Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na(+)-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [3H]NA release by SNC in normal Na(+) (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 microM desipramine, an NA transporter inhibitor. [3H]NA release in low Na(+) (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [3H]NA release by tyramine from both slices in normal and low Na(+) buffer was almost completely inhibited by desipramine. [3H]NA uptake into cerebral cortical slices was observed in low Na(+) buffer at 20-30% of normal Na(+) buffer levels. [3H]NA uptake in both normal and low Na(+) buffers was inhibited by desipramine and by SNC. Although [3H]NA uptake in normal Na(+) buffer was almost completely inhibited by 500 microM ouabain, the uptake in low Na(+) buffer was resistant to ouabain. These findings suggest the existence of a functional Na(+)-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.     

Role of Adenylate Cyclase in Presynaptic α2-Adrenoceptor- and μ-Opioid Receptor-Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ ionophore A 23187 (10 microM) in the presence of 1.2 mM Ca2+. Forskolin (10 microM), 8-bromo-cyclic AMP (300 microM), and dibutyryl-cyclic AMP (300 microM) facilitated both the electrically evoked and A 23187-induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX, 300 microM) and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771, 30 microM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM-1 microM) and the facilitatory effect of phentolamine (0.01-10 microM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8-bromo-cyclic AMP. Clonidine (1 microM) reduced and phentolamine (3 microM) enhanced A 23187-induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 microM) and [D-Ala2-D-Leu5]enkephalin (DADLE, 0.3 microM), like that of the Ca2+ antagonist Cd2+ (15 microM), on the electrically evoked release of [3H]noradrenaline were not affected by 8-bromo-cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187-induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic mu-opioid receptors, alpha 2-adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed-forward action of cyclic AMP on the secretion process.     

Redox Modulation of N-Methyl-D-Aspartate-Stimulated Neurotransmitter Release from Rat Brain Slices

Rat brain cortical slices released tritiated norepinephrine ([3H]NA) during a 2-min stimulation with N-methyl-D-aspartate (NMDA). Dithiothreitol (DTT; 0.1-5 mM), present for 6 min prior to stimulation, dose-dependently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 microM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose-response curve of NMDA to the left. Cortical slices released approximately the same amount of [3H]NA with 10 microM NMDA following DTT treatment (about 5%) as non-DTT-treated control slices did with 500 microM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA-stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7-chlorokynurenic acid, and the competitive NMDA antagonist, 2-aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA-stimulated responses.     

Hepatic inositol release upon hormonal stimulation of perfused rat liver.

A sustained increase in the hepatic release of 3H radioactivity was shown to occur upon hormonal stimulation of perfused rat liver 15-20 h after intraperitoneal injection of 100 microCi of myo-[2-3H]inositol. Hormone-released radioactive material was analysed by t.l.c. and was found to consist predominantly of [3H]inositol, without further metabolites. Vasopressin (14 nM), phenylephrine (1.7 microM), angiotensin II (15 nM), glucagon (0.5 nM) and dibutyryl cyclic AMP (5 microM) exert maximal effects on hepatic inositol efflux after 10-15 min of stimulation. Omission of Ca2+ from the perfusion medium abolishes the hormone-dependent inositol release. LiCl (10 mM) does not significantly affect the basal release of [3H]inositol, but suppresses vasopressin- and angiotensin-triggered inositol release. Inositol efflux induced by glucagon, dibutyryl cyclic AMP and phenylephrine, however, remains essentially unchanged by LiCl infusion. This establishes a further metabolic difference between these two groups of agonists in that stimuli that act through cyclic AMP produce a stimulated outflow of inositol, but apparently without a Li+-sensitive phosphatase being involved in the overall process.     

An investigation of noradrenaline uptake and release by the CATH.a cell line

The cell bodies of ascending noradrenergic neurons in the brain are located predominantly in the locus coeruleus. An in vitro model of locus coeruleus neurons could prove to be a useful tool in the investigation of noradrenergic neural networks and their associated pathophysiologies. The CATH.a cell line demonstrates some of the properties expected of locus coeruleus neurons, and the present study investigated the neurotransmitter uptake and release properties of the CATH.a cells. It was surprising that the CATH.a cells failed to accumulate [3H]noradrenaline ([3H]NA), suggesting the lack of a functional NA transporter. RT-PCR supported this finding by demonstrating the absence of NA transporter mRNA. Treatment of CATH.a cells with various differentiating agents failed to increase the [3H]NA uptake. Endogenous NA release was studied using HPLC detection, which revealed a lack of depolarisation-induced increases in endogenous NA release. A human NA transporter-transfected CATH.a cell line was generated (termed RUNT), and a study of the [3H]NA uptake revealed that the RUNT cells displayed significant uptake that could be blocked by cocaine (10 microM). Furthermore, the uptake capacity could be dramatically increased by differentiation of the cells with dibutyryl cyclic AMP (1 mM) for 24 h. Using dibutyryl cyclic AMP-differentiated RUNT cells, high K+ concentrations (50 mM) significantly increased [3H]NA release above basal levels.     

Comparison of dopamine uptake and release in vitro in sheep and rat striatum.

Cocaine inhibits tritium-labeled dopamine ([3H]DA) uptake in rat (IC50 approximately 400 nM) and sheep (IC50 approximately 1 microM) striatum. GBR 12909, a selective DA uptake inhibitor, potently inhibits [3H]DA uptake in rat (IC50 less than 10 nM), but is less effective (only 60% of the uptake is inhibited at a concentration of 10 microM) and less potent (IC50 approximately 300 nM) in sheep. [3H]DA release from slices of rat or sheep striatum is stimulated by potassium (15-50 mM). In the presence of nomifensine (10 microM), cocaine (10 microM) had no effect on potassium-stimulated [3H]DA release in either species. [3H]DA release is increased by N-methyl-D-aspartate (NMDA) (10-1000 microM) in rat striatum but NMDA did not stimulate [3H]DA release in sheep striatum. These findings suggest that NMDA receptors either are absent from or do not regulate release of preloaded [3H]DA in sheep striatum.     

Pharmacological effects of naltriben as a ligand for opioid mu and kappa receptors in rat cerebral cortex

Naltriben (NTB) has been used to differentiate the subtypes of delta opioid receptors, delta1 and delta2. However, there is considerable evidence suggesting that NTB may act on other types of opioid receptors too. We examined the effects of NTB on the specific binding of radiolabeled ligands for opioid mu and kappa2 receptors, and the effects on the release of [3H]norepinephrine ([3H]NE) in rat cerebral cortex slices. NTB displaced the specific binding of [3H]DAMGO with Ki value of 19.79 +/- 1.12 nM in rat cortex membranes. Specific binding of [3H]diprenorphine ([3H]DIP) was inhibited by NTB with Ki value of 82.75 +/- 6.32 nM in the presence of DAMGO and DPDPE. High K+ (15 mM)-stimulated release of [3H]NE was attenuated by DAMGO in rat cerebral cortex slices. NTB (30 nM) shifted the dose-response curve of DAMGO to the right and attenuated the maximal effect. In the meantime, NTB inhibited high K+-stimulated [3H]NE release at concentrations above 100 nM. The inhibitory effect of NTB was not attenuated by CTAP (10 nM) and naloxone (3 nM) but by higher concentration of naloxone (30 nM), nor-BNI (300 nM) and bremazocine (3 nM). These results indicate that NTB, depending on the dosage, could acts not only as an antagonist at delta but also as a noncompetitive antagonist for mu receptors, and as an agonist for kappa2 receptors in rat cerebral cortex.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.     

GABAB Receptor-Mediated Inhibition of Histamine H1-Receptor-Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex

Histamine-stimulated accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated slices of rat cerebral cortex was inhibited by gamma-aminobutyric acid (GABA) (IC50 0.30 +/- 0.03 mM). The maximum level of inhibition was 69 +/- 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAB agonist (-)-baclofen, IC50 0.69 +/- 0.04 microM, which was 430-fold more potent as an inhibitor than the (+)-isomer. (-)-Baclofen also inhibited histamine-induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H] inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (-)-and and (+)-baclofen had Hill coefficients greater than unity. (-)-Baclofen, at concentrations that caused inhibition of histamine-induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine-stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol-stimulated [3H]IP1 formation.     

Modulation of Phosphoinositide Hydrolysis by NaF and Aluminum in Rat Cortical Slices

NaF stimulated phosphoinositide hydrolysis in rat cortical slices. The production of [3H]inositol monophosphate was rapid for the first 15 min of incubation with NaF, followed by a plateau. The major product detected was [3H]inositol monophosphate, although significant amounts of [3H]inositol bisphosphate and [3H]inositol trisphosphate were also produced. The stimulation of [3H]inositol monophosphate production by NaF was concentration dependent between 2 and 20 mM NaF. Addition of 10 or 100 microM AlCl3 or aluminum maltol did not alter the effect of NaF, whereas at 500 microM, these aluminum preparations resulted in significant inhibition. Increasing the concentration of K+ from 5 to 20 mM potentiated [3H]inositol monophosphate production induced by carbachol but not by NaF. Incubation with 1 microM phorbol 12-myristate 13-acetate, a phorbol ester, inhibited carbachol-induced, but not NaF-induced, [3H]inositol monophosphate production. These results further support the hypothesis that a guanine nucleotide binding protein that can be activated by NaF is involved in phosphoinositide hydrolysis in brain. The use of NaF provides a means to bypass receptors to study intracellular regulatory sites of phosphoinositide metabolism without disrupting cells.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

Effects of Antidepressant Drug Treatments on α2-Adrenoceptor Control of [3H]Noradrenaline Release from Hypothalamic Synaptosomes

KCl (16 mM) stimulated the release of [3H]noradrenaline ([3H]NA) from rat hypothalamic synaptosomes in a Ca2+-dependent manner; this release was attenuated by clonidine (0.01-100 microM). Changes in the release of [3H]NA and the functional status of alpha 2-adrenoceptors in the medial hypothalamus of rats treated acutely and chronically with clorgyline (1 mg/kg/day) or desipramine (DMI, 10 mg/kg/day) were assessed using superfused synaptosomes in which the attenuating effects of clonidine (1 microM) or the potentiating effects of yohimbine (1 microM) on K+-evoked release of [3H]NA were measured. After acute administration of DMI, significantly less [3H]NA was accumulated into synaptosomes. Although total (spontaneous + K+-evoked) [3H]NA release from these synaptosomes was unchanged, a significant reduction was apparent in the K+-evoked release from the DMI-treated tissue. Attenuation of K+-evoked release by clonidine was abolished in both these acute treatment groups. Following the chronic antidepressant drug regimens, [3H]NA uptake into DMI-treated tissue remained significantly reduced although total percent and K+-evoked [3H]NA release were unchanged. The K+-evoked release of [3H]NA in S1 was significantly enhanced (by 22%) in the clorgyline treatment group. Attenuation of K+-evoked [3H]NA release by clonidine in both chronic antidepressant-treated tissues was not significantly changed. It is concluded that the functional sensitivity of alpha 2-adrenoceptors on nerve endings in the medial hypothalamus is unchanged by these chronic antidepressant drug regimens. In synaptosomes from untreated tissue, yohimbine significantly potentiated K+-evoked release of [3H]NA; this effect was unchanged after acute regimens and reduced after chronic administration of both the antidepressants.(ABSTRACT TRUNCATED AT 250 WORDS)     

4-Aminopyridine Stimulates B-50 (GAP43) Phosphorylation and [3H]Noradrenaline Release in Rat Hippocampal Slices

In situ phosphorylation of the presynaptic protein kinase C substrate B-50 was investigated in rat hippocampal slices incubated with the convulsant drug 4-aminopyridine (4-AP). Phosphorylation of B-50 was significantly enhanced 1 min after the addition of 4-AP (100 microM). This increase by 4-AP was concentration dependent (estimated EC50 30-50 microM). Concomitant with the changes in B-50 phosphorylation, 4-AP also dose-dependently stimulated [3H]noradrenaline [( 3H]NA) release from the slices. 4-AP stimulated [3H]NA release within 5 min to seven times the control level. The B-50 phosphorylation induced by 4-AP remained elevated after removal of the convulsant, this is contrast to B-50 phosphorylation induced by depolarization with K+. A similar persistent increase was observed for [3H]NA release after a 5-min incubation period with 4-AP. These results give more insight into the molecular mechanisms underlying 4-AP-induced epileptogenesis and provide further evidence for the correlation between B-50 phosphorylation and neurotransmitter release in the hippocampal slice.     

Involvement of a pertussis toxin-sensitive G protein in the action of gastrin on gastric parietal cells

The mechanism whereby gastrin triggers phosphoinositide breakdown was investigated in an enriched preparation of isolated rabbit parietal cells (approx. 75%). In a permeabilized preparation of myo-[3H]inositol-labelled cells, GTP[S], a non-hydrolysable GTP analogue, enhanced [3H]inositol trisphosphate ([3H]InsP3 accumulation in a dose-dependent manner; submaximal concentrations of GTP[S] (less than 10 microM), potentiated gastrin-induced [3H]InsP3 release; preincubation for 5 min with GDP[S], a non-hydrolysable GDP analogue, dose-dependently reduced [3H]InsP3 accumulation stimulated by gastrin even in presence of GTP[S]. Exposure of intact parietal cells for 3 h to pertussis toxin (PTx) (200 ng/ml) led to a 15-50% reduction in gastrin-induced [14C]aminopyrine [(14C]AP) uptake (an index of in vitro acid secretion) and [3H]inositol phosphate ([3H]InsP) accumulation. A decrease in the accumulation of the different [3H]inositol phosphate occurred in gastrin-stimulated parietal cells treated with PTx. A rightward shift of gastrin dose-response curves in the presence of PTx was observed for [14C]AP uptake (EC50 values: 0.125 +/- 0.045 nM without PTx and 1.05 +/- 0.63 nM with PTx), for [3H]InsP accumulation (EC50 values: 0.16 +/- 0.08 nM without PTx and 1.56 +/- 0.58 nM with PTx) and [125I]gastrin binding (IC50 values: 0.247 +/- 0.03 nM without PTx and 2.38 +/- 0.56 nM with PTx). In contrast, cholera toxin (CTx) treatment (100 ng/ml) for 3 h was without effect on gastrin-induced [3H]InsP accumulation. CTx induced a pronounced potentiation of gastrin-stimulated [14C]AP uptake; this effect can be mimicked by IBMX (a phosphodiesterase inhibitor) and by forskolin (an activator of adenylyl cyclase). We conclude that: (i) one or more than one G protein appeared to be involved in gastrin receptor coupling to phospholipase C (PL-C); (ii) these G proteins are not substrates for CTx; (iii) one of these appeared to be a PTx-sensitive 'Gi-like' protein which could be involved in hormone-induced acid secretion, (iiii) the potentiating effect of CTx observed on AP uptake stimulated by gastrin suggests the existence of a cooperative effect between cAMP pathway (CTx) and the gastrin-induced phosphoinositide breakdown in acid secretory activity of parietal cells.     

Studies on the Role of α2-Adrenoceptors in the Control of Synaptosomal [3H]5-Hydroxytryptamine Release: Effects of Antidepressant Drugs

The sensitivity of alpha 2-adrenoceptors on 5-hydroxytryptamine (5-HT) nerve endings obtained from rat cerebral cortex was investigated following treatment with the antidepressant drugs desipramine (10 mg/kg/day for 21-28 days) or clorgyline (1 mg/kg/day for 21-28 days). [3H]5-HT (100 nM) was used to load cortical synaptosomes (P2) after experiments with uptake inhibitors confirmed that this concentration of amine ensured exclusive uptake into 5-HT nerve terminals. The sensitivity of K+-stimulated release of [3H]5-HT to alpha 2-adrenoceptor occupancy was assessed in a superfusion system by means of the dose-dependent inhibition of [3H]5-HT release by clonidine. This is blocked by yohimbine (1 microM), which, when administered alone, enhances release, suggesting that endogenous catecholamines released from other synaptosomes act on these alpha 2-heteroreceptors. The effect of addition of citalopram (1 microM) to superfusates suggests that some reuptake of [3H]5-HT occurs during superfusion. Of the tritium released into superfusates during "background" and K+-stimulated release, 17 and 90%, respectively is [3H]5-HT. The attenuation of K+-stimulated release by clonidine is apparently diminished by the chronic clorgyline regimen but not by desipramine. However, clorgyline elevates catecholamine levels, and this might increase endogenous noradrenaline (NA) efflux, which by competition with clonidine could appear to alter alpha 2-adrenoceptor sensitivity. This possibility was investigated by depleting NA with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). These studies showed that the apparent effect of chronic clorgyline on alpha 2-adrenoceptor sensitivity to clonidine was due to competition with increased levels of endogenous NA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential presynaptic effects of hexachlorocyclohexane isomers on noradrenaline release in cerebral cortex.

To investigate presynaptic effects of hexachlorocyclohexane (HCH) isomers, the release of noradrenaline (NA) in brain tissue was analyzed using rat cerebral cortical slices preloaded with [3H]-NA. gamma-HCH (lindane) 50 microM significantly enhanced the [3H]-NA release evoked by 15-25 mM K+. alpha- and beta-HCH (50 microM) did not produce any significant effect on K(+)-evoked [3H]-NA release. delta-HCH (50 microM) induced a significant decrease of the 25 mM K(+)-evoked release of [3H]-NA. The effect of the gamma- and delta-HCH isomers on the presynaptic action of the alpha 2-agonist clonidine and the alpha 2-antagonist yohimbine was also studied. The presynaptic inhibitory effect of clonidine and the stimulatory effect of yohimbine on [3H]-NA release was attenuated by lindane and delta-HCH, respectively. These results are consistent with a presynaptic action of the HCH isomers on noradrenergic release processes.     

Neomycin inhibits K+- and veratridine-stimulated noradrenaline release in rat brain slices and rat brain synaptosomes

The possible involvement of phosphoinositides' turnover in the process of neurotransmitter release in the central nervous system (CNS) was studied using rat brain slices and synaptosomes. A depolarizing concentration of potassium chloride (25 mM) induces an 8.6 +/- 0.4% increase of [3H]noradrenaline [( 3H]NA) fractional release in cerebral cortical slices above spontaneous release, and 15 mM KCl induces a 3-fold increase of [3H]NA release in rat brain synaptosomes. Neomycin, an aminoglycoside which binds phosphoinositides, inhibits the potassium-induced release in cortical slices with an IC50 = 0.5 +/- 0.07 mM and with IC50 = 0.2 +/- 0.03 mM in synaptosomes. Veratridine, a veratrum alkaloid which increases membrane permeability to sodium ions and causes depolarization of neuronal cells, induces a net 13.4 +/- 0.3% increase of [3H]NA fractional release above spontaneous release in cortical slices. In analogy to K+ stimulation, neomycin inhibits the veratridine-stimulated release in cortical slices with an IC50 = 0.65 +/- 0.1 mM. It appears that the recycling of phosphoinositides, which is necessary for Ca2+ mobilization, participates in the Ca2+-dependent induced neurotransmitter release in the central nervous system.     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.     

Potentiation by γ-Aminobutyric Acid of α1-Agonist-Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex

Noradrenaline-induced accumulation of 3H-labeled inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium-treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by gamma-aminobutyric acid (GABA). However, the effect on [3H]IP2 accumulation was much greater than that on [3H]IP1 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration-response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of greater than 1 mM. Prazosin (1 microM) blocked the noradrenaline-induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mM GABA. 3H-IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 microM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3-aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (-)-Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline-induced 3H-IP formation was still apparent in the absence of Li+, but the increase of [3H]IP2 content was less than that of [3H]IP1 content.