Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N-ethylmaleimide-sensitive G-protein,but not to adenylate cyclase or dihydropyridine-sensitive Ca2+-channeIs

Electrically evoked [³H]acetylcholine ([³H]ACh) release from slices of the rat hippocampus was reduced in a dose-dependent manner by the adenosine A,-receptor agonist R-phenylisopropyladenosine (R-PIA) in the concentration range 0.1–10 μM. The maximal effect was observed with I μM R-PIA. Treatment with N-ethylmaleimide (NEM, 100 μ M, 10 min), which inactivates nucleotide-binding proteins (G-proteins), caused a slight increase in the basal overflow (0.17 ± 0.01%v. 0.10 ± 0.003% in the control slices), but did not affect stimulated release (0.73 ± 0.05%vs. 0.74 ± 0.03% in the control slices). N-ethylmaleimide pretreatment significantly reduced the prejunctional inhibitory effect of R-PIA on [³H]ACh release in a non-competitive manner. The S₂/S₁ ratio was 0.92 ± 0.03 in controls and was reduced to 0.32 ± 0.02 by I μ Mm R-PIA in the control slices and to 0.57 ± 0.03 after NEM pretreatment. Stimulation of cyclic AMP-accumulation by forskolin (I μ M) and rolipram (30 μ M) before the second stimulation (S₂) enhanced the S₂/S₁ ratio by about 30% to 1.26 ± 0.12, but did not reduce the inhibitory effect of R-PIA (I μ MM). The Ca²⁺-channel agonist Bay K 8644 (I μ MM), a concentration that increases K⁺-evoked noradrenaline release, did not affect the basal or electrically evoked [³H]ACh overflow, or the prejunctional effects of R-PIA (0.1 and I μ MM) on [³H]ACh release. Our results suggest that the presynaptic inhibitory effects of A₁-receptor agonists on [³H]ACh release are exerted via a nucleotide-binding protein that can be inhibited by NEM. However, the inhibitory effect is apparently not caused by a change in adenylate cyclase activity or by affecting dihydropyridine-sensitive Ca²⁺-channels.     

Effects of N-ethylmaleimide and forskolin on noradrenaline release from rat hippocampal slices. Evidence that prejunctional adenosine and alpha-receptors are linked to N-proteins but not to adenylate cyclase

N-ethylmaleimide (NEM) treatment has been shown to inactivate regulatory GTP-binding N (G)-proteins in many preparations, including slices of rat hippocampus. NEM-treatment (100 microM for 15 min) has been used to examine the possible involvement of a N-protein in the prejunctional inhibitory effect of an adenosine analogue, R-PIA acting on A1-receptors, and of clonidine acting on alpha 2-adrenoceptors in this tissue. NEM treatment significantly enhanced basal overflow of [3H]NA and the overflow stimulated by low (0.3 Hz) frequency stimulation, but not the overflow stimulated by higher (1-10 Hz) frequency stimulation. The prejunctional inhibitory effect of R-PIA (1 microM) on NA release, stimulated by a 3 Hz stimulation, was abolished by NEM pretreatment, which also eliminated the dose-dependent prejunctional effect of clonidine and reduced the facilitatory effect of yohimbine. Forskolin had a small, but significant stimulatory effect on NA overflow, but did not reduce the prejunctional inhibitory effect of R-PIA. The adenylate cyclase inhibitor SQ 22, 536 did not reduce NA overflow. These results show that NEM blocks a critical step in the prejunctional action of both adenosine- and alpha 2-receptor agonists, which may be a N-protein. The possibility is discussed that the prejunctional A1- and alpha 2-receptors couple to a N-protein that controls a different effector than adenylate cyclase.     

Effects of N-ethylmaleimide and forskolin on noradrenaline release from rat hippocampal slices. Evidence that prejunctional adenosine and a-receptors are linked to A/-proteins but not to adenylate cyclase

N-ethylmaleimide (NEM) treatment has been shown to inactivate regulatory GTP-binding N (G)-proteins in many preparations, including slices of rat hippocampus. NEM-treatment (100 μM for 15 min) has been used to examine the possible involvement of a N-protein in the prejunctional inhibitory effect of an adenosine analogue, R-PIA acting on A,-receptors, and of clonidine acting on a,-adrenoceptors in this tissue. NEM treatment significantly enhanced basal overflow of [³H]NA and the overflow stimulated by low (0.3 Hz) frequency stimulation, but not the overflow stimulated by higher (1–10 Hz) frequency stimulation. The prejunctional inhibitory effect of R-PIA (I μM) on NA release, stimulated by a 3 Hz stimulation, was abolished by NEM pretreatment, which also eliminated the dosedependent prejunctional effect of clonidine and reduced the facilitatory effect of yohimbine. Forskolin had a small, but significant stimulatory effect on NA overflow, but did not reduce the prejunctional inhibitory effect of R-PIA. The adenylate cyclase inhibitor SQ 22, 536 did not reduce NA overflow. These results show that NEM blocks a critical step in the prejunctional action of both adenosine-and a,-receptor agonists, which may be a N-protein. The possibility is discussed that the prejunctional A,- and a,-receptors couple to aN-protein that controls a different effector than adenylate cyclase.     

Adenosine A1-receptor-mediated inhibition of evoked acetylcholine release in the rat hippocampus does not depend on protein kinase C

The possible involvement of protein kinase C and/or a lipoxygenase product in the mechanism by which adenosine inhibits release of [3H]acetylcholine evoked by electrical pulses from [3H]choline-labelled hippocampal slices was examined. For comparison, the muscarinic autoreceptors were examined using carbachol. The order of potency of adenosine analogues (CHA = R-PIA greater than NECA much greater than CGS 21680, CV 1808) indicates that the adenosine receptor responsible is of the A1 subtype. Adenosine (10 microM) and R-PIA (0.1 microM) were virtually equiactive as inhibitors and were antagonized to an equal extent by 8-CPT with a potency (IC50 approximately 25 nM) which is also compatible with A1-receptor mediation. The effects of carbachol and of R-PIA were not antagonized by the lipoxygenase inhibitor NDGA (10 or 50 microM). Stimulation of protein kinase C by the phorbol ester 4 beta-phorbol 12,13-dibutyrate caused a concentration-dependent increase in stimulation-evoked 3H overflow, but did not antagonize the presynaptic inhibitory effect of R-PIA or carbachol (0.01-1 microM). Staurosporine (0.1 microM), which inhibited the stimulating effect of phorbol dibutyrate, did not alter the effects of carbachol or R-PIA. The presynaptic effects of phorbol dibutyrate, R-PIA and adenosine were reduced by pretreatment with N-ethylmaleimide (100 microM for 10 min), which inactivates G-proteins. The evoked transmitter release was unaffected by nifedipine (1 microM) in the presence and in the absence of phorbol dibutyrate. These results indicate that adenosine, by acting at presynaptic A1-receptors, reduces transmitter release by a mechanism that involves neither an NDGA-sensitive lipoxygenase nor protein kinase C. The results also indicate that the enhancement of transmitter release by phorbol esters is due to protein kinase C activation and that a G-protein may be involved in the effect but a dihydropyridine-sensitive L-type Ca2+ channel probably is not.     

Effects of N-ethylmaleimide and forskolin on glutamate release from rat hippocampal slices. Evidence that prejunctional adenosine receptors are linked to N-proteins,but not to adenylate cyclase

In the present experiments we have examined the effect of N-ethylmaleimide (NEM) on the release of [³H] glutamate from rat hippocampal slices. Pretreatment of slices with NEM in a concentration between 50 μM and 200 μM, can inhibit the GTP-binding protein (N) that transmitts receptor signals into inhibitions of adenylate cyclase, without affecting the N_s-protein, that transmits signals into stimulation of the cyclase, or the cyclase. The adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, I μM) caused an approximately 50% inhibition of the evoked [³H] glutamate release. This effect was completely prevented by NEM treatment, which did not affect basal or stimulated release of the amino acid. By contrast, the effect of R-PIA was unaffected by adding an adenylate cyclase stimulator (forskolin 1 μM) and a phosphodiesterase inhibitor (rolipram, ZK 62.711, 30 μM) which raised the cyclic AMP content of the slices approximately 1 o-fold. In conclusion, these results suggest that the adenosine receptor that mediates prejunctional inhibition of glutamate release is coupled to a protein similar to the N₁-protein, but that another effector than adenylate cylase is involved.,     

Effects of oxiracetam on neurotransmitter release from rat hippocampus slices and synaptosomes.

The effects of the nootropic drug oxiracetam on the K(+)-evoked overflow of [3H]D-aspartic acid ([3H]D-ASP), [3H]acetylcholine ([3H]ACh), [3H] gamma-aminobutyric acid ([3H]GABA), [3H]noradrenaline ([3H]NA) and [3H]5-hydroxytryptamine ([3H]5-HT) have been studied in superfused rat hippocampal slices. The overflow of [3H]D-ASP was enhanced by low concentrations of oxiracetam (0.01-1 microM) but not by high concentrations (10-100 microM) which showed some tendency to inhibit it. Similarly, low concentrations of oxiracetam increased, although less effectively, the depolarization-evoked overflow of [3H]ACh, whereas higher concentrations were without effect. At the concentrations active on [3H]D-ASP and [3H]ACh overflow oxiracetam did not affect that of [3H]GABA, [3H]NA or [3H]5-HT. The oxiracetam effects present in slices could not be observed in hippocampal synaptosomes. Thus oxiracetam may selectively increase the release of glutamate and acetylcholine in hippocampus by a mechanism which appears not to be sited in the releasing nerve terminals.     

Alpha 2-adrenoceptor agonist-mediated inhibition of [3H]noradrenaline release from rat hippocampus is reduced by 4-aminopyridine, but that caused by an adenosine analogue or omega-conotoxin is not

The inhibitory effect of an adenosine analogue, R-PIA, and an alpha 2-adrenoceptor agonist, UK 14,304, on [3H]NA efflux from field-stimulated rat hippocampal slices was examined. The effect of 0.1 microM UK 14,304 was mimicked by 30 nM omega-conotoxin and by 10 microM cadmium chloride, inhibitors of N- and L-type Ca2+ channels. R-PIA (1 microM) had no effect per se, but caused a clear-cut inhibition after blockade of the pre-synaptic alpha 2-receptor by yohimbine. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release. At 30 microM 4-AP did not affect the actions of omega-conotoxin or cadmium chloride. The pre-synaptic effect of R-PIA was similarly unaffected by 30 microM 4-AP. The pre-synaptic effect of UK 14,304 was virtually abolished by 4-AP (30 microM). The effect of UK 14,304 (0.1 microM) could be partly restored by reducing the Ca2+ concentration during treatment with 4-AP (22% inhibition compared to 42% with normal Ca2+). The magnitude of increase in evoked [3H]NA efflux by yohimbine (1 microM) was decreased by 4-AP in a concentration-dependent manner from 142% increase in controls to 21% at 100 microM 4-AP. The present results indicate that NA release is reduced by somewhat different mechanisms by pre-synaptic alpha 2- and adenosine A1-receptors. Furthermore, the results indicate that pre-synaptic A1-receptors on hippocampal NA neurons do not primarily regulate 4-AP-dependent potassium channels, but they might act directly on a Ca2+ conductance.     

Quantitative evaluation of alpha- and beta-adrenoceptor modulation of [3H]choline release in guinea pig superior cervical ganglia

[3H] Overflow evoked by 5 min supramaximal preganglionic stimulation at 1 pps has been studied in isolated guinea pig superior cervical ganglion preparations preincubated with [3H]choline. At 15 microM norepinephrine (NE) reduced both the [3H]choline overflow and endogenous acetylcholine release by 59.4 and 54.1% respectively; the dose-response curve for NE inhibitory action is described. Evidence is given that endogenous catecholamines effectively reduce ACh release from the ganglia. After blocking the inhibitory action of endogenous NE, a significant beta-adrenoceptor-mediated facilitatory effect on ACh release could be observed. Preincubation of the ganglia with different combinations of alpha 1 and alpha 2 agonists (phenylephrine, 10 microM and clonidine, 1 microM respectively) and antagonists (prazosin, 10 microM and yohimbine, 3 microM) showed that the adrenoceptors involved in alpha-mediated NE inhibition of ACh output are exclusively of the alpha 2-type.     

Autoinhibition of histamine synthesis mediated by presynaptic H3-receptors

The regulation of histamine synthesis was studied on rat brain slices or synaptosomes labeled with L-[3H]histidine. Depolarization by increased extracellular K+ concentration enhanced by about twofold the [3H]histamine formation in slices of cerebral cortex. This stimulation was also observed, although to a lesser extent, in synaptosomes from cerebral cortex and slices from the posterior hypothalamus where most histaminergic cell-bodies are located, suggesting that it may occur in nerve endings as well as in perikarya. In the presence of exogenous histamine in increasing concentrations the K+-induced stimulation was progressively reduced by up to 60-70%. The effect of exogenous histamine appears to be receptor-mediated as shown by its saturable character, high pharmacological specificity and competitive reversal by histamine antagonists. The EC50 value of histamine for synthesis reduction (0.34 +/- 0.03 microM) was similar to its EC50 value for release inhibition known to be mediated by H3-receptors. In addition, whereas mepyramine and tiotidine, two potent antagonists at H1- and H2-receptors, respectively, were poorly effective, the H3-receptor antagonists burimamide and impromidine reversed the histamine effect in an apparently competitive manner. These effects were observed in slices of cerebral cortex or posterior hypothalamus as well as in cortical synaptosomes. Furthermore, even in the absence of added histamine, H3-receptor antagonists enhanced the depolarization-induced stimulation of [3H]histamine synthesis, indicating a participation of released endogenous histamine in the synthesis control process. The potencies of H3-receptor antagonists were similar to those of these agents at presynaptic autoreceptors controlling [3H]histamine release. It is concluded that H3-receptors control not only release but also synthesis of histamine at the level of nerve endings and also, presumably, of perikarya. A relationship between the two regulatory processes, possibly via intracellular calcium, seems likely but remains to be investigated at the molecular level.     

Suppressing effect of pertussis toxin on clonidine-induced inhibition of noradrenaline release from cerebral cortical slices of rats

Clonidine at 1.0 microM significantly decreased 20 mM K+-evoked release of L-[3H]noradrenaline (NA) from rat cerebral cortical slices preloaded with L-[3H]NA. Inhibitory effects of clonidine, however, were not observed in slices pretreated with 20 micrograms/ml pertussis toxin, an islet-activating protein, together with NAD and adenosine triphosphate. It is suggested that the inhibitory guanine nucleotide-binding protein (Ni) could be involved in alpha 2-adrenoceptor-mediated inhibition of NA release from nerve terminals in the central nervous system.     

Specific inhibitory effect of amyloid-β on presynaptic muscarinic receptor subtypes modulating neurotransmitter release in the rat nucleus accumbens

In this study we investigate on the effect of amyloid-beta1-40 (Aβ1-40) on the oxotremorine (OXO)-induced release of [³H] dopamine (DA), [³H]GABA and [³H]acetylcholine (ACh) from synaptosomes in the rat nucleus accumbens (NAc). OXO in presence of himbacine (HIMBA) was able to increase the basal release of [³H]GABA. The OXO-elicited [³H]GABA overflow was significantly antagonized by atropine (A; 94%), by the M3 antagonists DAU5884 (96%) and 4-DAMP (70%), and by Aβ1-40 (65%). Exposure of NAc synaptosomes to OXO produced a dose-dependent increase of [³H]DA overflow which was antagonized by A, partially inhibited by Aβ1-40 (100 nM) but unaffected by DAU5884 and 4-DAMP. The K⁺-evoked [³H]ACh overflow was inhibited by OXO. This effect was counteracted by the M2 antagonist AFDX-116 but not by the selective M4 antagonist mamba toxin 3 (MT3). The K⁺-evoked [³H]GABA overflow was also inhibited by OXO but conversely, this effect was counteracted by MT3 and not by AFDX-116. Aβ1-40 (100 nM) did not modify the inhibitory effect of OXO both on the K⁺-evoked [³H]ACh and [³H]GABA overflow. The results show that in the rat NAc, Aβ1-40 selectively inhibits the function of the muscarinic subtypes which stimulate neurotransmitter release and not those which modulate negatively the stimulated release.     

Insulin-like growth factor 1 stimulates the release of acetylcholine from rat cortical slices

The effect of somatomedin, or insulin-like growth factors (IGF-1 and IGF-2), on the basal and potassium induced release of [3H]acetylcholine ([3H]Ach) from rat cortical slices, previously preincubated with [3H]choline ([3H]Ch), was studied in vitro. IGF-1 (1.4 x 10(-9) to 1.4 x 10(-8) M) had no effect on the basal release of [3H]ACh, while IGF-1 (1.4 x 10(-9) to 4.3 x 10(-8) M) increased the potassium induced release of [3H]ACh from rat brain slices in a concentration-dependent manner. However IGF-2 (1.4 x 10(-8) M) had no effect. Insulin (1.8 x 10(-8) to 5.3 x 10(-8) M), similarly, did not have any influence on the release of [3H]ACh, demonstrating that the facilitatory effect of IGF-1 on [3H]ACh release is not mediated via insulin receptors. This report demonstrates for the first time that IGF-1 has an effect on neurotransmission in the adult brain.     

Histamine H3 receptor activation inhibits dopamine D1 receptor-induced cAMP accumulation in rat striatal slices

In striatal membranes bearing significant levels of histamine H3 receptors (72 +/- 14 fmol/mg protein), the H3 agonist immepip (1 microM) increased [35S]GTPgammaS binding to 119 +/- 2% of basal, an effect prevented by the H3 antagonist clobenpropit and by pre-treatment with pertussis toxin. In slices labelled with [3H]adenine and in the presence of 1 mM isobutylmethylxantine (IBMX), the selective dopamine D1-like (D1/D5) receptor agonist SKF-81297 stimulated cyclic [3H]AMP ([3H]cAMP) accumulation (maximal stimulation 205 +/- 24% of basal, EC50 113 +/- 12 nM), an effect fully blocked by the D1/D5 antagonist SCH-23390. The accumulation of [3H]cAMP induced by 1 microM SKF-81297 was inhibited in a concentration-dependent manner by the selective H3 receptor agonist immepip (maximal inhibition 60+/-5%, IC50 13 +/- 5 nM). The inhibitory action of 100 nM immepip was reversed in a concentration-dependent manner by the H3 antagonist thioperamide (EC50 13 +/- 3 nM, Ki 1.4 +/- 0.3 nM). Forskolin-induced [3H]cAMP accumulation (726 +/- 57% of basal) was also reduced by H3 receptor activation, although to a lesser extent (19.1 +/- 3.2% inhibition), an action not affected by the absence of either IBMX or Ca2+ ions in the incubation medium. Neither the density of [3H]SCH-23390 binding sites (D1 receptors) nor the inhibition by SKF-81297 were affected by 1 microM immepip, ruling out a direct interaction between D1 and H3 receptors. These results indicate that through H3 receptors coupled to Galphai/o proteins, histamine modulates cAMP formation in striatal neurones that possess D1 receptors, most probably GABAergic striato-nigral neurones.     

Muscarinic receptors mediate direct inhibition of GABA release from rat striatal nerve terminals

The effects of acetylcholine (ACh) on the depolarization-evoked release of [3H]gamma-aminobutyric acid ([3H]GABA) have been investigated using synaptosomes prepared from rat corpus striatum and depolarized by superfusion with 9 mM KCl. Acetylcholine inhibited the [3H]GABA overflow in a concentration-dependent manner. The maximal effect was about 50%. The IC50 value (concentration producing half-maximal effect) amounted to 1 microM, in the absence of acetylcholinesterase inhibitors. The effect of ACh on the K(+)-evoked [3H]GABA release was counteracted by the muscarinic receptor antagonist atropine, but not by the nicotinic receptor antagonist mecamylamine or by the selective M1 antagonist pirenzepine. The data show that muscarinic receptors with low affinity for pirenzepine are localized on GABAergic nerve endings in rat corpus striatum where they may directly inhibit the release of GABA.     

Muscarinic receptor activation attenuates D2 dopamine receptor mediated inhibition of acetylcholine release in rat striatum: indications for a common signal transduction pathway

In the present investigations, we used a superfusion system to study the effect of simultaneous activation of D2 dopamine receptors and so-called muscarinic "autoreceptors" on the K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. Activation of D2 receptors with the selective agonist LY 171555 (0.01-1 microM) clearly decreased the evoked release of [3H]acetylcholine. This effect was markedly attenuated in the presence of either the selective muscarinic receptor agonist oxotremorine (3 microM) or the cholinesterase inhibitor physostigmine (1 microM). Conversely, D2 receptor activation with LY 171555 (1 microM) completely abolished the muscarinic receptor mediated inhibition of evoked [3H]acetylcholine release induced by oxotremorine (0.03-10 microM). These results show that the inhibitory effects of D2 dopamine receptor and muscarinic receptor activation on striatal acetylcholine release are non-additive and therefore are interdependent processes. In addition, we investigated some aspects of the signal transduction mechanism by which the muscarinic receptor mediates inhibition of K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. It appeared that the effect of muscarinic receptor activation was not significantly influenced either by a lowering of the extracellular Ca2+ concentration from the usual 1.2-0.12 mM or by an increase of the intracellular cyclic adenosine-3',5'-monophosphate content. However, increasing extracellular K+ strongly decreased the inhibition of evoked [3H]acetylcholine release mediated by activation of muscarinic receptors. This set of results indicates that the muscarinic "autoreceptor" mediates the decrease of depolarization induced [3H]acetylcholine release from rat striatum to a large extent through stimulation of K+ efflux (opening of K+ channels) in a cyclic adenosine-3',5'-monophosphate independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine modulation of neuroeffector transmission in guinea-pig uterine smooth muscle.

Effects of adenosine and adenosine analogues on neuroeffector transmission in separate layers of the guinea-pig uterine smooth muscle during different phases of the oestrus cycle were studied. Adenosine (ADO), N6-[(R)-1-methyl-2-phenylethyl]- adenosine (R-PIA) and 5'-N-ethylcarboxamideadenosine (NECA) enhanced spontaneous contractile activity as well as contractile responses to nerve stimulation or direct muscle stimulation in the longitudinal and circular muscle layers both at the time of ovulation and implantation. The agonist potency order was R-PIA greater than or equal to NECA greater than ADO. Furthermore, in the circular muscle layer inhibitory effects were seen at time of implantation and the agonist potency for this inhibitory effect was NECA greater than R-PIA greater than or equal to ADO. 8-p-sulphophenyltheophylline (8-PSOT) antagonized both the stimulatory and inhibitory effects of the purines and 8-p-sulphophenyltheophylline applied to untreated preparations inhibited nerve-induced contractile activity. NECA inhibited stimulation-induced release of 3H in preparations incubated with [3H]noradrenaline. We suggest that adenosine and its analogues can modulate adrenergic neuroeffector transmission in guinea-pig uterine smooth muscle via action at postjunctional receptors stimulating contractile activity and prejunctional receptors inhibiting transmitter release. In the circular muscle layer postjunctional receptors inhibiting contractile activity were evident at time of implantation. Furthermore, endogenous purines exerting a stimulatory action on the neuroeffector transmission were likely formed during the present experimental conditions.     

Inactivation of presynaptic 5-HT autoreceptors by lithium in rat hippocampus

The effect of lithium ion on the electrically stimulated 5-[3H]hydroxytryptamine (5-HT) release from the rat hippocampal slices preloaded with [3H]5-HT was studied. Electrically stimulated [3H]5-HT release decreased when the slices were exposed to 5-HT in a concentration-dependent manner. Lithium (2.5 mM) did not affect [3H]5-HT release when added alone to the superfusion medium. However, the inhibitory effect of 5-HT (1 microM) on [3H]5-HT release was abolished by lithium. The results suggest that lithium may inhibit the regulation of 5-HT release via presynaptic 5-HT autoreceptors in rat hippocampus.     

Cyclothiazide binding to the GABAA receptor

In order to explore the molecular interaction between cyclothiazide (CTZ) and gamma-aminobutyric acidA (GABAA) receptors, possibly underlying inhibition of GABAA receptor currents, [3H]-CTZ was synthesized. Binding of [3H]-CTZ to rat brain synaptic membranes could be observed only in the presence of the GABAA receptor antagonist (-)[1S,9R]-bicuculline methiodide (BMI) (EC(50,BMI)=500+/-80microM). GABA decreased [(3)H]-CTZ binding induced by the presence 300microM and 3mM BMI with IC(50,GABA) values of 300+/-50microM and 5.0+/-0.7mM, respectively. Binding of CTZ to [3H]-CTZ labeled sites was characterized by IC(50,CTZ) values of 0.16+/-0.03muM ([BMI]=300microM) and 7.0+/-0.5microM ([BMI]=3mM). Binding of the diastereomeric fraction [3H]-(3R,1'S,4'S,5'R+3S,1'R,4'R,5'S)-CTZ induced by 3mM BMI was quantitatively the more significant in cerebrocortical and hippocampal membranes. It was characterized by IC(50,CTZ)=80+/-15nM and IC(50,GABA)=13+/-3mcapital EM, Cyrillic. In the absence of BMI, CTZ (1mM) significantly decreased GABA-induced enhancement of [3H]-flunitrazepam binding. Our findings suggest that functional inhibition may occur through binding of CTZ to an allosteric site of GABAA receptors. This allosteric site is possibly emerged in the receptor conformation, stabilized by BMI binding.     

Nicotinic antagonist-produced frequency-dependent changes in acetylcholine release from rat motor nerve terminals.

1. The frequency (0.5-150 Hz) and calcium dependence (0.5-2.0 mM) of the effects of the nicotinic antagonist tubocurarine (0.2 microM) on acetylcholine (ACh) liberation from motor nerve terminals has been examined using binomial analysis of quantal transmitter release. 2. At an extracellular calcium ion concentration ([Ca2+]o) of 2.0 mM, tubocurarine produced a decrease in the endplate current (EPC) quantal content of approximately 30% at high frequencies of motor nerve stimulation (50-150 Hz). In contrast, at low frequencies of stimulation (0.5-1.0 Hz), tubocurarine enhanced the EPC quantal content by approximately 20%. 3. The enhancement of EPC quantal content produced by tubocurarine at low frequencies of motor nerve stimulation was [Ca2+]o dependent, being abolished when [Ca2+]o was lowered from 2.0 to 0.5 mM. In contrast, the decrease in quantal content produced by tubocurarine at high frequencies of motor nerve stimulation was independent of [Ca2+]o, being approximately 30% at all calcium ion concentrations studied. 4. In direct contrast to tubocurarine, the nicotinic antagonist vecuronium (1.0 microM) produced no increase in EPC quantal content at low frequencies of nerve stimulation. However, at high frequencies of nerve stimulation it decreased EPC quantal content to a similar extent to 0.2 microM tubocurarine. The frequency-dependent decrease in EPC quantal content produced by 1.0 microM vecuronium in 2.0 mM [Ca2+]o was very similar to that seen with 0.2 microM tubocurarine in 0.5 mM [Ca2+]o. 5. Binomial analysis revealed that all the changes in EPC quantal content associated with both nicotinic antagonists were due to changes in the size of the pool of quanta in the nerve terminal available for immediate release with no effect on the probability of release of an individual quantum. 6. The results are interpreted in terms of two separately identifiable prejunctional actions of the nicotinic antagonists, both involving an action at nicotinic ACh receptors situated on the motor nerve terminal. Thus, at high frequencies of motor nerve stimulation tubocurarine and vecuronium produce a [Ca2+]o-independent decrease in ACh release, probably through an inhibitory action on a positive-feedback prejunctional nicotinic autoreceptor closely related to the muscle-type nicotinic ACh autoreceptor. However, at low frequencies of motor nerve stimulation we suggest that tubocurarine, but not vecuronium, produces a [Ca2+]o-dependent increase in ACh release through an action at a negative-feedback prejunctional neuronal-type nicotinic ACh autoreceptor.     

Role of dynorphin-containing neurons in the presynaptic inhibitory control of the acetylcholine-evoked release of dopamine in the striosomes and the matrix of the cat caudate nucleus.

The roles of acetylcholine and dynorphin (1-13) in the presynaptic control of the release of [3H]dopamine continuously synthesized from [3H]tyrosine were examined in a prominent striosomal enriched area and in an adjacent matrix enriched area of the cat caudate nucleus. This was achieved using microsuperfusion devices applied vertically onto coronal slices of cat brain. These devices were placed in a striosomal enriched area located in the core of the structure (acetylcholinesterase-poor zone) and in an adjacent matrix enriched area (acetylcholinesterase-rich zone). [3H]Tyrosine was delivered continuously to each microsuperfusion device and [3H]dopamine released was estimated in the superfusate. As previously shown, in the presence of tetrodotoxin (1 microM), acetylcholine (50 microM) induces a prolonged stimulation of [3H]dopamine release in both compartments through an interaction with muscarinic receptors. Our present study indicates that both dynorphin 1-13 (1 microM) and the selective kappa agonist trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]benzeneace tamine (U50488) (1 microM) inhibit the tetrodotoxin-resistant acetylcholine-evoked release of [3H]dopamine, these effects being slightly more pronounced in the matrix than in the striosomal enriched area. Naloxone (1 microM) reversed the inhibitory effect of U50488 in both areas. These results suggest that dynorphin exerts an inhibitory presynaptic control of dopamine release through kappa opioid receptors located on dopamine nerve terminals in the striosome as well as in the matrix. However, the presynaptic cholinergic control of dopamine release is much more complex in the matrix than in the striosomal enriched area. Besides its tetrodotoxin-resistant stimulatory effect, acetylcholine exerts two opposing tetrodotoxin-sensitive effects on [3H]dopamine release, one facilitatory and the other inhibitory. We demonstrate here that in the superfused matrix enriched area, the indirect acetylcholine inhibitory response is mediated by dynorphin-containing neurons. Indeed, the short-lasting stimulatory effect of acetylcholine on [3H]dopamine release was converted into a long-lasting response in the presence of naloxone (1 microM), and, in this latter condition, the co-application of dynorphin 1-13 (1 microM) restored the short-lasting stimulatory effect.