Inhibition of [3H] γ-aminobutyric acid release from guinea-pig hippocampal synaptosomes by serotonergic agents

We studied the effects of (m-trifluoromethyl-phenyl)piperazine (TFMPP) and quipazine on the K(+)-evoked [3H]GABA release from guinea-pig hippocampal synaptosomes loaded with [3H]GABA.TFMPP and quipazine inhibited the K(+)-evoked release of [3H]GABA dose-dependently (IC50 = 153 and 123 microM, respectively). Serotonergic antagonists such as methiothepin (0.1, 0.3 and 1 microM), ketanserin (0.1, 0.3 and 1 microM), dihydroergotamine (0.1 microM), metergoline (0.1 and 0.3 microM), methysergide (0.3 microM), propranolol (1 microM) and yohimbine (1 microM) did not significantly alter the inhibitory effect of TFMPP on [3H]GABA release suggesting that neither 5-HT1 nor 5-HT2 receptors are involved in this process. By contrast, the effect of TFMPP was diminished by selective 5-HT3 receptor antagonist: MDL 72222 (0.3 microM), tropisetron (0.3 and 1 microM), ondansetron (0.3 microM) and metoclopramide (1 microM). Tropisetron (1 microM) and ondansetron (0.3 microM) also inhibited significantly the quipazine effect whereas methiothepin (1 microM), dihydroergotamine (0.1 microM), yohimbine (1 microM) and ketanserin (1 microM) were ineffective on the quipazine inhibition of [3H]GABA release. Our results show a serotonergic modulatory effect on the K(+)-evoked [3H]GABA release from guinea-pig hippocampal synaptosomes by receptors which are neither 5-HT1, 5-HT2 or 5-HT4. They appear to be pharmacologically related to the 5-HT3 type but different from the 5-HT3 ionic channel receptors.     

Interaction acetylcholine-glutamate in rat hippocampus: involvement of two subtypes of M-2 muscarinic receptors

The effects of acetylcholine (ACh) on the release of endogenous glutamic acid (GLU) and of [3H]ACh have been investigated comparatively in superfused rat hippocampal synaptosomes. Exogenous ACh added to the superfusion fluid inhibited the Ca++-dependent K+ (15 mM)-evoked release of GLU in a concentration-dependent manner (the maximal inhibition was about 50%). Carbachol and oxotremorine mimicked, although less potently, the action of ACh. The inhibition of GLU release caused by 10 microM ACh was antagonized by 0.1 microM atropine but not by 10 microM mecamylamine. It also was insensitive to the M-1 receptor antagonists pirenzepine or dicyclomine (both at 1 microM). In contrast, the novel M-2 muscarinic antagonist AF-DX 116 [(11-[(2-[diethylamino]methyl)-1-piperidinyl]acetyl)-5-11-dihydro-6 H-pyrido-[2-3-b][1,4]benzo-diazepine-6-one] was as potent as atropine in blocking the inhibition of GLU release brought about by ACh. The autoreceptor-mediated inhibition of [3H]ACh release observed in presence of ACh (10 microM) was totally antagonized by atropine (0.1 microM). It was insensitive to mecamylamine (10 microM), dicyclomine (1 microM) or pirenzepine (1 microM). However, it was much less sensitive to AF-DX 116 (80-100 times) than the cholinergic inhibition of GLU release. It is concluded that 1) the release of GLU in rat hippocampus can be inhibited through a muscarinic receptor and 2) this novel muscarinic receptor belongs to the M-2 subtype but it seems to differ pharmacologically from the M-2 autoreceptor.     

gamma-Aminobutyric acid-induced modulation of acetylcholine release from the guinea pig lung

gamma-Aminobutyric acid (GABA) content was measured biochemically and the effect of GABA on the release of [3H]acetylcholine (ACh) was studied in strips of the guinea pig lung preloaded with [3H]choline. GABA contents were highest in the middle sections of the lung, as compared with proximal and distal areas. GABA evoked the release of [3H]ACh from the strips of the lung. The effect of GABA was mimicked by muscimol and antagonized by bicuculline and furosemide. Perfusion with Ca++-free medium and tetrodotoxin, but not nipecotic acid, inhibited the GABA- and muscimol-evoked release of [3H]ACh, thereby indicating that the released ACh was of neuronal origin. Diazepam and pentobarbital potentiated the muscimol-evoked [3H]ACh release. On the other hand, GABA reduced the KCl (40 mM)-evoked release of [3H]ACh in the presence of tetrodotoxin and bicuculline and baclofen mimicked the inhibitory effect of GABA. The effects of GABA and baclofen were not altered by alpha and beta adrenergic antagonists. These findings provide evidence for two types of GABA receptors in the lung of the guinea pig, and these receptors are involved in regulating the release of ACh.     

Pharmacologic characterization and functional role of muscarinic autoreceptors in the rabbit striatum

The objectives of the present studies were 1) to pharmacologically characterize the muscarinic autoreceptors in the striatum and 2) to examine their role in the regulation of physiologic acetylcholine (ACh) release. Schild plots were generated for atropine and pirenzepine against oxotremorine-induced inhibition of [3H]ACh release. Atropine, a nonselective antagonist, yielded a pA2 of 8.92. The pA2 for pirenzepine, a purported M1-selective antagonist, was 7.14. Both Schild plots had slopes not significantly different from one. Four agonists [oxotremorine, carbachol (CARB), McN-A-343 and pilocarpine] were tested for their effectiveness in inhibiting [3H]ACh release. McN-A-343 and pilocarpine have been reported to be selective for M1 receptors. Oxotremorine and carbachol were effective and potent inhibitors of [3H] ACh release, whereas McN-A-343 and pilocarpine were weak. Although the existence of muscarinic receptor subtypes remains an open question, these data are consistent with the "low" pirenzepine affinity (M2) subtype. Chronic treatments (14 days) with several agents were carried out (in vivo) to assess the role of muscarinic autoreceptors in the regulation of physiologic ACh release. Scatchard analyses of binding studies with [3H]quinuclidinyl benzilate were also performed to assess changes in the muscarinic receptor population in the striatum. Chronic treatment with scopolamine caused a 100% increase in the Bmax for [3H]quinuclidinyl benzilate binding but had no effect on the sensitivity of [3H]ACh release to inhibition by CARB. Fourteen-day treatment with physostigmine (3 mg/kg) produced a decrease in the sensitivity of [3H]ACh release to CARB plus a 42% decrease in Bmax and a 48% decrease in Kd for [3H]quinuclidinyl benzilate binding. Chronic haloperidol treatment caused an increase in the sensitivity of [3H]ACh release to CARB accompanied by a 46% increase in Bmax for 3H quinuclidinyl benzilate binding. These data suggest that muscarinic autoreceptors in the striatum do not regulate physiologic ACh release in the presence of intact acetylcholinesterase and that the interaction of dopaminergic and cholinergic neurons in the striatum may not be simple trans-synaptic inhibition.     

Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M(2) and M(4) muscarinic receptors in rat brain

We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [(3)H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M(2) and M(4) autoreceptors, respectively, immediate application of 10 microM xanomeline had no effect on evoked [(3)H]ACh release or its inhibition by 10 microM carbachol. In contrast, preincubation with 1, 10, or 100 microM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 microM xanomeline) or partially (at 100 microM xanomeline) blocked by 1 microM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 microM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N-methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic alpha(2)-adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M(2) and M(4) receptors with both competitive and allosteric components of action.     

Presence of a gamma-aminobutyric acid (GABA) uptake system on cholinergic terminals of rat hippocampus: evidence for neuronal coexistence of acetylcholine and GABA?

The effect of gamma-aminobutyric acid (GABA) on the basal release of [3H]acetylcholine ([3H]ACh) was investigated using synaptosomes prepared from rat hippocampus and superfused after prelabeling with [3H]choline. Exogenous GABA added to the superfusion medium caused a long-lasting and concentration-dependent enhancement of the basal efflux of [3H]ACh. The effect of GABA was not antagonized by bicuculline or picrotoxin. Muscimol increased slightly but not significantly the release of [3H]ACh, whereas (+/-)-baclofen or (-)-baclofen were ineffective. The effect of GABA was counteracted by SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid], SK&F 100330A [N-(4,4-diphenyl-3-butenyl)-guvacine] and SK&F 100561 [N-(4,4-diphenyl-3-butenyl)-homo-beta-proline], three novel inhibitors of GABA uptake, but was unaffected by hemicholinium-3 or by beta-alanine. Nipecotic acid, a substrate-inhibitor of the GABA transporter, mimicked GABA and enhanced [3H]ACh release. The results indicate that a GABA transport system is present on cholinergic terminals.     

Comparative anticholinergic properties of thioridazine, mesoridazine and sulforidazine

The anticholinergic properties of thioridazine (THD) and its metabolites mesoridazine (MES) and sulforidazine (SUL) were compared to the antimuscarinics atropine and quinuclidinylbenzilate (QNB). THD, MES and SUL were virtually inactive in antagonizing the carbachol-induced inhibition of evoked ACh release from perfused rabbit striatal slices. This lack of effect was seen even when dopamine influences were abolished by treatment with reserpine and alpha-methyl-p-tyrosine. The lack of functional anticholinergic potency contrasted with the affinity of THD for muscarinic receptors measured as competition for [3H]QNB binding sites in striatal homogenates (Ki values: atropine, 2.7 nM; THD 14 nM; SUL, 66 nM; and MES, 90 nM). Both atropine and QNB blocked carbachol-induced inhibition of ACh release in a dose-dependent manner (IC50 values vs. 3 microM carbachol: 0.5 nM for QNB; 1.25 nM for atropine). THD, only 5 times less potent than atropine in competing for [3H]QNB binding sites, was inactive in antagonizing carbachol-induced ACh release. At very high concentrations (3-30 microM), THD, MES and SUL did enhance dopamine efflux and inhibit ACh release. In summary, the lack of effect of THD on release modulatory muscarinic receptors suggest that THD is selective for the M1 subtype. Because the M2 subtype is a small fraction of the total population in the striatum, it is not surprising that they would escape recognition in the QNB binding assays. These data suggest that inhibition of ACh release may contribute to the actions of THD only at very high doses, or when drug accumulation is abnormal.     

Ca2(+)-evoked [3H]dopamine release from synaptosomes is dependent on neuronal type Ca2+ channels and is not mediated by acetylcholine, glutamate or aspartate release

Elevation of potassium concentrations ([K+]) in the presence of Ca2+ is the most common method of evoking neurotransmitter release from synaptosomes. However, we have been investigating a method of releasing dopamine from synaptosomes that does not involve using elevated [K+]. In this paradigm of neurotransmitter release, dopamine is released from synaptosomes, previously exposed to micromolar or lower [Ca2+], by 1.25 mM Ca2+ in the presence of non-depolarizing [K+] (4.5 mM). The present experiments characterize the Ca2+ channel(s) involved in the Ca2(+)-evoked release of dopamine from synaptosomes, and determine whether the release is mediated by acetylcholine, glutamate or aspartate. omega-Conotoxin (10 nM), which blocks N-, L- and possibly T-type voltage-sensitive Ca2+ channels (VSCC), inhibited the Ca2(+)-evoked [3H]dopamine release from either striatal or olfactory tubercle synaptosomes to less than 50% of control. Neither 1 microM nifedipine nor 1 microM verapamil, which block L-type VSCC, affected Ca2(+)-evoked release. The N- and T-type VSCC blocker neomycin and the nonspecific Ca2+ antagonist, cobalt2+, inhibited release to a greater extent than omega-conotoxin. At 1 mM, both compounds inhibited release to approximately 30% of control. Neither the excitatory neurotransmitter glutamate nor aspartate (2mM) affected 1 microM LY-171555 (a dopamine D2 agonist) inhibition of Ca2(+)-evoked [3H]dopamine release. Also, the glutamate antagonist, glutamic acid diethyl ester, did not affect either Ca2(+)-evoked release or 1 microM LY-171555 inhibition thereof. The nicotinic antagonist hexamethonium (10 microM) and the muscarinic antagonist atropine (1 microM) were also ineffective in inhibiting Ca2(+)-evoked release or LY-171555 inhibition of release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation between muscarinic receptor-mediated inhibition of adenylate cyclase and autoreceptor inhibition of [3H] acetylcholine release in rat hippocampus

Activation of muscarinic cholinergic receptors (mAChRs) in the central nervous system reduces the catalytic activity of membrane-bound adenylate cyclase and attenuates depolarization-dependent release of acetylcholine (ACh). Inasmuch as reports have indicated that these mAChR-mediated responses exhibit pharmacological profiles similar to the M2 subclass of mAChR, the present studies were undertaken to ascertain whether attenuation of presynaptic adenylate cyclase activity [and concurrent reduction of intraneuronal cyclic AMP (cAMP) levels] underlies mAChR-mediated autoinhibition of electrically evoked ACh release. In [3H]choline-prelabeled rat hippocampal slices, the mAChR agonists oxotremorine (EC50 = 15 microM) and carbachol (EC50 = 80 microM) caused atropine-reversible inhibition of [3H]ACh release up to a maximum of 80% reduction. The rank order of potency for antagonist reversal of this inhibitory action (N-methylatropine = atropine greater than scopolamine much greater than pirenzepine) was generally consistent with an M2 mAChR-mediated response although pirenzepine was ineffective up to 1 mM. Under these assay conditions, forskolin (1-10 microM) and 8-bromo-cAMP (30-300 microM) enhanced electrically evoked [3H]ACh release maximally by 50 to 60%; however, neither agent significantly reversed mAChR agonist-induced inhibition of [3H]ACh release. Additional studies were undertaken to determine the consequences of chemically uncoupling mAChR from their G protein-adenylate cyclase effector system in this tissue. Whereas brief pretreatment with the sulfhydryl alkylating agent N-ethylmaleimide (30 microM) or pertussis toxin (1 microgram/ml) markedly attenuated carbachol inhibition of adenylate cyclase activity in hippocampal tissue, there was no concurrent reduction of carbachol-inhibited [3H] ACh release.(ABSTRACT TRUNCATED AT 250 WORDS)     

gamma-Aminobutyric acidB receptors mediate inhibition of somatostatin release from cerebrocortex nerve terminals.

The effects of gamma-aminobutyric acid (GABA) and of various GABA receptor agonists and antagonists on the calcium-dependent depolarization-evoked release of somatostatin (SRIF) from rat cerebrocortex synaptosomes have been studied by a superfusion technique. GABA (0.3-30 microM) decreased the K+ (15 mM)-evoked overflow of SRIF-like immunoreactivity (SRIF-LI) in a concentration-dependent manner (EC50 = 1.3 microM; maximal inhibition, 45% reached at 10 microM GABA). The effect of the amino acid was insensitive to the GABAA receptor antagonist bicuculline. Accordingly, the K(+)-evoked SRIF-LI release was not affected by muscimol, a GABAA receptor agonist, up to 100 microM. The effect of GABA was mimicked by the GABAB receptor agonist (-)-baclofen (EC50 = 1.2 microM; maximal effect, about 45% reached at 10 microM). The effect of baclofen was stereoselective, the (+)-enantiomer being inactive up to 100 microM. The inhibition of SRIF-LI release brought about by GABA was sensitive to the GABAB receptor antagonists 2-hydroxy-saclofen and CGP 35348 [3-aminopropyl(diethoxymethyl)phosphinic acid]. Also, the effect of (-)-baclofen was antagonized by CGP 35348 (IC50 = 4.8 microM). It is concluded that GABA can inhibit the depolarization-evoked release of SRIF by activating receptors which are located on SRIF-releasing nerve terminals and belong to the GABAB type.     

Inhibitory effects of amphetamine on potassium-stimulated release of [3H]dopamine from striatal slices and synaptosomes

Amphetamine, 10(-7) M or greater, evoked the release of [3H]dopamine ([3H]DA) and inhibited subsequent K+-evoked [3H]DA release from striatal synaptosomes superfused at a flow rate (1 ml/min) that prevented reuptake. Amphetamine inhibited the K+-evoked release of [3H]DA to a lesser extent in striatal slices or in synaptosomes superfused at a flow rate (0.35 ml/min) that allowed reuptake. The observed decrease in amphetamine inhibition of K+-evoked release was primarily due to amphetamine blocking [3H]DA reuptake. Interneuronal interactions may account for some of the inhibitory effects of amphetamine on K+-evoked release in the slice. Inhibition of K+-evoked release from either slices or synaptosomes was still evident when 10(-6) M amphetamine was removed from the superfusion buffer and the spontaneous release had returned to control levels. The presence of Ca++ during amphetamine exposure was required for subsequent inhibition of K+-evoked release in synaptosomes. Amphetamine in the presence of Ca++ did not affect the subsequent release of [3H]DA evoked by the Ca++ ionophore, A23187. Therefore, amphetamine inhibition of the K+-evoked release of [3H]DA cannot be explained by prior depletion of Ca++-releasable pools. Nifedipine, 1 microM, failed to block either the Ca++-dependent release of [3H]DA or the inhibition of K+-evoked release by amphetamine. However, 1 mM cobalt inhibited the Ca++-dependent release of [3H]DA by amphetamine and antagonized the inhibition of K+-evoked release after amphetamine exposure. This suggests that amphetamine may open voltage-dependent Ca++ channels sensitive to cobalt but not nifedipine. Amphetamine may desensitize these voltage-dependent Ca++ channels and inhibit their activation by K+ depolarization.     

Modulation of the Ca++-evoked release of [3H]dopamine from striatal synaptosomes by dopamine (D2) agonists and antagonists

Release of [3H]dopamine ([3H]DA) from striatal synaptosomes is evoked most commonly by elevating potassium levels in the presence of calcium. However, it has been difficult to show that DA agonists or antagonists can modify K+-evoked release of [3H]DA. DA. In this study [3H]DA release evoked by exposure of synaptosomes (isolated and superfused previously with 0.0 mM Ca++ and 0.1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) to 1.25 mM Ca++ can be modulated by the DA (D2) agonists apomorphine, pergolide and quinpirole and antagonists l-sulpiride and domperidone. The release was evoked under low potassium (6 mM or less) concentrations and the potassium concentration in the superfusion medium was not elevated before or during Ca++ exposure. Analysis of the superfusates obtained during Ca++ exposure revealed that approximately 80% of the tritium released was [3H]DA. The ability of DA (D2) agonists to inhibit the Ca++-evoked release from synaptosomes superfused with 9 mM K+ was greatly reduced. Therefore, prolonged depolarization may block DA (D2) regulation of [3H]DA release from synaptosomes. The Ca++-evoked release of [3H]DA was reduced greatly when 1 microM tetrodotoxin was present indicating sodium channels play a role in triggering the processes involved in Ca++-evoked [3H]DA release.     

Frequency-dependent muscarinic receptor modulation of acetylcholine and dopamine release from rabbit striatum

The effects of muscarinic receptor activation on the electrically evoked release of [3H]dopamine (DA) and [14C]acetylcholine (ACh) or [3H]ACh were investigated in rabbit striatal slices. Release was measured in the presence of 10 microM hemicholinium and 1 microM sulpiride to block choline uptake and prevent the effects of released DA on DA receptors modulating release. Stimulation (120 pulses, 20 mA, 2 msec) at 0.3, 3 and 10 Hz produced (3H or 14C) ACh release that sharply declined with increasing stimulation frequency. A flat frequency-release curve was obtained for DA. Oxotremorine (OXO), a direct muscarinic agonist (1-100 microM), produced a concentration-dependent inhibition of ACh release, inversely related to stimulation frequency, at a fixed number of pulses (120). When the number of pulses was modified to produce similar amounts of ACh release (20 pulses at 0.1 Hz, 39 pulses at 0.3 Hz, 120 pulses at 3 Hz and 350 pulses at 10 Hz), much greater inhibition of ACh release by OXO (0.3 and 3 microM) was obtained with lower frequencies and lower number of pulses. Physostigmine, an acetylcholinesterase inhibitor, decreased ACh release with an inverse relationship to stimulation frequency. Atropine (1 microM), a selective muscarinic antagonist, enhanced the release of ACh more at 10 Hz than at 0.3 and 3 Hz and completely antagonized the effects of OXO (10 microM) and physostigmine (1 microM) at all three stimulation frequencies. OXO (3 and 10 microM) enhanced DA release at 3 Hz. Physostigmine (1 microM) and atropine (1 microM) had no effect on DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the presence in the cerebral cortex of muscarinic receptor subtypes which differ in neuronal localization, function and pharmacological properties

The existence in rat frontal cerebral cortex of subtypes of muscarinic receptors was investigated by using as a receptor-mediated functional response the release of neurotransmitters from isolated nerve endings. Synaptosomes prelabeled with [3H]choline or [3H]dopamine were depolarized with 15 mM KCl. Acetylcholine (ACh) concentration-dependently decreased the release of [3H]ACh and increased that of [3H]dopamine. Both actions of ACh were counteracted by the classical muscarinic antagonists atropine and quinuclidinyl benzylate. The two antagonists did not discriminate between the muscarinic presynaptic receptors sited on cholinergic terminals (muscarinic autoreceptors) and those located on dopamine nerve endings (muscarinic heteroreceptors). The apparent affinity (pA2) values for the two receptors were: 8.41 and 8.57 with atropine and 8.55 and 8.34 with quinuclidinyl benzylate. However, other muscarinic antagonists behaved differently. Dicyclomine strongly antagonized ACh at the heteroreceptors (pA2 = 8.69), whereas it was ineffective at the autoreceptors when tested at 5 microM. Pirenzepine had a similar behavior, although its affinity at the heteroreceptors was lower (pA2 = 6.33). In contrast, secoverine antagonized ACh at the autoreceptors with an affinity (pA2 = 7.58) higher than that showed at the heteroreceptors (pA2 = 6.51). The data support the existence in the frontal cortex of muscarinic receptors that are located on different neurons, mediate different functional responses and are pharmacologically distinguishable.     

Effects of selected muscarinic cholinergic antagonists on [3H]acetylcholine release from rat hippocampal slices

A number of cholinergic muscarinic (M) agonists and antagonists were studied for their ability to enhance tritiated acetylcholine ([3H]ACh) release from electrically field-stimulated rat hippocampal slices. A Ca++-free medium and carbachol, but not nicotine, inhibited [3H]ACh release. Atropine, methylatropine and dexetimide produced concentration-dependent increases in [3H]ACh release to a maximum of about 50% above control. Aprophen and benactyzine produced a maximal response 25 to 35% above control. The selective M1 antagonist pirenzepine had the least effect on [3H]ACh release. Of the nonspecific M1-M2 antagonists studied, benactyzine produced the least amount of [3H]ACh release. The order of potency of the M antagonists in promoting a 15% increase in [3H]ACh release was aprophen greater than benactyzine greater than methylatropine greater than dexetimide greater than pirenzepine greater than atropine. However, the order of promoting maximal release of [3H]ACh was atropine greater than dexetimide greater than methylatropine greater than aprophen greater than benactyzine greater than pirenzepine.     

Effects of amiloride and 5-(N,N-hexamethylene)amiloride on dopaminergic and cholinergic neurotransmission

In order to establish the role of the Na+/H+ exchange transport on neurotransmission, we investigated the effects of amiloride and of 5-(N,N-hexamethylene)amiloride (HMA) on dopamine (DA) and acetylcholine (ACh) release and on receptor-mediated modulation of DA and ACh release. Superfused rabbit striatal slices prelabeled with [3H]DA and [14C]choline were stimulated electrically in the presence and absence of several concentrations of these agents. Amiloride (3-10 microM) and HMA (0.3-10 microM) reduced the basal efflux and the stimulation evoked overflow of total 3H and of [3H]-3,4-dihydroxyphenylacetic acid and inhibited monoamine oxidase activity. The inhibition of stimulation evoked overflow of total 3H was blocked by pretreatment with nomifensine but not by sulpiride. Amiloride had no effect on the basal efflux and the stimulation evoked overflow of ACh or it did modify apomorphine-induced inhibition of DA and ACh release. However, at 3 to 10 microM, HMA enhanced the basal efflux of 3H; this effect was not prevented either by uptake inhibition with nomifensine or by low extracellular calcium. These results suggest that amiloride-sensitive Na+ transport and the amiloride and HMA-sensitive Na+/H+ antiporter play no role on the secretion of DA and ACh, or on the mechanisms by which activation of pre- and postsynaptic DA receptors lead to inhibition of neurotransmitter release. Amiloride- and HMA-induced monoamine oxidase inhibition accounts for the effects of amiloride and HMA on DA efflux and overflow. The guanidine moiety present in the amiloride and HMA molecules is most likely responsible for these effects.     

Prejunctional inhibitory effects of prostanoids on sympathetic neurotransmission in the rabbit iris-ciliary body

Both naturally occurring and synthetic prostaglandins (PGs) caused concentration-dependent inhibition of electrically evoked [3H]norepinephrine (NE) overflow from the isolated, superfused rabbit iris-ciliary body without affecting basal tritium efflux. The rank order of potencies of the agonists was: sulprostone greater than 16, 16-dimethyl-PGE2 greater than PGE2 greater than 11-deoxy-PGE1 greater than iloprost (stable PGl2 analog) greater than PGF2 alpha greater than or equal to PGD2. However, the Tx-mimetic, U-46619, was without effect on transmitter release at concentrations up to 1 microM. The selective EP1-receptor antagonists, AH 6809 (30 microM) or SC-19220 (10 microM) had no effect on basal or field-stimulated [3H]NE secretion, nor did they antagonize the PGE2-mediated reduction of evoked [3H]NE overflow. Indomethacin (3 microM) and the 5-lipoxygenase inhibitor, BW A4C (1 microM) were without effect on basal or evoked [3H]NE release, suggesting that endogenously formed arachidonic acid metabolites have no significant modulatory role in this in vitro system. Inhibitory effects of submaximal or maximal concentrations of PGE2 combined with corresponding concentrations of clonidine or carbachol were not additive, suggesting that prejunctional PGE2 receptors coexist with alpha-2 adrenergic and muscarinic receptors at neurotransmitter release sites. In the presence of yohimbine (100 nM) and/or atropine (100 nM), however, the inhibition produced by PGE2 was enhanced markedly, suggesting that tonic activation of prejunctional alpha-2 adrenergic or muscarinic receptors by endogenously released transmitters may impair the response to exogenous PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic mechanism of dopaminergic and muscarinic auto- and heteroreceptor activation in superfused striatal slices: role of extracellular chloride

The Cl dependency of agonist-induced ionic mechanisms involved in the receptor-mediated modulation of electrically stimulated release of dopamine (DA) and acetylcholine (ACh) was examined in superfused rabbit striatal slices prelabeled with [3H] DA (3,4-[8-3H]dihydroxyphenylethylamine) and [14C]choline ([ methyl-14C]choline Cl). Cl- was substituted in the superfusion medium to varying degrees with the impermeant anions isethionate, methanesulfonate or gluconate. The sodium concentration was held constant. Apomorphine (30 nM), a DA receptor agonist, inhibited the stimulation-evoked (1 Hz, 2 min) release of both DA and ACh in Krebs-Ringer-bicarbonate medium (KRB; 125.4 mM Cl-). The inhibitory effects of the agonist were not altered significantly in media containing 66.4 mM Cl-. In 7.4 mM Cl- medium (isethionate replacement), apomorphine-induced inhibition of DA release was reduced (40% inhibition vs. 67% inhibition in KRB). Similarly, apomorphine inhibition of ACh release was lowered from 38% in KRB to 25% in 7.4 mM Cl-. The muscarinic receptor agonist carbachol (10 microM) inhibited the stimulation-evoked release of ACh while enhancing the evoked release of DA in normal Cl- (125.4 mM) medium. Inhibition of ACh release was not altered in 66.4 mM Cl- media but was increased in 7.4 mM Cl- (63% inhibition in low Cl- vs. 50% in KRB). The enhancing effects of carbachol on stimulated DA release were potentiated in 66.4 mM Cl- (88% enhancement vs. 57% in KRB), whereas no change in the agonist effect was observed in the lower Cl- medium (7.4 mM Cl-).(ABSTRACT TRUNCATED AT 250 WORDS)     

[3]PIRENZEPINE BINDING IN RAT CORPUS STRIATUM DECREASES AFTER HEMITRANSECTION OF THE NIGRO-STRIATAL PATHWAY

The localization and pharmacologic characterization of muscarinic receptors possibly regulating the release of dopamine (DA) in rat corpus striatum were investigated by in vitro binding with [3H]pirenzepine ([3H]PZ) after hemitransection of the nigro-striatal pathway. DA levels in the corpus striatum ipsilateral to the lesion were substantially reduced by 66% compared with the unlesioned side after 8 days. The uptake of [3H]DA was also diminished by 63%. A significant decrease in the specific binding of [3H]PZ of 42% was seen in the corpus striatum ipsilateral to the lesion. The data indicate a loss of binding sites, whereas the lesion caused no change in the affinity constant for the muscarinic antagonist. The results support those previously obtained in studies of the muscarinic modulation of [3H]DA release from striatal synaptosomes and favor the idea that at least part of the muscarinic receptors regulating striatal DA release are localized on the nigro-striatal axon terminals and belong to the pirenzepine-sensitive subtype.     

M2, M3, and M4 receptor subtypes contribute to muscarinic potentiation of GABAergic inputs to spinal dorsal horn neurons

The spinal cholinergic system and muscarinic receptors are important for regulation of nociception. Activation of spinal muscarinic receptors produces analgesia and inhibits dorsal horn neurons through potentiation of GABAergic inputs. To determine the role of receptor subtypes in the muscarinic agonist-induced synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons using whole-cell voltage-clamp recordings in rat spinal cord slices. The muscarinic receptor agonist oxotremorine-M dose-dependently (1-10 microM) increased GABAergic sIPSCs but not miniature IPSCs. The potentiating effect of oxotremorine-M on sIPSCs was completely blocked by atropine. In rats pretreated with intrathecal pertussis toxin to inactive inhibitory G (i/o) proteins, 3 microM oxotremorine-M had no significant effect on sIPSCs in 31 of 55 (56%) neurons tested. In the remaining 24 (44%) neurons in pertussis toxin-treated rats, oxotremorine-M caused a small increase in sIPSCs, and this effect was completely abolished by subsequent application of 25 nM 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), a relatively selective M(3) subtype antagonist. Furthermore, himbacine (1 microM), a relatively specific antagonist for M(2) and M(4) subtypes, produced a large reduction in the stimulatory effect of oxotremorine-M on sIPSCs, and the remaining effect was abolished by 4-DAMP. Additionally, the M(4) receptor antagonist MT-3 toxin (100 nM) significantly attenuated the effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that M(2) and M(4) receptor subtypes play a predominant role in muscarinic potentiation of synaptic GABA release in the spinal cord. The M(3) subtype also contributes to increased GABAergic tone in spinal dorsal horn by muscarinic agonists.