Phospholipid Methylation Increases [3H]Diazepam and [3H]GABA Binding in Membrane Preparations of Rat Cerebellum

The effect of phospholipid methylation on both [3H]diazepam and [3H]GABA ( [3H]gamma-aminobutyric acid) binding to crude synaptic plasma membrane from rat cerebellum has been studied. S-Adenosylmethionine (SAM) stimulates [3H]methyl group incorporation into membrane phospholipids and enhances [3H]diazepam binding by increasing the apparent Bmax. Conversely, inhibition of [3H]methyl group transfer from [3H]SAM to phospholipids by preincubation with SAM at 0 degrees C or with SAH abolishes the increase of binding. After preincubation with SAM, analysis of the GABA binding reveals the presence of binding sites with high affinity, a property absent in control membranes preincubated without SAM. Among the neurotransmitter bindings tested, only those of GABA and benzodiazepine in the cerebellum and beta-adrenergic ligands in the cerebral cortex are enhanced upon stimulation of phospholipid methyltransferase activity. [3H]Dihydromorphine, [3H]dihydro-alpha-ergokryptine and [3H]spiroperidol bindings are not affected by SAM. The present data suggest an involvement of phospholipid methylation in regulation of both [3H]GABA and [3H]-diazepam binding.     

Dihydropyridine [methyl-3H]PN 200–110 Binding and Myogenesis in Intact Muscle Cells In Vitro

The radioligand dihydropyridine [methyl-3H]PN 200-110 binds to contracting myotubes in culture derived from chick embryo pectoralis muscle. [methyl-3H]PN 200-110 binds specifically to high-affinity sites, with nonspecific binding only between 15 and 30% of the total binding. A Scatchard plot of the specific binding revealed a single high-affinity binding site with a KD (dissociation constant) of 0.5 nM +/- 0.2 nM and Bmax (number of binding sites) of 100 fmol/10(6) nuclei. We employed this sensitive assay to probe the appearance of high-affinity [methyl-3H]PN 200-110 binding sites during myogenesis. The time course of appearance of high-affinity binding sites lags behind that of fusion. Low-calcium media prevented the differentiation of myoblasts and blocked the appearance of high-affinity sites. Chelation of intracellular calcium before or after fusion of myoblasts with the calcium indicator Quin 2 prevented the appearance of dihydropyridine binding sites. These findings are consistent with the view that the expression of dihydropyridine receptors is modulated by the intracellular calcium.     

THE EFFECT OF CORDYCEPIN ON THE APPEARANCE OF [3H]RNA IN THE GOLDFISH OPTIC TECTUM FOLLOWING INTRAOCULAR INJECTION OF [3H]URIDINE

Abstract— Although biochemical and electron microscopic evidence has shown that RNA molecules may be found within axons, the origin of this RNA is not known. In order to determine if the RNA found in axons is synthesized in the nerve cell body and axonally transported, we have studied the effect of the RNA inhibitor cordycepin (3′-deoxyadenosine) on the retinal synthesis and axonal migration of radioactive RNA. Ten μg of cordycepin was injected into the right eye of 11 fish and 3 h later [³H]uridine was injected into the same eye. Twelve control fish were injected with [³H]uridine only and all fish were sacrificed 6 days later. Results of RNA extraction of retina and tecta showed that cordycepin decreased retinal RNA synthesis by approx 24%, while inhibiting the amount of [³H]RNA appearing in the contralateral tectum by 74%. Since the transport of RNA precursors was depressed by only 50%, (significantly different from the effect on RNA, P < 0.01) it seems unlikely that the action of cordycepin in decreasing tectal [³H]RNA levels was due solely to a decrease in the availability of labeled precursors for tectal RNA synthesis. For the purpose of blocking tectal RNA synthesis, 200 μg of cordycepin was injected intracranially several days after the intraocular injection of [³H]uridine. This route of cordycepin administration failed to significantly block the appearance of [³H]RNA in the tectum, suggesting that at least some of the [³H]RNA in the tectum was synthesized before arrival in the tectum itself. To be sure that cordycepin itself was not being transported, we injected cordycepin into the right eye of fish and 5 days later, injected fish intracranially with [³H]uridine. Autoradiograms were prepared and grains were counted over the fiber layers of left (experimental) and right (control) tecta. No significant difference was observed in the number of grains of left vs right tecta indicating that cordycepin itself is not axonally transported. These experiments support earlier findings from our laboratory which suggest that RNA may be axonally transported in goldfish optic fibers.     

Characterization of the binding of S-adenosyl-L-methionine to plasma membranes of HL-60 promyelocytic leukemia cells

S-Adenosyl-L-methionine (AdoMet) has been found to bind specifically to the plasma membrane of promyelocytic leukemia cells, HL-60. The Kd for AdoMet is 4.2.10(-6) M and the Bmax is 4.0.10(-12) mol/10(7) HL-60 cells. The binding is not related to the adenosine receptor since neither adenosine, ADP, nor ATP affect the ligand-receptor reaction. When HL-60 cells were incubated with physiological concentrations of [methyl-3H]AdoMet (20 microM) at 36 degrees C, AdoMet did not equilibrate with the intracellular pool, nor were any [3H]methyl groups incorporated into nucleic acids or proteins. In contrast, significant amounts of [3H]methyl groups were incorporated into membrane phospholipids. When cells were incubated with 20 microM [methyl-3H]AdoMet, [3H]methyl groups were transferred to phosphatidylethanolamine, -monomethylethanolamine, and -dimethylethanolamine yielding phosphatidylcholine. However, the rate of methyl transfer with AdoMet was only 22% of that observed when cells were incubated with a comparable amount of [methyl-3H]methionine. Both the binding of AdoMet and the methylation of phospholipids were inhibited by exogenous S-adenosyl-L-homocysteine. Therefore, the binding may be linked to a phospholipid methyltransferase.     

Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

Comparison of the In Vitro Receptor Binding Properties of N-[3H]Methylspiperone and [3H]Raclopride to Rat and Human Brain Membranes

The aim of the present investigation was to study and compare the in vitro binding properties of the two radioligands N-[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug-naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5-hydroxytryptamine 2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10-fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half-life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The Ki values were, however, two- to four-fold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.     

CATECHOLAMINE METABOLISM IN THE DOG: COMPARISON OF INTRAVENOUSLY AND INTRAVENTRICULARLY ADMINISTERED [14C]DOPAMINE AND [3H]NOREPINEPHRINE

—The urinary excretion of labelled metabolites was measured in dogs which had been injected intravenously or intraventricularly with [³H]norepinephrine or [¹⁴C]dopamine. [³H]Norepinephrine injected by either route produced more labelled 3-methoxy-4-hydroxy-phenylglycol than 3-methoxy-4-hydroxymandelic acid, as did [¹⁴C]dopamine after intravenous administration. In contrast, following the intraventricular injection of [¹⁴C]dopamine, more [¹⁴C]3-methoxy-4-hydroxymandelic acid was formed than [¹⁴C]3-methoxy-4-hydroxyphenylglycol. These observations suggest that the metabolism of exogenously-administered and endogenously-formed norepinephrine may proceed through different routes and that the predominant metabolite of norepinephrine in canine brain may be 3-methoxy-4-hydroxymandelic acid rather than 3-methoxy-4-hydroxyphenylglycol.     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.     

DIFFERENT LABELLING PATTERNS IN MOUSE LYMPHOID TISSUES WITH [3H]DEOXYCYTIDINE AND [3H]THYMIDINE

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [³H]CdR, or tritiated thymidine, [³H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [³H]CdR, whereas with [³H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [³H]TdR. Thus, labelling patterns with [³H]CdR differed from those with [³H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [³H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [³H]CdR. This radioactivity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of [3H]Inositol Metabolites in Rat Hippocampal Formation Slices Prelabelled with [3H]Inositol and Stimulated with Carbachol

Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion-exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4-trisphosphate, [3H]inositol 1,4,5-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate. The levels of [3H]inositol 1,4,5-trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5-trisphosphate was incubated with hippocampal formation homogenates in an ATP-containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4-bisphosphate, [3H]inositol 1,3,4-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5-tetrakisphosphate is metabolised to [3H]inositol 1,3,4-trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4-bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5-bisphosphate selective phospholipase C.     

Rapid Release of [3H]Dopamine from Median Eminence and Striatal Synaptosomes

Release of preaccumulated, tritium-labeled dopamine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1-20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+ resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+- and the veratridine-stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+-induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]o and the [Ca2+]o dose-response curves, consistent with actions of Mg2+ on screening surface membrane charges and blocking voltage-dependent Ca2+ channels. In all studies, steady-state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.     

Transport and Metabolism of D-[3H]Adenosine and L-[3H]Adenosine in Rat Cerebral Cortical Synaptoneurosomes

The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D-[3H]adenosine and L-[3H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent KT and Vmax values for adenosine transport. For D-[3H]adenosine, high- and low-affinity accumulation processes were present. For the high-affinity system an inverse relationship was found between transport reaction times and KT and Vmax values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D-[3H]adenosine to nucleotides, apparent KT values were 9.4, 8.4, and 4.5 microM, respectively, and Vmax values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an adenosine deaminase inhibitor, and 5'-iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D-[3H]adenosine to 6% with 5-s and 9% with 15-s incubations. This resulted in significantly higher KT values: 36 microM at 5 s and 44 microM at 15 s. At 10-min incubations in the presence of these inhibitors, metabolism of accumulated D-[3H]adenosine was 32%, and apparent KT and Vmax values at this time were not significantly different from those obtained without inhibitors. For L-[3H]adenosine, apparent KT and Vmax values for 20-s incubations were 38.7 microM and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L-[3H]adenosine was observed only at incubations of greater than 30 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrasynaptosomal Sequestration of [3H]Amphetamine and [3H]Methylenedioxyamphetamine: Characterization Suggests the Presence of a Factor Responsible for Maintaining Sequestration

We examined the incorporation of [3H]methylenedioxyamphetamine ([3H]MDA) and [3H]amphetamine into rat brain synaptosomes. Saturation studies, using increasing concentrations of nonradioactive ligand, revealed that [3H]-MDA interacted with two saturable sites that were sensitive to boiling of the tissue. Eadee-Scatchard plots of [3H]MDA saturation data were curvilinear; nonlinear curve-fitting analysis of these data suggested the presence of high- and low-affinity [3H]MDA sites of association: KD high = 295 nM, Bmax high = 32 pmol/mg of protein; KD low = 45 microM, Bmax low = 5.2 nmol/mg of protein. Association of [3H]MDA to the low-affinity site was dependent on the presence of isotonic sucrose in the incubation medium. The high capacities of these sites argue against a bimolecular interaction of [3H]MDA with monovalent protein binding sites. [3H]MDA incorporation was reduced under conditions that disrupt the integrity of plasma membranes, such as sonication, incubation in hypotonic media, and incubation in the presence of the detergent digitonin. These data indicate that [3H]MDA incorporation into synaptosomes may represent an internalization and sequestration phenomenon. [3H]MDA incorporation was also reduced by preincubation of the synaptosomal preparation at 37 degrees C or in hypotonic buffer at 4 degrees C, a result suggesting that this sequestration is maintained by an intrasynaptosomal component that is lost under the preincubation conditions described above. [3H]MDA incorporation was pH dependent (maximal at pH 7.5) and temperature sensitive (maximal incorporation occurred at 21 degrees C and was substantially reduced at 37 degrees C). [3H]Amphetamine was also incorporated into synaptosomes, and this incorporation was sensitive to the same physical manipulations of the tissue preparation as [3H]MDA incorporation. The synaptosomal sequestration of both [3H]MDA and [3H]amphetamine was inhibited by permeant cations, such as sodium and potassium, a result suggesting that the proposed intrasynaptosomal component that maintains the sequestration is anionic. Preliminary pharmacological profiles of [3H]MDA and [3H]amphetamine sequestration were identical. The rank order of inhibitor potencies for the incorporation of both ligands was desipramine greater than amphetamine greater than MDA greater than methylphenidate. This order of potency does not correspond to the lipophilicity of the test drugs. The synaptosomal incorporation and sequestration of [3H]MDA, [3H]methylenedioxymethamphetamine, and [3H]amphetamine described in the present report may be important in the molecular mechanism of action of monoamine release induced by the amphetamines.     

Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices

The uptake of [3H]adrenaline and [3H]noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high-affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose-dependent manner, but the selective dopamine and 5-hydroxytryptamine uptake inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]-noradrenaline and by unlabelled noradrenaline with [3H]-adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline-containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high-affinity sites on noradrenergic nerve terminals.     

Studies on [3H]Diazepam and [3H]Ethyl-β-Carboline Carboxylate Binding to Rat Brain In Vivo. II. Effects of Electroconvulsive Shock

In vivo specific binding of [3H]diazepam was not altered by a single electroconvulsive shock given 5, 30, or 60 min, or 24 h previously, nor 24 h after the last of 10 daily shocks. Similarly, in vivo [3H]ethyl-beta-carboline carboxylate binding was not changed in the brains of animals that had been given a single electroconvulsive shock 30 min previously or a series of 10 daily shocks. Brain areas examined included cerebral cortex, hippocampus, cerebellum, and striatum. However, cortical binding of [3H]diazepam was increased by 32% in animals which were present in the same room while another was being injected and killed. This may represent a response to stress and/or anxiety.     

ON THE RELATIONSHIP BETWEEN [3H]CHOLINE UPTAKE ACTIVATION AND [3H]ACETYLCHOLINE RELEASE

The depolarization-induced, calcium-dependent release of [³H]ACh from hippocampal synaptosomes was studied in a superfusion system. Release increased, with increasing depolarization. Barium and strontium effectively substituted for calcium during the depolarization, but magnesium inhibited the release. Releasable [³H]ACh is derived from the sodium-dependent component of the [³H]choline uptake which points out the physiologic importance of sodium-dependent choline transport. It is concluded that [³H]ACh release in this system has the same properties as neurotransmitter release in many other systems. Previous studies have shown that treatments which alter the activity of cholinergic neurons in vivo result in parallel changes in sodium-dependent choline uptake in vitro. When synaptosomes were utilized from animals treated to reduce cholinergic activity, there was a reduced release following the reduced uptake. Conversely, when synaptosomes were taken from animals treated to increase sodium-dependent choline uptake, there was an increase in the release. It is concluded that the changes in sodium-dependent choline uptake in vitro consequent to changes in neuronal activity in vivo result in parallel changes in releasable ACh. A comparison was made between the effect of a number of ions and agents on release and their effect on the in vitro, depolarization-induced activation of sodium-dependent choline uptake. Barium and strontium, ions which substitute for calcium in the release process, support the in vitro activation of uptake. Vinblastine and Bay a 1040, compounds which block release, prevented the in vitro activation of sodium-dependent choline uptake. However, magnesium blocked release in a dose-dependent manner, but did not block the activation of uptake in vitro. Rather, magnesium substituted for calcium and supported the activation of uptake in a dose-dependent fashion. It is concluded that acetylcholine release is not necessary for the activation of choline uptake.     

Bacterial [methyl-3H]thymidine incorporation in substrate-amended estuarine sediment slurries

Abstract Five different bacterial communities were enriched in substrate-amended slurries of sediment from the Tay Estuary, Scotland. During incubation of the slurries, concentrations of volatile fatty acids, sulphate, sulphide and methane were monitored to clearly define the activity of the stimulated populations. An aerobic population, a ‘microaerophilic’ population and three anaerobic populations (fermentative heterotrophs, sulphate-reducing bacteria and methanogens plus acetogens) were established to reflect community growth and metabolism both in surface oxic and deeper anoxic layers. Similar numbers of cells involved in division were observed in all five slurries, demonstrating the potential for bacterial production. Thymidine incorporation rates in glucose-stimulated slurries under both aerobic and fully anaerobic conditions were similar, confirming the ability of fermentative anaerobic heterotrophs to incorporate [ methyl -³H]thymidine into DNA during growth. Although anaerobic communities of sulphate-reducing, acetogenic plus methanogenic bacteria were stimulated and actively growing, they did not incorporate [ methyl -³H]thymidine into DNA. Since the thymidine technique does not measure the growth of these important groups, calculated productivity values based upon thymidine incorporation within anoxic sediment systems will be substantially underestimated, even if growth substrates are not limiting.     

Inhibition by Calmodulin Antagonists of [3H]MK-801 Binding in Brain Synaptic Membranes

In brain synaptic membranes not extensively washed, (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) binding was markedly inhibited in a concentration-dependent manner (at concentrations above 1 microM) by several compounds having antagonistic activity at the Ca(2+)-binding protein calmodulin. Scatchard analysis revealed that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the binding through a significant decrease in the density of binding sites without affecting the affinity at 10 microM. In membranes extensively washed and treated with a low concentration of Triton X-100, L-glutamic acid (Glu) drastically accelerated the initial association rate of [3H]MK-801 binding with glycine (Gly), almost doubling the initial association rate found in the presence of Glu alone. The addition of W-7 invariably reduced the initial association rate observed in the presence of either Glu alone or both Glu and Gly, without significantly altering the dissociation rate of bound [3H]-MK-801, irrespective of the presence of the two stimulatory amino acids. The maximal potencies of Glu, Gly, and spermidine in potentiating the binding were all attenuated by W-7. These results suggest that calmodulin antagonists may interfere with opening processes of an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in rat brain.