triggers no additional response that acts synergistically with depolarizing or nicotinic stimuli to augment catecholamine release.

Notes: : The role of nerve growth factor (NGF) and its receptor (NGFR) in the regulation of cholinergic activity has been studied during the aging process. NGFRs were quantified in cortical membranes using a radioactive binding assay. NGF levels and choline acetyltransferase (ChAT) activity were determined in cortex, hippocampus, neostriatum, and septum. These assays were performed in both adult (6-month-old) and aged (36-month-old) rats. High-and low-affinity 125I-NGF binding sites were present in cortex of adult and aged rats. Furthermore, we observed a decrease in number and affinity of both NGFRs in aged rats. ChAT activity in these rats was lower (⋍30%) than in adult rats in all the brainregions examined. NGF levels were not modified in cortex and hippocampus and were decreased in neostriatum (55%) and septum (35%). In conclusion, our results suggest that, during the aging process, the cholinergic impairment is related to a decrease in NGF levels in neostriatum but not in cortex and hippocampus. The reduction in level of NGF protein in septum could be due to a decrease in number of high-affinity 125I-NGF binding sites.

Notes: The incorporation of enkephalin-containing peptides (ECPs) derived from proenkephalin into chromaffin vesicles was examined in primary cultures of adrenal medullary chromaffin cells. Cells were pulse-labeled with [35S]methionine and chased for periods up to 24 h. Chromaffin vesicles in cell homogenates were then fractionated by density gradient centrifugation and the presence of [35S]Met-enkephalin sequences in gradient fractions determined. 35S-ECPs were incorporated into particles suggestive of immature vesicles within 1–2 h after radiolabeling. Vesicle maturation, measured by co-equilibration of 35S-ECPs and total ECPs in the gradients, was complete within 9–12 h and was unaffected by treatments that increase proenkephalin synthesis. Incorporation of [35S]chromogranin A into chromaffin vesicles followed a similar time course, but 35S-labeled dopamine β-hydroxylase was much more slowly incorporated, possibly reflecting differences in incorporation of membrane and soluble components. In summary, the data demonstrate that ECPs are rapidly sequestered in immature chromaffin vesicles, a process unaltered by changing rates of proenkephalin synthesis.

Notes: Abstract: The efficacy of funnel-freezing of rat brain to inactivate metabolic processes and preserve in vivo tissue glucose concentration was validated by comparing the results obtained by funnel-freezing with those obtained with freeze-blowing of brain. The arterial plasma glucose level was clamped at 9 mM in halothane-anesthetized rats to produce identical glucose levels in brain tissue prior to freeze fixation. In funnel-frozen and freeze-blown brains, tissue glucose concentrations were 2.47 ± 0.05 and 2.47 ± 0.06 μMol/g (means ± SEM), respectively. Lactate levels in funnel-frozen brains were slightly but significantly higher than those in freeze-blown brains, i.e., 1.56 ± 0.05 μMol/g versus 1.30 ± 0.05 μMol/g (means ± SEM; p 〈 0.05). Regional analysis in funnel-frozen brains revealed that glucose concentrations in superficial and basal brain areas remained approximately equal at 2.30 ± 0.1 μMol/g and 2.31 ± 0.09 μMol/g (means ± SEM), respectively. Our findings indicate that in the anesthetized rat, funnel-freezing of brain is suitable for the measurement of regional in vivo glucose concentrations.

Notes: Abstract: Binding characteristics of the selective dopamine uptake inhibitor [3H]GBR 12935 have been described for the striatum but not for the frontal cortex. We have developed assay conditions for quantifying [3H]GBR 12935 binding in the frontal cortex. In both the rat and human frontal cortex, the assay required four times more tissue (8 mg/ml) than in the striatum (2 mg/ml).[3H]GBR 12935 binding in the frontal is complex, as it involves multiple binding sites. The high-affinity binding site is sodium dependent and is inhibited by sodium. In human but not in rat frontal cortex, addition of K+ reversed the sodium inhibition. The pharmacological profile of the high-affinity [3H]GBR 12935 binding site is consistent with that of the dopamine transporter, because drugs with the most selective dopamine reuptake blocking activities are the most potent displacers of [3H]GBR 12935 binding. There is a positive correlation between the rat and human inhibitory constants, a finding indicating that there are similar pharmacological profiles across at least these two species. Rats with a 6-hydroxydopamine lesion had a 47% decrease in number of [3H]GBR 12935 binding sites, a result indicating that at least a portion of these sites had been on presynaptic dopamine terminals.

Notes: Abstract: Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]–G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]–G protein complex.

Notes: Abstract: The effect of adenosine on 45Ca2+ uptake by rat brain synaptosomes stimulated by electrical pulses was investigated. 45Ca2+ uptake was voltage dependent. Adenosine (1 μM-1 μM) decreased the uptake of 45Ca2+ induced by electrical stimulation (amplitude, 20 V; duration, 400 pμs; frequency, 10 pulses/s) in a concentration-dependent manner. At a concentration of 1 μM, adenosine almost abolished the 45Ca2+ uptake induced by electrical stimulation (92.9 ± 5.3% inhibition), but when the calcium uptake was induced by high-K+ (60 mM) medium, the effect of adenosine (1 μM) was smaller (43.8 ± 5.2% inhibition). The inhibitory effect of 1 μM adenosine on calcium uptake induced by electrical stimulation was antagonized by 1,3-dipropyl-8-p-sulfophenylxanthine (5 μM). The possibility that adenosine interacts with the calcium channels opened by electrical stimulation is discussed.

Notes: Abstract: The major 68-kDa protein found selectively in the faster of the two subcomponents of slow axonal transport [group IV or slow component b (SCb)] in the rat sciatic nerve has been characterized. It was found to contain two distinct classes of proteins, S1 and S2, both of which have isoelectric points of 5.7, but differ in their solubility in the presence of calcium. The S1 protein, which contributes up to 70% of the 68-kDa component, was soluble in the presence or absence of calcium, whereas the S2 protein was bound to the cytoskeleton in a calcium-dependent manner. Further characterization of the two proteins by peptide mapping and immunological methods revealed that the S1 protein belonged to a family of proteins related to the 70-kDa heat shock protein, whereas the S2 protein was identical to 68-kDa calelec-trin (annexin VI). Selective occurrence in SCb of these proteins with potential abilities to regulate protein-protein or protein-membrane interactions suggests that they may play important roles in the control of cytoskeletal organization in the axon, because SCb contains mainly cytoskeletal proteins in a more dynamic form compared with the slowest rate component, slow component a, which is enriched in the stably polymerized form of these proteins.

Notes: Abstract: This study explored further the function of the metabotropic excitatory amino acid receptor in the rat brain. The trans and cis isomers of (±)-1-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were characterized for relative affinities at ionotropic and metabotropic excitatory amino acid receptors in vitro, as well as ability to produce in vivo excitatory or excitotoxic effects in rats. trans-ACPD was about 12 times more potent in vitro as an agonist for metabotropic excitatory amino acid receptors when compared to its ability to displace N-methyl-D-aspartate (NMDA) ([3H]CGS-19755) receptor binding. cis-ACPD was about 30 times more potent as a displacer of [3H]CGS-19755 binding than as a stimulant of phosphoinositide hydrolysis. When administered intra-peritoneally to neonatal rats, both cis- and trans-ACPD produced convulsions that were prevented by the competitive NMDA receptor antagonists, LY233053 and LY274614. cis-ACPD was six times more potent as a convulsant when compared to trans-ACPD. Both compounds were examined for excitotoxic effects in vivo following stereotaxic injection into the mature or neonatal rat striatum. Doses of trans-ACPD of up to 5,000 or 1,200 nmol produced few signs of striatal neuronal degeneration in the mature or neonatal brain, respectively. However, cis-ACPD produced extensive dose-related neuronal degeneration at doses of 100–1,000 nmol in the mature brain and 50–200 nmol in the neonatal brain. These studies suggest that, unlike the ionotropic excitatory amino acid receptors, activation of the metabotropic excitatory amino acid receptor does not result directly in excitatory effects, such as excitotoxicity.

Notes: Abstract: The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki= 0.33 nM), hexahydrosiladifenidol (Ki= 18 nM), and pirenzepine (Ki= 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists meth-octramine (10-4M) and AFDX-116 (10-4M), by ∼14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10-4M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.

Notes: Abstract: The expression of protein kinase C (PKC) subspecies in synaptosomes prepared from a number of adult brain regions was compared. Cerebral cortical and thalamic/striatal synaptosomes were found to express three peaks of enzyme activity upon hydroxyapatite chromatography, corresponding to the type I(γ), type II(β), and type III(α) subspecies. Synaptosomes prepared from either the hippocampus or the cerebellar cortex, however, contained only two major peaks, corresponding to the α- and β-subspecies, with barely detectable levels of the γ-subspecies, even though these tissue areas were enriched in the latter enzyme. When the ontogenic pattern of hippocampal synaptosomal PKC subspecies was examined, it was found that at postnatal day 7, significant quantities of the γ-subspecies were present and that this subspecies reached its peak levels at around postnatal day 14, before steadily declining to its adult level. Similar changes were observed also for the γ-subspecies in cerebellar cortex synaptosomes. The dynamic changes in the synaptosomal PKC subspecies take place at a critical period in the development of the rat brain, concomitant with an active period of synaptogenesis, suggesting that it may play a role in synaptogenesis.

Notes: Abstract: Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphon-ovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p 〈 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA. These increases were selective for SS mRNA and were not accompanied by any change in β-actin mRNA. By contrast, glutamate at the single dose tested was without effect on both SSLI and SS mRNA. These results demonstrate that Quin induces a selective NMDA receptor-mediated stimulation of SS gene expression and SS biosynthesis in vitro and suggest a similar mechanism for the augmented striatal SSLI in HD.

Notes: Abstract: Extracellular concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid were measured by microdialysis in rat striatum 1 month after a unilateral infusion via a dialysis probe of a high concentration (10 mM) of 1-methyl-4-phenylpyridinium ion (MPP+) into the substantia nigra. The basal extracellular DA concentration at the lesioned side was about 20% of the concentration at the nonlesioned side. However, basal DOPAC dialysate levels from the lesioned striatum represented only 2.4% of those from the contralateral side. Intrastriatal infusion with nomifensine increased the dialysate content of DA about twofold and eightfold at the lesioned and nonlesioned sides, respectively. Co-infusion of nomifensine with (–)-sulpiride caused an additional pronounced rise of the DA output on top of the nomifensine-induced increase at the nonlesioned side, whereas no effect was observed at the lesioned side. Finally, MPP+ (10 mM) was infused for 45 min into both striata. The increase in the dialysate content of DA in response to MPP+ (considered as an index of the total striatal DA content) from the lesioned side was only 0.6% of the MPP+-induced DA increase from the nonlesioned side. A strong compensatory response to increased extracellular dopamine was observed in the ipsilateral striatum. This effect was achieved by a severe suppression of reuptake mechanisms, as well as of the autoreceptor feedback response. It is concluded that infusion of MPP+ into the substantia nigra can be used as a chronic biochemical model for clinically manifest parkinsonism.

Notes: Abstract: Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum-free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Ham's F-12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbecco's modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change.

Notes: Abstract: This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence-activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH-containing nerve terminals or the concentration of TH protein were found in subjects 15–63 years of age. In light of previous studies showing an age-related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH-containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age-related differences in the relative abundance of these forms were found.

Notes: Abstract: The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the γ-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10−4M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 μM, or the agonist lorazepam, 10 μM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10−4M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ. Administration of EEDQ to senescent mice (20 months) led to a similar decrease in benzodiazepine sites compared to young mice, but receptor density returned more slowly to control levels (calculated receptor t1/2: young, 25.3 h; senescent, 75.1 h). EEDQ modifies all three major sites on the GABAA receptor, with the greatest effect at the benzodiazepine site. This compound may be useful in assessing benzodiazepine receptor half-life in vivo.

Notes: Abstract: A single intraventricular injection of tetanus toxin produced a time-dependent elevation of serotonin levels in brain and spinal cord of adult rats. This tetanus toxin-induced increase was produced in areas of high density of serotonergic innervation, such as the hypothalamus, hippocampus, and spinal cord. Little or no effect was found in the thalamus, cerebellum, and frontal cortex, areas that are poorly innervated by serotonergic terminals. The responses of catechol-amines (no change in dopamine level and generalized decrease in norepinephrine) pointed to a specific action of tetanus toxin on the serotonergic system. Stereotaxic injections of tetanus toxin in dorsal or magnus raphe nuclei did not have an evident effect on biogenic amine levels in the brain and spinal cord, respectively. Because direct stereotaxic injections of the toxin in the hypothalamus or hippocampus produced significant serotonin increases in both areas, it is proposed that tetanus toxin interacts with presynaptic targets to produce serotonin accumulation; this is probably due in part to an activation of tryptophan 5-hydroxylase.

Notes: Abstract: The pharmacological study of the blood-brain barrier has often been hampered by the unavailability of a large number of pure and fully differentiated brain capillary endothelial cells. Here we describe a homogeneous culture of brain capillary endothelial cells isolated from bovine brain (BBECs), which retain at least some phenotypic characteristics of the functional blood-brain barrier: intercellular tight junctions and monoamine oxidase activity. These cells were subcultured in vitro, in the absence of any neuronal or glial influences, for 〉 100 doublings without any sign of senescence. The present study is focused on the expression of ß-adrenergic receptors on BBECs. By Northern blot hybridization, subtype-specific ligand binding, and cyclic AMP accumulation experiments, we demonstrate that ß1- and ß2-adrenergic receptors are coexpressed (in the respective proportions of 42 and 58%) on BBEC membranes and are functionally coupled to adenylate cyclase. This is the first report documenting a significant number of ß1-adrenergic receptors on brain capillary endothelial cells. The results are discussed in light of the known noradrenergic innervation of brain capillaries.

Notes: Abstract: “Bound’ and “free’ RNA polymerase activities were assessed in the nuclear fraction of cerebral cortical, neuronal, astroglial, and oligodendroglial cells obtained from rats of young, adult, and old ages. Significant decreases in both the bound and free polymerase II activities were noticed in old brain, as compared to adult brain, in neuronal and oligodendroglial nuclei. In astroglia, only the free polymerase II was found to be affected. No effect of aging could be seen on the activity of bound RNA polymerase I + III. The free RNA polymerase I + III activity was increased from adult to old age in neuronal nuclei, but unchanged in oligodendroglial and astroglial nuclei. The age-dependent reduction in RNA polymerase II was maximum in oligodendroglial cells, whereas it was least, although still significant, in neuronal cells. DNA isolated from old brain was unable to enhance the transcriptional activity when added to chromatin preparations obtained from rat brains of any of the above ages, and the “old’ chromatin was unable to accept even the “young’ DNA as additional exogenous template. It is concluded that the reduced gene expression noticed in old brain nuclei is due to both altered chromatin/DNA structure and inadequate levels of free RNA polymerase II.

Notes: Abstract: Gene expression for myelin proteolipid protein (PLP) and myelin basic protein (MBP) in the dysmyelinating mutant mice shiverer and jimpy was analyzed by nuclear run-off transcription assay, in situ hybridization, and immunohistochemistry. The level of PLP transcription in shiverer brains was lower than that in controls at postnatal day 18 but relatively higher at later stages. In spite of the considerable amount of hybridization with PLP cDNA, immunoreaction for PLP was greatly reduced in shiverer mice throughout their lives, probably owing to a defect in the assembly of PLP into myelin. Abnormal deposition of PLP in oligodendroglial cell bodies suggested that transport of PLP to myelin is delayed in shiverer brains. The number of oligodendrocytes expressing PLP mRNA was drastically reduced in jimpy mice. MBP mRNA in jimpy mice is localized preferentially in oligodendroglial cell bodies, a result suggesting that oligodendrocytes in jimpy are mostly the immature type. Although transcriptional activity of the MBP gene in jimpy was greatly reduced, a finding reflecting the decrease in the number of mature oligodendrocytes, that of the PLP gene remained high at early stages. The discrepancy of the two gene expressions is discussed relative to the role of PLP transcripts at early stages of myelination.

Notes: Abstract: Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hi-rudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10-6M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.

Notes: Abstract: Direct intrastriatal injection of N-methyl-D-aspartate (NMDA; 100 μg/rat) increased striatal dopamine (DA) release in vivo. However, parenteral administration of (±)-3-(2-carboxypiperizin-4-yl)propyl-1-phosphonic acid (CPP) and cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS-19755) did not alter DA metabolism and release in several brain regions in the rat and mouse. Intracerebroventricular administration of the competitive NMDA antagonists CPP, CGS-19755, 2-amino-5-phosphonopentanoate, and 2-amino-7-phosphonoheptanoate did not alter rat striatal DA metabolism and release but profoundly reduced cerebellar cyclic GMP (cGMP) levels in the same animals. CPP and CGS-19755 decreased basal cerebellar cGMP levels in the mouse with ED50 values of 6 and 1 mg/kg, i.p., respectively. CPP antagonized the harmaline-induced increases in cGMP levels with an ED50 value of 5.0 mg/kg, i.p. CPP (25 mg/kg, i.p.) also decreased basal cGMP levels in mouse cerebellum for up to 3 h, a result suggesting brain bioavailability and a long duration of NMDA receptor antagonism in vivo. These contrasting patterns suggest that NMDA receptors exert a tonic excitatory tone on the guanine nucleotide signal transduction pathway in the cerebellum while exerting a phasic control over nigrostriatal dopaminergic neurotransmission. These results also indicate that competitive NMDA antagonists, unlike phencyclidine receptor agonists, may not mediate biochemical and behavioral effects via dopaminergic mechanisms.

Notes: Abstract: In immature rodent brain, the glutamate receptor agonist N-methyl-D-aspartate (NMDA) is a potent neurotoxin. In postnatal day (PND)-7 rats, intrastriatal injection of 25 nmol of NMDA results in extensive ipsilateral forebrain injury. In this study, we examined alterations in high-affinity [3H]glutamate uptake (HAGU) in NMDA-lesioned striatum. HAGU was assayed in synaptosomes, prepared from lesioned striatum, the corresponding contralateral striatum, or unlesioned controls. Twenty-four hours after NMDA injection (25 nmol), HAGU declined 44 ± 8% in lesioned tissue, compared with the contralateral striatum (mean ± SEM, n = 6 assays, p 〈 0.006, paired t test). Doses of 5–25 nmol of NMDA resulted in increasing suppression of HAGU (5 nmol, n = 3; 12.5 nmol, n = 3; and 25 nmol, n = 5 assays; p 〈 0.01, regression analysis). The temporal evolution of HAGU suppression was biphasic. There was an early transient suppression of HAGU (−28 ± 4% at 1 h; p 〈 0.03, analysis of variance, comparing changes at 0.5, 1, 2, and 3 h after lesioning); 1 or 5 days postinjury there was sustained loss of HAGU (at 5 days, −56 ± 11%, n = 3, p 〈 0.03, paired t test, lesioned versus contralateral striata). Treatment with the noncompetitive NMDA antagonist MK-801 (1 mg/kg i.p.) attenuated both the early and subsequent irreversible suppression of HAGU (1 h postlesion −28 ± 4%, n = 6 assays versus −12.6 ± 5% with MK-801, n = 4, p= 0.005; 24 h postlesion, −44 ± 8%, n = 5, versus +2.4 ± 6%, n = 3 with MK-801, p= 0.01, Wilcoxon ranked sum tests). In immature brain excitotoxic lesions produce an acute reversible suppression of HAGU, and a delayed long-lasting reduction in HAGU secondary to brain injury. These data suggest that accumulation of endogenous glutamate, as a consequence of the acute disruption of HAGU, could contribute to the pathogenesis of excitotoxic neuronal injury.

Notes: Abstract: Histochemical and biochemical determinations of total iron, iron (II), and iron (III) contents in brain regions from Parkinson's and Alzheimer's diseases have demonstrated a selective increase of total iron content in parkinsonian substantia nigra zona compacta but not in the zona reticulata. The increase of iron content is mainly in iron (III). The ratio of iron (II):iron (III) in zona compacta changes from almost 2:1 to 1:2. This change is thought to be relevant and may contribute to the selective elevation of basal lipid peroxidation in substantia nigra reported previously. Iron may be available in a free state and thus can participate in autooxidation of dopamine with the resultant generation of H2O2 and oxygen free radicals.

Notes: Abstract: Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.

Notes: An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50= 9.8 ± 2.9 nM) for displacement of [3H] flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50= 4.0 ±0.3 nM). However, at concentrations from 0.1 to 10 μM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton ±-100, a result suggesting that ∼7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1–4 h at 37°C) of neurons with 100μMγ-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H] flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154–176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.

Notes: The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with I25I-insulin resulted in the specific labeling of insulin receptor α-subunits of ∼ 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (αβ-heterodimer) receptors affinity-labeled with I25I-insulin indicated the presence of a disulfide-linked β-subunit of ∼95 kDa. Endoglycosidase F digestion of the affinity-labeled a-subunits increased their mobility by reducing their apparent mass to ∼83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657–670 of the carboxy terminal of the human insulin receptor α-subunit, whereas this antibody did label insulin receptor a-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor β- subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose a-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.

Notes: Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1-14C] isopentenyl pyrophosphate ([1-MC] I-P-P) into long-chain polyisoprenyl pyrophosphates (Poly-P-P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly-P-P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 μM I-P-P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (S) was stimulated by dithiothreitol. The major products were identified as C90and Q95 allyhic Poly-P-P on the basis of the following chemical and chromatographic properties: (1) the intact product cochromatographed with authentic Poly-P-P on silica-gel-impregnated paper, (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly-P) by alkaline hydrolysis; (3) treatment of the labeled Poly-P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly-P from lipophilic Sephadex LH-20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (Q95) isoprene units when analyzed by reverse-phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long-chain Poly-P-P. The enzymatic reactions by which the Poly-P-P intermediate is converted to dolichyl phosphate remain to be elucidated.

Notes: Although considerable evidence supports a role for excitatory amino acids in the pathogenesis of ischemic neuronal injury, few in vivo studies have examined the effect of increasing durations of ischemia on the extracellular concentrations of these agents. Recently, other neurotransmittiers (e.g., glycine and doparaine) have been implicated in the mechanism of ischemic neuronal injury. Accordingly, this study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, glycine concentrations in the hippocampus, and dopamine, serotonin, and dopamine metabolites in the caudate nucleus with varying durations (5, 10, or 15 minutes) of transient global cerebral ischemia as evidence to support their pathogenetic roles. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. Glutamate and aspartate concentrations in the dialysate increased from baseline by 1-, 5-, and 13-fold and by 4-, 9-, and 31-fold, respectively, for the three ischemic durations. The concentrations returned to baseline rapidly after reperfusion. The peak concentrations of glutamate and aspartate were significantly higher with increasing ischemic duration. Dopamine concentrations increased by approximately 700-fold in response to all three ischemic durations and returned to baseline within 10 min of reperfusion. Glycine, in contrast, increased during ischemia by a mean of 4-fold, but remained elevated throughout the 80-min period of reperfusion. The final concentrations of glycine were significantly higher than baseline levels (p = 0.0002, Mann-Whitney test). That glutamate and aspartate concentrations in the hippocampus co-vary with the duration of global ischemia is taken as supportive evidence of their pathogenetic role in ischemic neuronal injury. The observation that dopamine concentrations increased independently of ischemic duration indicates a maximal release with only S min of ischemia and suggests that dopamine's role in the incremental injury seen with increasing ischemic duration is limited to prolonged high concentrations rather than increasing peak levels as seen with glutamate and aspartate. The sustained elevation of glycine concentrations after ischemia may explain the ability of excitatory amino acids to produce delayed toxicity in the reperfusion phase, as glycine has been shown to facilitate glutamate's activity at the N- methyl-D-aspartate receptor.

Notes: Bovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4, 5-bisphosphate. Approximately 60–70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10-fold higher than that of resealed ROS enzyme activity. Peptide-specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y-region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of ∼ 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140-kDa antigen from a Sephadex G-150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140-kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.

Notes: Rat sciatic nerve contains a membrane-bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 μg of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg ot protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M, phosphatidylethanol represents about one-half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.

Notes: Abstract: A small acidic polypeptide, termed thymosin β10, has been identified and is present in the nervous system of the rat by the ninth day of gestation. Thymosin β10 levels rise during the remaining days of life in utero, and then decline to nearly undetectable values between the second and fourth week post partum. The present study investigates the possible developmental signals and mechanisms that might regulate the expression of thymosin β10 during neuroembryogenesis. Many cell lines derived from tumors of the central nervous system express thymosin β10, as well as its homologue gene product, thymosin β4. Because some of these cell lines respond to exogenously applied agents by increasing their apparent state of differentiation, we have determined whether thymosin β10 levels are coordinately modulated. In several neuroblastomas, including the B103 and B104 lines, retinoic acid elicits a time- and dose-dependent increase in the content of thymosin β10, but not that of thymosin β4. The increase in thymosin β10 polypeptide is associated with a marked increase in the specific mRNA encoding this molecule. The mRNA for thymosin β4 is unaffected by retinoic acid. This is in contrast with the situation in vivo, where the expression of both genes decreases after birth. Other agents that influence the morphology of B104 cells, such as phorbol esters and dibutyryl cyclic AMP, have no influence on β-thymosin levels. A range of steroids, which like retinoids act upon nuclear receptors, was also inactive. The stimulatory action of retinoic acid is detectable within 4 h, and thymosin β10 peptide levels continue to rise for at least 4 days. The influence of the isoprenoid is fully reversible and exhibits structural specificity. We believe that this culture system is mimicking the early rising phase of thymosin β10 levels in brain and that endogenous retinoids may be candidate physiological regulators of this gene.

Notes: Abstract: Zinc is essential for the normal development and function of the CNS, although little is known about brain zinc homeostasis. Therefore, in this investigation we have studied 65Zn uptake by brain from blood and have measured the blood-brain barrier permeability to 65Zn in the anaesthetised rat in vivo. Adult male Wistar rats within the weight range 500–600 g were used. 65ZnCl2 and 125I-albumin, the latter serving as a vascular marker, were injected intravenously in a bolus of normal saline. Sequential arterial blood samples were taken during experiments that lasted between 5 min and 5 h, after which the whole brain was removed, dissected, and analysed for radioisotope activity. Data have been analysed by graphical analysis, which suggests that after 30 min of circulation, 65Zn uptake by brain from blood is unidirectional with an influx rate constant, Kin, of ∼5 × 10−4 ml/min/g. At circulation times of 〈30 min, 65Zn fluxes between blood and brain are bidirectional, where influx has a K value of 〉5 × 10−4 ml/min/g. In addition to the blood space, the brain appears to contain a rapidly exchanging compartment(s) for 65Zn of ∼4 ml/100 g, which is not CSF.

Notes: Abstract: Single cell Ca2+ mobilization was studied by non-parametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 μM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 μM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 μM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1–10 μM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.

Notes: Abstract: The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromo-cytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 μM and 7.1 μmol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36–46), for example, exhibited a Km of 108 μM and a Vmax of 6.93 μmol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.

Notes: Abstract: In order to study the possible involvement of dopamine receptors in the pathophysiology of various neurological and psychiatric disorders, we have isolated the human D2A gene. Like the rat D2A gene, the human gene contains at least eight exons and spans at least 52 kb. Exons 2–8 are clustered within 14 kb of genome. Exon 1 is separated from exon 2 by at least 38 kb. We and others have shown that alternative utilization of exon 6 gives rise to alternative D2A transcripts. Despite the extreme size of intron 1, no alternative transcription between exons 1 and 2 can be detected in basal ganglia and pituitary using polymerase chain reaction analysis. The relative abundance and tissue distribution of the alternative D2A transcripts were examined in 18 human brain regions. The relative expression of the transcripts varied by at least 70-fold across the brain regions surveyed. As expected, high levels of transcripts were detected in caudate, putamen, and pituitary. Moderate levels were detected in regions of catecholamine-containing cell bodies and in the amygdala. In contrast to the rat brain, very low levels of transcripts were detected in cortical regions.

Notes: Abstract: We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3-phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72-kDa protein (3PG-PP72). This system was separated into the substrate, 3PG-PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1-32P]1,3-bisphosphoglycerate ([1-32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [γ-32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1-32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG-PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG-PP72 is G1,6P2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG-PP72 preparation.

Notes: Abstract: A series of genomic clones containing DNA that encodes the chicken γ-aminobutyric acidA (GABAA) receptor β4 subunit have been isolated. These have been restriction mapped and partially sequcnced to determine the structural organization and the size of the β4-subunit gene. This gene, which comprises nine exons, spans more than 65 kb. The organization of the chicken GABAA receptor β4-subunit gene has been compared to that of the murine GABAA receptor δ-subunit gene and to those of the genes that encode other members of the ligand-gated ion-channel superfamily, namely muscle and neuronal nicotinic acetylcholine receptors (AChRs). Although the positions of the intron/exon boundaries of GABAA receptor sub-unit genes are seen to be highly conserved, there are significant differences between the genes that encode GABAA receptor and AChR subunits. These results are discussed in relation to the proposal that this superfamily of ligand-gated ion-channel receptor genes arose by duplication of an ancestral receptor gene.

Notes: Abstract: The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline-containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water-soluble ethanolamine-containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10−8M and 10−5M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2-hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10−6M and 10−5M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 × 10−5M propranolol.

Notes: Abstract: Neurite-promoting activities of lipids were assessed using serum-free cultures of fetal rat septal neurons. The most potent one was phosphatidylinositol (PI), followed by PI 4-phosphate, phosphatidylserine, sphingomyelin, and phosphatidylcholine. The EC50 value for PI was 1.5 μg/ml (1.8 μM), and activity was maximal at 4 μg/ml (56% of total cells had neurites after 24 h). Cerebroside, sulfatide, and di- and triacylglycerols showed relatively low activities. Synthetic dipalmitoyl phosphatidylcholine was also active, with a maximal activity (47%) at 100 μg/ml, a finding implying that the unsaturated fatty acid moiety is not released and further used as substrate for the arachidonic acid cascade. Lysophospholipids, phosphatidylglycerol, and cardiolipin were rather cytotoxic and lacked activity, an observation suggesting that membrane perturbation is not responsible for the neuritepromoting activity. Treatment with a protein kinase C inhibitor, H-7, or an Na+,K+-ATPase inhibitor, ouabain, inhibited the PI-induced neurite outgrowth, but the cyclic AMP- and cyclic GMP-dependent protein kinase inhibitor HA1004 did not inhibit this activity, a result indicating that multiple elements (protein kinase C and Na+,K+-ATPase) are involved in the induction of neurites. Because phospholipids can be provided either as lipid vesicles or as lipoproteins produced by macrophages at regeneration sites, they may play an important role in the regeneration of certain populations of neurons.

Notes: Abstract: The secretion of catecholamines and ATP induced by cholinergic agonists and its dependence on extracellular Ca2+ were studied in cultured porcine adrenal chromaffin cells. Both nicotine and methacholine (a selective muscarinic agonist) induced secretion and increases in cytosolic free Ca2+ concentration ([Ca2+]in), although the activation of nicotinic receptors produced responses that were larger than those produced by activation of muscarinic receptors. The secretion and the increase in [Ca2+]in evoked by nicotine were completely dependent on extracellular Ca2+ and were blocked by prior depolarization of the cells with high extracellular K+ levels. In addition, nicotine induced significant 45Ca2+ influx. In contrast, the secretion and the increase in [Ca2+]in evoked by methacholine were partially dependent on extracellular Ca2+; methacholine also induced 45Ca2+ influx. Prior depolarization of the cells with high extracellular K+ levels did not block methacholine-induced secretion. In general, nicotinic responses were mediated by Ca2+ influx through voltage-dependent pathways. In contrast, muscarinic responses were dependent on both Ca2+ influx through an unknown mechanism that could not be inactivated by high K+ concentration-induced depolarization and presumably also intracellular Ca2+ mobilization.

Notes: Abstract: Studies were conducted to ascertain the temporal and dose-dependent effects of nicotinic ligand exposure on functional activity of different nicotinic acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat pheochromocytoma (ganglia-type nAChR) or the TE671/RD human (muscle-type nAChR) clonal line. Chronic (3–72-h) agonist (nicotine or carbamylcholine) treatment of cells led to a complete (TE671) or nearly complete (PC12) loss of functional nAChR responses, which is referred to as “functional inactivation.”Some inactivation of nAChR function was also observed for the nicotinic ligands d-tubocurarine (d-TC), mecamylamine, and decamethonium. Half-maximal inactivation of nAChR function was observed within 3 min for TE671 cells and within 10 min for PC12 cells treated with inactivating ligands. Functional inactivation occurred with dose dependencies that could not always be reconciled with those obtained for acute agonist activation of nAChR function or for acute inhibition of those responses by d-TC, decamethonium, or mecamylamine. Treatment of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium alone had no effect on levels of expression of functional nAChRs. However, evidence was obtained that either of these antagonists protected TE671 cell muscle-type nAChRs or PC12 cell ganglia-type nAChRs from functional inactivation on long-term treatment with agonists. Recovery of TE671 cell nAChR function following treatment with carbamylcholine, nicotine, or d-TC occurred with half-times of 1–3 days whether cells were maintained in situ or harvested and replated after removal of ligand. By contrast, 50% recovery of functional nAChRs on PC12 cells occurred within 2–6 h after drug removal. In either case the time course for recovery from nAChR functional inactivation is much slower than recovery from nAChR “functional desensitization,”which is a reversible process that occurs on shorter-term (0–5-min) agonist exposure of cells. These results indicate that ganglia-type and muscle-type nAChRs are similar in their sensitivities to functional inactivation by nicotinic ligands but differ in their rates of recovery from and onset of those effects. The ability of drugs such as the agonists d-TC, decamethonium, and mecamylamine to induce functional inactivation may relate to their activities as partial/full agonists, channel blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and alcuronium are likely to reflect simple competitive inhibition of primary ligand binding at functional activation sites. Agonist-induced functional inactivation of TE671 cell nAChRs occurs under conditions previously shown to induce increases in numbers of nAChR ligand binding sites expressed on the cell surface and in total membrane pools and contrasts with coordinate down-regulation of nAChR ligand binding and function following chronic agonist treatment of nAChR-bearing cells in the periphery.

Notes: Abstract: [3H]Kainate bound to chick cerebellar membranes with a KD of 0.6 μM and with an exceptionally high Bmax of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [3H]kainate was reduced (KD= 2.7 μM), but the Bmax was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate 〉 kainate 〉 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 〉 glutamate. Binding sites for α-[3H]amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) were much less abundant, with KD and Bmax values in membranes of 86 nM and I pmol/mg of protein, respectively. The affinity of [3H]AMPA binding was also reduced on solubilisation (KD= 465 nM), but there was an increase in the Bmax (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [3H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [3H]kainate, domoate was markedly less potent than kainate at displacing [3H]AMPA. These results suggest that [3H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [3H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non-N-methyl-D-aspartate receptors.

Notes: A 103-kDa protein present in membrane cytoskeletal preparations from bovine brain has been identified. We have purified this protein to 〉95% homogeneity using gel filtration and ion-exchange chromatography. This protein, p 103, is an asymmetric dimer in dilute solution and has two major variants that can be distinguished by isoelectric focussing, pI 5.60 and 5.75. Using subcellular fractionation, it is most enriched in postsynaptic densities. Immunolocalization with anti-p 103-specific antibodies reveals that it is confined to the dendrites and perikarya; it is apparently absent from spinal cord axons. It coextracts from brain membrane-skeletal preparations with brain spectrin and actin, but in vitro, it does not interact with them.

Notes: In primary cultures of neurons from rat cerebral cortex and neostriatum, excitatory amino acids stimulate the translocation of protein kinase C (PKC) from the cytoplasm to the membrane. In the presence of a physiological concentration of Mg2+ in the extracellular medium, glutamate induces PKC translocation by binding to both TV-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) excitatory amino acid receptors. Quisqualate translocates the enzyme by stimulating primarily AMPA receptors and possibly metabotropic receptors. NMDA receptor-induced PKC translocation is sodium independent, whereas quisqualate receptor-induced PKC translocation is sodium dependent; none of the agonists is active in the absence of calcium from the extracellular medium. Muscimol does not modify excitatory amino acid stimulation; however, blockade of γ-aminobutyric acidA receptors by bicuculline greatly enhances glutamate-induced PKC translocation. This enhancement is blocked by the NMDA receptor antagonist (+)-5-methyl-10, 11-dihydro-5H- dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK-801) and by tetrodotoxin.

Notes: Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and the cervical sympathetic trunk in which 〉99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed I25I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelinand non-myelin-forming Schwann cells. Endopeptidase-24. 11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.

Notes: The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-l-methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced, (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation, CCh-stimulated IP3 production and contraction are attenuated, (c) The increase in tension by a maximal effective dose of NaF (20 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation, (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX-induced cAMP formation increased in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively, (e) Isoproterenol (5 μM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively, (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh-stimulated IP3 production and contraction of the sphincter by ∼30% of their respective controls, (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are causal by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to tie hypothesis that there are reciprocal interactions between the cAMP and IP3-Ca2+ signaling systems and the contractile response in the iris smooth muscle.

Notes: Isolated rat brain synaptosomes accumulated L-asparagine with a Km value of 348 μM and a Kmax value of 3.7 nmol/mg of protein/min at 28°C. Uptake of L-asparagine was inhibited by the presence of L-glutamine, whereas transport of L-glutamine was blocked by L-asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas α-(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L-asparagine had a substantial sodium-dependent component, whereas that of the D-stereoisomer was almost unaffected by the presence or absence of the cation. L-Asparaginc was accumulated to a maximal gradient, [L-Asn]i/[L-Asn]o, of 20–30, and this value was reduced to 5–6 by withdrawal of sodium or addition of high [KG]. A plot of log [Na+]o/{Na+]i against the log [L-Asn]i/[L-Asn]o had a slope close to 1, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L-asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L-asparagine and L-glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [GIn]i maintained by the brain in vivo.

Notes: Abstract: The effects of loop diuretics and ion substitution on the 2-min uptake of K (86Rb as marker) were analyzed to obtain evidence for K cotransport with Na and Cl in the choroid plexus epithelium. The isolated plexuses, which were excised from lateral ventricles of adult rats, were bathed in artificial cerebrospinal fluid (aCSF). At concentrations of 10-6 to 10-4M, the specific cotransport inhibitors, bumetanide and piretanide, suppressed uptake of K in a dose-dependent manner. Ouabain-insensitive K uptake was stimulated by preincubating the choroid plexus in aCSF very low in [Na] and [K], then incubating it in much higher concentrations of these cations; bumetanide (10-4M) blocked this stimulated uptake by 52%. Moreover, tissue preincubation in Na- or Cl-free medium, followed by incubation with normal concentrations of both ions, stimulated the ouabain-insensitive uptake of K from 15 (baseline) to 35 nmol/mg dry weight. This stimulation of K transport depended on the simultaneous presence of both Na and Cl in aCSF, and replacing either ion alone did not stimulate the ouabain-insensitive K uptake. Collectively, these findings, together with those from a previous pharmacological study of 22Na and 36Cl transport, constitute strong evidence for the cotransport of Na, K, and Cl in rat choroid plexus.

Notes: Abstract: DNA sequences encoding two variants of a novel γ-aminobutyric acidA (GABAA) receptor β subunit were isolated from an embryonic chicken whole-brain cDNA library and a chicken genomic library. The coding regions of these variants only differ from each other by the absence or presence of 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4; the encoded subunits have been named β4 and β4′, respectively. The predicted mature polypeptides are 72–77% identical to the previously characterized mammalian and chicken β1,β2, and β3 subunits. Analysis of the β4-subunit gene reveals that the different transcripts encoding the two variants arise by the use of one of two 5′-donor splice sites that are separated by 12 bp. This is the first demonstration of alternative splicing of a GABAA receptor subunit gene transcript and represents a further mechanism for the generation of GABAA receptor heterogeneity.

Notes: Abstract: Book reviewed in this article: Nutrition and the Brain, Vol. 8 by R. J. Wurtman and J. J. Wurtman. Glossary of Biochemistry and Molecular Biology by D. M. Glick. Antiepileptic Drugs (Third Edition) edited by R. H. Levy, F. E. Dreifuss, R. H. Mattson, B. S. Meldrum, and J. K. Penry. Developmental Neurobiology (Nestlé Nutrition Workshop Series, Vol. 12) edited by P. Evrard and A. Minkowski. Allosteric Modulation of Amino Acid Receptors: Therapeutic Implications (Fidia Research Foundation Symposium Series, Vol. 1) by E. A. Barnard and E. Costa. Neurochemical Pharmacology—A Tribute to B. B. Brodie (Fidia Research Foundation Symposium Series, Vol. 2) edited by E. Costa. Neuromethods Vol. 11: Carbohydrate and Energy Metabolism edited by A. A. Boulton, G. B. Baker, and R. F. Butterworth. Adenosine and Adenosine Receptors edited by M. Williams. Laughing Death: The Untold Story of Kuru by V. Zigas. Psychoactive Drugs: Tolerance and Sensitization edited by A. J. Goudie and M. W. Emmett-Oglesby. The NMDA Receptor edited by J. C. Watkins and G. L. Collingridge. Basic and Clinical Aspects of Neuroscience (Vol. 3): The Role of Brain Dopamine edited by E. Flückiger, E. E. Müller, and M. O. Thorner. Glycine Neurotransmission edited by O. P. Otterson and J. Storm-Mathisen. The Neuropeptide Cholecystokinin (CCK): Anatomy and Biochemistry, Receptors, Pharmacology and Physiology edited by J. Hughes, G. Dockray, and G. Woodruff.

Notes: Abstract: The effect of the anxiogenic β-carboline methyl-β-carboline-3-carboxyamide (FG 7142) on dopamine release in prefrontal cortex and striatum in the awake freely moving rat was determined using the technique of microdialysis. FG 7142 (25 mg/kg, i.p.) caused a time-dependent increase in dopamine release in prefrontal cortex which was statistically significantly greater than the response to vehicle administration. Dopamine release in striatum was unaltered by FG 7142. Pretreatment of animals with the benzodiazepine antagonist Ro 15-1788 (30 mg/kg, i.p., 15 min prior to FG 7142 administration) completely abolished the increase in dopamine release caused by FG 7142 in prefrontal cortex. These data indicate that the anxiogenic benzodiazepine inverse agonist FG 7142 can selectively increase dopamine release in prefrontal cortex, and that this effect appears to be mediated via the γ-aminobutyric acid/benzodiazepine receptor complex.

Notes: Abstract: The release of endogenous histamine (HA) from the hypothalamus of anesthetized rats was measured by in vivo microdialysis coupled with HPLC with fluorescence detection. Freshly prepared Ringer's solution was perfused at a rate of 1 μl/min immediately after insertion of a dialysis probe into the medial hypothalamus, and brain perfusates were collected every 30 min into microtubes containing 0.2 M perchloric acid. The basal HA output was almost constant between 30 min and 7 h after the start of perfusion, with the mean value being 7.1 pg/30 min. Thus, the extracellular HA concentration was assumed to be 7.8 nM, by a calculation from in vitro recovery through the dialysis membrane. Perfusion with a high K+ (100 mM)-containing medium increased the HA output by 170% in the presence of Ca2+. Systemic administration of either thioperamide (5 mg/kg, i.p.), a selective H3 receptor antagonist, or metoprine (10 mg/kg, i.p.), an inhibitor of HA-N-methyltransferase, caused an approximately twofold increase in the HA output 30–60 min after treatment. The combined treatment with thioperamide and metoprine produced a marked increase (650%) in the HA output. The HA output decreased by ∼70% 4–5 h after treatment with α-fluoromethylhistidine (α-FMH; 100 mg/kg, i.p.), an inhibitor of histidine decarboxylase. Furthermore, the effect of combined treatment with thioperamide and metoprine was no longer observed in α-FMH-treated rats. These results suggest that both HA-N-methyltransferase and H3 autoreceptors are involved in maintaining a constant level of extracellular HA and that their blockade effectively results in a higher activity level of the endogenous histaminergic system in the CNS.

Notes: Abstract: We have isolated cDNA clones coding for the human homologue of the neuronal cell adhesion molecule L1. The nucleotide sequence of the cDNA clones and the deduced primary amino acid sequence of the carboxy terminal portion of the human L1 are homologous to the corresponding sequences of mouse L1 and rat NILE glycoprotein, with an especially high sequence identity in the cytoplasmic regions of the proteins. There is also protein sequence homology with the cytoplasmic region of the Drosophila cell adhesion molecule, neuroglian. The conservation of the cytoplasmic domain argues for an important functional role for this portion of the molecule.

Notes: Abstract: The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5′-O-(3-thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half-maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.

Notes: Abstract: We describe a simple, rapid, and efficient method, based on separation on a Percoll centrifugation gradient, to purify glial progenitor cells from newborn rat brains. Cytofluorimetry analysis of the isolated cell population showed that 75 ± 8 and 86 ± 7% of the cells were A2B5- and R24-positive, respectively. Transmission electron microscopy examination of the purified cell population confirmed their homogeneity and illustrated their typical morphology, as previously described in situ. Assay of UDP-galactose-ceramide galactosyltransferase, 3′-phosphoadenosine 5′-phosphosulfate galactosylceramide sulfotransferase, and 2′,3′-cyclic nucleotide 3′-phosphohydrolase activities showed that the levels of these enzymes were 446, 76, and 11 times lower, respectively, than the levels measured in mature oligodendrocytes. Low levels of mRNA coding for 2′,3′-cyclic nucleotide 3′-phosphohydrolase and myelin proteolipid protein, but not for myelin basic protein, were present in the glial progenitor cells. At the time of isolation, 40% of the cells in the population were dividing, and the cells could easily be expanded in culture. After 3 weeks of culture in the presence of 1% fetal calf serum, 75% of the cells had differentiated into galactosylceramide-positive oligodendrocytes. When the culture took place in the presence of 10% fetal calf serum, only 2% of the cells expressed galactosylceramide, and 60% were glial fibrillary acidic protein-positive astrocytes; half of them were also A2B5 positive.

Notes: Abstract: Concurrent cocaine and alcohol use is common practice in the general population, as indicated by recent prevalence studies. In the presence of ethyl alcohol, cocaine is metabolized to its ethyl homolog, cocaethylene. The transesterification of cocaine and ethanol to cocaethylene takes place in the liver and represents a novel metabolic reaction. Cocaethylene was detected in postmortem blood, liver, and neurological tissues in concentrations equal to and sometimes exceeding those of cocaine. In vitro binding studies demonstrate that cocaethylene has a pharmacological profile similar but not identical to that of cocaine at monoamine transport sites assayed in the human brain. Cocaethylene was equipotent to cocaine at inhibiting [3H]mazindol binding to the dopamine transporter. The blockade of dopamine reuptake in the synaptic cleft by cocaethylene may account for the enhanced euphoria associated with combined alcohol and cocaine abuse.

Notes: Abstract: γ-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (α, β, γ, and δ). The γ2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine γ2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids, LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the γ2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.

Notes: Abstract: Cell surface binding, internalization, and biological effects of insulin-like growth factors (IGFs) I and II have been studied in primary neuronal cultures from developing rat brain (embryonic day 15). Two types of IGF binding sites are present on the cell surface. The IGF-I receptor α-subunit (Mr 125,000) binds IGF-I with a KD of 1 nM and IGF-II with 10 times lower affinity. The mannose-6-phosphate (Man-6-P)/IGF-II receptor (Mr 250,000) binds IGF-II with a KD of 0.5 nM and IGF-I with 100 times lower affinity. Surface-bound IGF-I and IGF-II are internalized by their respective receptors and degraded to amino acids. Man-6-P increases the receptor binding and internalization of IGF-II but not those of IGF-I. Neuronal synthesis of RNA and DNA is increased twofold by IGF-I with 10 times higher potency than IGF-II. Antibody 3637, which blocks receptor binding of IGF-II, has no effect on the DNA response to IGF-I or IGF-II. Double immunocytochemical staining with antibodies to bromodeoxyuridine and neurofilament shows that 〉80% of the bromodeoxyuridine-positive cells become neurofilament positive. It is concluded that IGF-I and IGF-II bind to two receptors on the surface of neuronal precursor cells that mediate endocytosis and degradation of IGF-I and IGF-II. Proliferation of neuronal precursor cells is stimulated by IGF-I and IGF-II via activation of the IGF-I receptor.

Notes: Abstract: In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor sub-type, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.

Notes: Abstract: The release of putative neurotransmitters [aspartate, glutamate, and γ-aminobutyric acid (GABA)] was studied in hippocampal slices from adult normal C57BL/6J (B6) and El (epileptic) mice. The El mice, a genetic model of temporal lobe epilepsy, had an average of 86 seizures. Sets of B6 and El hippocampal slices (400 μm thick) were incubated in a series of normal and high potassium (60 mM) buffers in the presence or absence of calcium. The calcium-dependent and calcium-independent potassium-induced release of amino acids was compared in each mouse strain. Release of endogenous amino acids was measured using liquid chromatog-raphy with electrochemical detection and was expressed as picomoles of amino acid released per milliliter of incubation buffer per minute of incubation per slice ± SEM. No significant differences were found between the El and B6 mice for the calcium-dependent potassium-evoked release of glutamate (18.20 ± 2.62 and 15.41 ± 3.56), or GABA (17.28 ± 2.90 and 12.73 ± 1.37), respectively. Aspartate release, however, was significantly higher in the El mice (6.62 ± 0.69) than in the B6 mice (3.31 ± 0.72). These findings suggest that enhanced aspartate release may be related to seizure expression in El mice.

Notes: Abstract: One of the major clinical findings in Alzheimer's disease (AD) is the formation of deposits of β-amyloid protein in amyloid plaques, derived from the β-amyloid precursor protein (β-APP). To determine the possible use of β-APP as a diagnostic marker for AD in CSF, a monoclonal antibody-based immunoassay specific for this protein was developed. The assay does not differentiate between β-APP695 and β-APP751 forms but does preferentially recognize β-APP751 complexed with a protease. Of the two sets of CSF samples tested, one set, obtained from living patients, gave a slightly lower level of β-APP in AD and Parkinson's disease patients relative to controls, whereas the other set, composed of postmortem samples, showed no significant differences between the AD and control groups.

Notes: Abstract: Dopaminergic neurons that project to the striatum from the substantia nigra are thought to modulate methionine-enkephalin (Met-Enk) metabolism in the striatum. We administered a dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that produces a moderate depletion of dopamine in striatum, about 50%, without overt motor deficits, and found that Met-Enk-like immunoreactivity and preproenkephalin mRNA content increased in the tissue. Pretreatment with the monoamine oxidase B inhibitor deprenyl or the dopamine transport blocker nomifensine prevented these changes, suggesting that the changes were related to the partial loss of dopaminergic neurons rather than to MPTP. Moreover, administering GM1 ganglioside, which partially restores the MPTP-induced dopaminergic deficit, partially corrected the Met-Enk changes in the striatum as well. These findings are consistent with the hypothesis that dopaminergic input to the striatum, in part, modulates Met-Enk metabolism. Moreover, they show that moderate nigrostriatal lesions are sufficient to elevate Met-Enk and preproenkephalin mRNA contents and that restoration of dopaminergic function, as in our studies with GM1 ganglioside, restores the content of Met-Enk.

Notes: Book Reviewed in this article: Neuromethods, Volume 16: Molecular Neurobiological Techniques edited by A. A. Boulton, G. B. Baker, and A. T. Campagnoni.

Notes: Abstract: In a previous work we have shown that histidine decarboxylase (HD) activity is found in a soluble and a membrane-bound form. A major part (82%) of the membrane-bound HD activity in the crude mitochondrial fraction (P2) was present in the synaptic plasma membrane-containing subfraction. Physiological concentrations of Ca2+ had no direct effect on HD activity but caused a solubilization of ∼50% of membrane-bound HD in the P2 fraction. Mg2+ had similar but lower effects (20% solubilization) than Ca2+. Incubation with depolarizing concentrations of K+ in the presence of 1 mM CaCl2 caused a significant (30%) solubilization of HD.

Notes: Abstract: A cDNA probe specific to microtubule-associated protein 2 (MAP2) was used to study the expression of the mRNAs encoding the high- and low-molecular-weight MAP2 variants in cultured neurons and astrocytes. The timing and relative abundance of these MAP2 transcripts and of their encoded proteins were also studied in the developing cerebral hemispheres and cerebellum of the mouse. A 9-kb mRNA, known to encode high-molecular-weight MAP2, was expressed in cultured astrocytes, albeit at a lower level than in neurons. The 6-kb transcript, recently shown to encode low-molecular-weight MAP2 (MAP2c), was expressed in neurons and was the predominant MAP2 transcript of the astrocytes. The level of the 9- and 6-kb transcripts decreased at late stages of astroglial and neuronal cell culture. The 9-kb mRNA was detected in the cerebellum and cerebral hemispheres at every developmental stage. Although the levels of this mRNA varied slightly in the cerebral hemispheres, its expression was biphasic in the cerebellum. This might be explained by the differences in timing of development of the various neuronal cell types formed in these two brain areas. The 6-kb transcript was detected only at early developmental stages in the two brain areas. Correlating the temporal expression of the 9-kb mRNA to that of high-molecular-weight MAP2 indicates that the accumulation of this protein is in part regulated at a cytoplasmic level.

Notes: Abstract: The changes in the levels of protein kinase C [PKC(α, βII, γ)] were studied in cytosolic and particulate fractions of striatal homogenates from rats subjected to 15 min of cerebral ischemia induced by bilateral occlusion of the common carotid arteries and following 1 h, 6 h, and 48 h of reperfusion. During ischemia the levels of PKC(βII) and -(γ) increased in the particulate fraction to 390% and 590% of control levels, respectively, concomitant with a decrease in the cytosolic fraction to 36% and 20% of control, respectively, suggesting that PKC is redistributed from the cytosol to cell membranes. During reperfusion the PKC(βII) levels in the particulate fraction remained elevated at 1 h postischemia and decreased to below control levels after 48 h reperfusion, whereas PKC(γ) rapidly decreased to subnormal levels. In the cytosol PKC(βII) and -(γ) decreased to 25% and 15% of control levels at 48 h, respectively. The distribution of PKC(α) did not change significantly during ischemia and early reperfusion. The PKC activity in the particulate fraction measured in vitro by histone IIIS phosphorylation in the presence of calcium, 4β-phorbol 13-myristate 12-acetate, and phosphatidylserine (PS) significantly decreased by 52% during ischemia, and remained depressed over the 48-h reperfusion period. In the cytosolic fraction PKC activity was unchanged at the end of ischemia, and decreased by 47% after 6 h of reperfusion. The appearance of a stable cytosolic 50-kDa PKC-immunoreactive peptide or an increase in the calcium-and PS-independent histone IIIS phosphorylation was not observed. Consequently, during ischemia PKC, preferentially PKC(γ) and PKC(βII), is translocated from the cytosol and inserted into cell membranes, concomitant with a decrease in PKC activity. In the reperfusion phase the depression of PKC activity persists and the enzyme is degraded. The observed translocation and downregulation of PKC during ischemia and reperfusion may be of significance for the development of ischemic neuronal damage.

Notes: Abstract: Enhanced cocaine concentrations in brain and blood observed after an intraperitoneal challenge dose in rats exposed to cocaine for 10 days by subcutaneous administration are traced to a change in the absorption process from the site of an intraperitoneal injection to general circulation. This conclusion is reached by three sets of corroborating results: (a) Adipose tissue of rats treated for 10 days with repeat subcutaneous injections of cocaine did not reveal a buildup of cocaine in sufficient concentrations to account for the twofold increase in brain and blood concentrations seen during intraperitoneal administration; (b) administration of the drug by an intravenous route after 10-day cocaine treatment did not show a significant difference between treatment and control groups; (c) nonlinear regression on the intravenous and intraperitoneal data sets using a two-compartment open model indicated a difference in the absorption process but not in the metabolic and blood-brain transfer processes.

Notes: Abstract: The annexins are a group of highly related Ca2+-dependent membrane-binding proteins that are present in a wide variety of cells and tissues. We have examined the subcellular distribution of five members of the annexin family in the adrenal medulla. Bovine adrenal medullary tissue was homogenized in buffers containing EGTA and fractionated on sucrose gradients. p36 (the large subunit of calpactin I) was found to be predominantly membrane associated, with ∼20% present in fractions enriched in chromaffin granules. In contrast, lipocortin I was localized primarily to the cytosol, with only a small proportion found in plasma membrane-containing fractions. Like lipocortin I, endonexin I was found to be present almost entirely in the soluble fractions. The 67-kDa calelectrin was localized primarily to the plasma membrane fractions, with a small amount present in the chromaffin granule and cytoplasmic fractions. Synexin was present in both membranous and cytoplasmic fractions. p36 appeared to be a peripherally associated granule membrane protein in that it was dissociated from the membrane by addition of base and it partitioned with the aqueous phase when granule membranes were treated with Triton X-114. Antiserum against p10 (the small subunit of calpactin I) reacted with a protein of 19 kDa that is specifically localized in chromaffin granule membrane fractions. The differences in subcellular distributions of the annexins suggest that these proteins have distinct cellular functions. The finding that p36 is associated with chromaffin granule and plasma membrane fractions provides further support for a possible role of calpactin in exocytosis.

Notes: Abstract: HPLC analysis of guanidinium hydrochloride extracts of neonatal and adult rat brain revealed a polypeptide that is present in high concentration in the immature nervous system, but whose levels decline dramatically in the adult. This polypeptide has been isolated and its complete amino acid sequence determined by gas-phase Edman degradation following specific chemical and enzymatic cleavages. The molecule is identified as thymosin β10, a member of a multigene family that encodes a structurally conserved series of small acidic polypeptides of uncertain function. Thymosin β10 is present in the developing nervous system as early as embryonic day 9. Levels subsequently increase to peak values between embryonic day 15 and postpartum day 3, before falling to adult values (about a 20-fold reduction) by postpartum day 14. The elevated levels of thymosin β10 in fetal and neonatal brain correlate with high levels of thymosin β10 mRNA, whereas the low values of the polypeptide in the adult and juvenile are mirrored by an approximate 15-fold reduction in specific mRNA. In comparison, the levels of thymosin β4 polypeptide, a homologue of thymosin β10, only decline by about 20% during the same developmental period. However, the mRNA encoding thymosin β4 is elevated in fetal brain, and its levels decrease approximately four-fold to a stable value around the time of birth. The reason for this discrepancy between thymosin β4 protein and mRNA levels is unknown. Thymosin β10 can also be detected by HPLC in fetal liver, where levels are approximately 5% of those in brain. In liver, thymosin β10 also declines following birth. It is concluded that β-thymosin expression (as measured by steady-state mRNA and polypeptide levels) is both up-and down-regulated during different phases of maturation of the mammalian nervous system.

Notes: Abstract: [3H]Harman (1-[3H]methyl-β-carboline) was used in a novel radioligand binding assay to label selectively and with high affinity monoamine oxidase (MAO) type A. The concentration of the enzyme was determined in six CNS regions of the primate species marmoset (Callithrix jacchus) and of the rat: hypothalamus, hippocampus, cerebellum, cerebral cortex, striatum, and spinal cord. The specific [3H]harman binding in the CNS of the marmoset reveals the same pharmacological profile and other characteristics (affinity, saturability, and reversibility) as in the CNS of the rat. The regional distribution of the [3H]harman binding density (Bmax) in the CNS exhibits a distinct pattern in the marmoset and the rat and a 35 (hypothalamus) to 75% (hippocampus) lower Bmax in the marmoset than in the rat. The Bmax values of [3H]harman binding in the CNS of the marmoset and the rat combined as well as those from visceral organs of the rat (liver, heart, lung, thymus, spleen, and kidney) correlated positively and highly significantly with the respective Vmax values of specific MAO activity of the A type but not of the B type, determined with kynuramine as the substrate. In subcellular fractionation experiments with rat cerebral cortex, the highest [3H]harman binding density (Bmax) and MAO-A activity (Vmax) were detected in mitochondrial fractions and severalfold lower values in the synaptosomal membrane fraction. In conclusion, we suggest that [3H]harman binding is a biochemical tool as a selective marker to quantify MAO-A in the CNS of different mammalian species as well as in extraneuronal tissues.

Notes: Abstract: In PC12 pheochromocytoma cells whose phospholipids had been prelabeled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1–2 min, before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells; bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.

Notes: Abstract: The stereoenantiomers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 μM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 ± 14 and 75 ± 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 ± 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthioinosine, both at a concentration of 100 μM, reduced the levels and rates of L-[3H]adenosine accumulation by 〉44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.

Notes: Abstract: The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 μM Pb2+ to intact synaptosomes in Ca2+-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2+-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1–50 nM (EC50± 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2–5 μM (EC50± 0.5 μM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.

Notes: Abstract: Intact human neuroepithelioma SK-N-MC cells bound the β-adrenergic antagonist (–)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to β-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol norepinephrine 〉 epinephrine, which is indicative of a β1-subtype receptor. Membranes prepared from the cells bound (–)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the β1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the β2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only β1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the β1- and β2-adrenergic receptors revealed the presence of a very strong β1-adrenergic receptor mRNA signal, while under the same conditions no β2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of β1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.

Notes: Abstract: In earlier studies, a 75,000-dalton glycoprotein (gp75) has been identified as a component of both low- and high-affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF-induced up-regulation of fos oncogene expression, and did not efficiently internalize NGF. LANS is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high-affinity NGFRs but no detectable low-affinity sites. The transformant line LAN5/NGFR expressed both high- and low-affinity NGFRs, displayed NGF-induced up-regulation of fos oncogene, and efficiently internalized NGF. The number of high-affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high-affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high-affinity NGFRs, the putative second factor may also limit NGF responsiveness.

Notes: Abstract: Significant differences were demonstrated between the long-sleep (LS) and short-sleep (SS) selected mouse lines in the abilities of barbiturates and γ-aminobutyric acid (GABA) to inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to well-washed cerebral cortical membranes. Thus, using phenobarbital to initiate the dissociation of [35S]TBPS, the extent of inhibition was significantly greater in LS mice (but not SS mice) than would be predicted using equilibrium conditions. Pentobarbital had the opposite effect, causing [35S]TBPS to dissociate to a greater extent in SS than LS membranes. [35S]TBPS binding was dissociated from LS and SS membranes by GABA to a greater and lesser extent, respectively, than would be predicted from equilibrium studies. Because no line differences in the potencies of these drugs to inhibit [35S]TBPS binding were found using equilibrium conditions, these results indicate that the association rates of barbiturates and GABA may be different between these lines. These findings are consistent with neurochemical studies indicating differences in the benzodiazepine/GABA receptor-chloride channel complex in these selected lines and may explain their differential sensitivities to certain agents acting through this supramolecular complex.

Notes: Abstract: An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is substantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7: 1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.

Notes: Abstract: We have studied the metabolic and functional effects of two new platelet-activating factor (PAF) antagonists (BN 50726 and BN 50739) and their diluent (dimethyl sulfoxide; DMSO) during reoxygenation of the 14-min ischemic isolated brain. Blood gases, EEG, auditory evoked potentials, cerebral metabolic rate for glucose (CMRglc), and cerebral metabolic rate for oxygen (CMRO2) were monitored throughout the study. Frozen brain samples were taken for measurement of brain tissue high-energy phosphates, carbohydrate content, and thiobarbituric acid-reactive material (TBAR, an indicator of lipid peroxidation) at the end of the study. Following 60 min of reoxygenation in the nontreated 14-min ischemic brains, lactate, AMP, creatine (Cr), intracellular hydrogen ion concentration [H+]i), and TBAR values were significantly higher and ATP, creatine phosphate (PCr), CMRglc, CMRO2, and energy charge (EC) values were significantly lower than the corresponding normoxic control values. PCr and CMRO2 values were significantly higher, and glycogen, AMP, and [H+]i values were significantly lower in the BN 50726-treated ischemic brains than in DMSO-treated ischemic brains. In brains treated with BN 50739, ATP, ADP, PCr, CMRO2, and EC values were significantly higher, and lactate, AMP, Cr, and [H+]i values were significantly lower than corresponding values in the DMSO-treated ischemic brains. TBAR values were near control levels in all brains exposed to DMSO. There was also marked recovery of EEG and auditory evoked potentials in brains treated with DMSO. Treatment with BN 50726 or BN 50739 in DMSO appeared to improve brain mitochondrial function and energy metabolism partly as the result of DMSO action as a free radical scavenger. The PAF antagonists act at a different level, possibly restoring normal mitochondrial function or preventing some of the adverse effects of increased excitatory amino acid and/or intracellular Ca2+ levels. This is the only drug combination currently known to promote such complete short-term metabolic and functional recovery during postischemic reoxygenation.

Notes: Abstract: Heterogeneity of rat brain angiotensin II receptors was revealed by quantitative autoradiography after incubation with 125I-Sar1-angiotensin II and displacement with the angiotensin II antagonists CGP 42112 A and DuP-753 and by receptor sensitivity to dithiothreitol. Receptors in areas involved in cardiovascular and fluid control—the subfornical organ, nucleus of the solitary tract, paraventricular nucleus, and area postrema—are displaced by DuP-753 with an IC50 of 1 × 10−7M, are sensitive to 5 mM dithiothreitol, and thus are angiotensin II type-1. Receptors in the inferior olive are displaced by CGP 42112 A (IC50, 1 × 10−9M) but not by DuP-753 in concentrations up to 10−4M, are insensitive to 5 mM dithiothreitol, and thus are angiotensin II type-2.

Notes: The release of γ-aminobutyric acid (GABA) in rat dorsolateral striatum was studied using in vivo microdialysis. Dialysis was conducted 2 days after probe implantation in awake, freely moving rats using a modified Ringer solution. Calcium induced a reversible increase in GABA release that was abolished by tetrodotoxin but was only slightly attenuated by a maximally effective dose of pergolide, a D2 receptor agonist. It was thus concluded that pergolide inhibits calcium-stimulated release of GABA presynaptically by a mechanism distinct from that of tetrodotoxin.

Notes: In l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-treated mouse brain, there was no significant increase or decrease in the content of an endogenous amine, 1,2,3,4-tctrahydroisoquinoline (TIQ), which is well noted for inducing parkinsonism, whereas another endogenous amine, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ), was markedly reduced. This result agrees with the finding in human idiopathic parkinsonianism, confirmed by our previous research. In addition, pretreatment with 1-MeTIQ completely prevented MPTP- or TIQ-inducing bradykinesia, a symptom of parldnsonism. This study confirmed that 1-MeTIQ plays an important role in preventing the pathogenesis of parldnsonism and is a possible leading compound of anti-parkinsonism agents.

Notes: An extract of the whole brain of the frog Rana ridibunda contained high concentrations of substance P-like immunoreactivity, measured with an antiserum directed against the COOH-terminal region of mammalian substance p and neurokinin b-like immurtoreactivity, measured with an antiserum directed against the NH2-terminus of neurokinin B. The primary structure of the substance p-related peptide (ranakinin) was established as: Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Gly-Leu-Met-NH2. Mammalian substance P was not present in the extract. The primary structure of the neurokinin b-related peptide was established as: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. This amino acid sequence is the same as that of mammalian neurokinin B.Ranakinin was equipotent with substance p and [Sar9,-Met(O2)11]substance p in inhibiting the binding of 125I-Bolton-Hunter-[Sar9, Met(O2)11]substance p, a selective radio-ligand for the NK1 receptor, to binding sites in rat subman-dibular gland membranes (IC50 1.6 ± 0.3 nM; n = 5). It is concluded that ranakinin is a preferred agonist for the mammalian NK1 tachykinin receptor subtype.

Notes: Elevated iron concentrations in the substantia nigra (SN) pars compacta have been implicated in the development of idiopathic Parkinson's disease. Because, as a transitional metal, iron promotes free radical formation, the role of iron in the degeneration of the nigrostriatal dopamine neurons in Parkinson's disease has received much attention. This study further investigates the cytotoxic effects of iron in the SN. Various concentrations of FeCl3 (1, 5, and 50 μg of Fe3+ in 5 μl) were unilaterally injected into the SN of adult rats. The two lower doses of iron had no effect on striatal dopamine levels or on the behavioral responses of the rats. However, injection of 50 μg of Fe3+ resulted in a substantial selective decrease of striaial dopamine (95%), 3,4-dihydroxyphenylacetic acid (82%), and homo-vanillic acid (45%), without any change in norepinephrine concentration. Dopamine-related behavioral responses, such as spontaneous movements in a novel space and rearing, were significantly impaired, whereas amphetamine administration induced ipsilatcral rotation in the iron-treated rats. The present study indicates that the nigrostriatal dopamine neurons are susceptible to the presence of ionic iron and thus supports the assumption that iron initiates dopaminergic neu-rodegeneration in Parkinson's disease.

Notes: The study of the regulation of low-abundance blood-brain barrier (BBB) transcripts either in isolated brain microvessels or in endothelial cells in tissue culture (ECL cells) requires isolation of poly(A)+ mRNA. Therefore, we describe here a single-step method for isolation of polyfA)+ mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo-de-oxythymidine cellulose affinity chromatography. The yield of poly(A)+ mRNA was—15-19 #g/g of brain or choroid plexus, 14-17 μg per batch of isolated capillaries in a single bovine forebrain (190 g), and 6-12 μg/107 ECL cells. Northern blot analysis showed characteristic and undegraded 2.1-and 1.7-kb actin transcripts in brain capillaries and a 2.1-kb actin mRNA in brain and ECL cells. Northern analysis was also used to quantify the glucose transporter type I transcript, which is very rare in basal ECL cells, and this mRNA was shown to be up-regulated by glucose deprivation. This method represents a significant improvement in the mRNA yield for brain capillaries or cultured endothelial cells compared with the conventional two-step method.

Notes: : The effect of hypothermia on the ischemia-induced changes in the subcellular distribution of protein kinase C (PKC)(γ), -(βII), and -(α) and the activity of PKC was studied in striatal homogenates of rats subjected to 20 min of cerebral ischemia. The effect of post-ischemic cooling was also studied. During normothermic ischemia, PKC(γ) and -(βII) increased 3.9-and 2.9-fold, respectively, in the particulate fraction, signifying a translocation of PKC to cell membranes. The levels of PKC(α) did not change significantly. PKC activity decreased during ischemia by 52% and 47% (p 〈 0.05) in the paniculate and cytosolic fractions, respectively, and remained inhibited for the 1 h recovery period. In hypothermic animals, there was no evidence of translocation, and the inhibition of PKC activity was completely abolished. Hypothermia induced in the recovery phase, however, did not affect PKC distribution or activity. The protective effect of intraischemic hypothermia may in part be due to the prevention of the ischemia-induced translocation and subsequent downregulation of PKC, possibly through a temperature-dependent modification of the cell membranes.

Notes: Two rabbit arylamine N-acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, ρ-aminosalicylic acid, and ρ-aminobenzoic acid. In the pineal gland, serotonin is metabolized to N-acetylserotonin by an unknown N-acetyltransferase. Similarity of the liver enzymes and the pineal gland arylalkylamine N-acetyltransferase (AA-NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as ρ-phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed the N-acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressed rNAT1 and rNAT2 genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross-hybridization of rNAT1 or rNAT2 to the gene for the pineal gland AA-NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step of N-acetylation of serotonin.