Release-enhancing pre-synaptic muscarinic and nicotinic receptors co-exist and interact on dopaminergic nerve endings of rat nucleus accumbens

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR–mAChR agonists acetylcholine (ACh) and carbachol provoked [³H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 μmol/L, together with 3, 10, or 100 μmol/L (−)nicotine provoked synergistic effect on [³H]DA overflow. The [³H]DA overflow elicited by 100 μmol/L (−)nicotine plus 30 μmol/L oxotremorine was reduced by atropine down to the release produced by (−)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (−)nicotine/oxotremorine evoked [³H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (−)nicotine/oxotremorine. Similarly to (−)nicotine, the selective α4β2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, α4β2 nAChRs exert a permissive role on the releasing function of reportedly M₅ mAChRs co-existing on the same dopaminergic nerve endings.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad.

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied. 2. CA secretion was induced by ACh or nicotine, but not by muscarine. 3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response. 4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine. 5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M2, which may play a regulatory function.     

Actions of cholinergic drugs in the nematode Ascaris suum. Complex pharmacology of muscle and motorneurons

The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d- tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic- like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied.2. CA secretion was induced by ACh or nicotine, but not by muscarine.3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response.4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine.5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M₂, which may play a regulatory function.     

Pharmacology of the isolated foregut of the locust Schistocerca gregaria—IV. Characterization of a muscarinic acetylcholine receptor

1. Acetylcholine (ACh; 10⁻⁶ M—7 × 10⁻⁵ M), in the presence of neostigmine (10⁻⁵ M), caused contraction of the locust isolated foregut.2. The effect of ACh was mimicked by carbachol, propionylcholine (PCh), butyrylcholine (BCh), nicotine, SD35651, oxotremorine and muscarine.3. The contractions caused by ACh, BCh and carbachol were abolished by atropine (10⁻⁶M) and reduced by d-tubocurarine (10⁻⁵ M) and decamethonium (5 × 10⁻⁵ M). Hexamethonium and α-bungaro-toxin had no effect on contractions caused by the above agonists.4. None of the antagonists used in this study blocked the contractile effects of nicotine.5. It is concluded that the foregut contains a neuronal nicotinic receptor which, when activated, causes release of ACh which acts on a neuromuscular muscarinic receptor.     

The nature of the acetylcholine and 5-hydroxytryptamine receptors in buccal smooth muscle of the pest slug Deroceras reticulatum

The characteristics of the acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) receptors of Deroceras buccal muscle were examined using specific pharmacological probes and sucrose gap electrophysiological analysis. ACh induced concentration-dependent smooth tonic contractures coupled with considerable depolarisation from the normal resting membrane potential of -30.6 mV. The use of choline ester analogues such as carbachol, propionylcholine and butyrylcholine, specific cholinergic agonists such as nicotine, muscarine, bethanecol and pilocarpine and antagonists such as d-tubocurarine, succinylcholine, hexamethomium, atropine, gallamine, pirenzepine and scopolamine indicated that the ACh receptor showed both nicotinic and muscarinic characteristics; the muscarinic activity resembled that of a mammalian M(2)-like receptor. Alternatively, it can not be ruled out that both mammalian types of receptor may be present in this preparation since both nicotine and muscarine induced noticeable tension. 5-HT application induced characteristic dose-dependent phasic contractions accompanied by small but quite consistent depolarisations. Serotonergic agonist and antagonist experiments using 1-(3-chlorophenyl) piperazine, 1-(m-chlorophenyl) biguanide, methiothepin, methysergide and metoclopramide strongly suggested that the 5-HT receptor showed closest pharmacological affinity with the 5-HT(1) receptor class of mammals but with some 5-HT(2) activity. In view of the phylogenetic gap between molluscs and mammals it is not surprising that the ACh and 5-HT receptors of Deroceras can not be properly classified by conventional mammalian terminology.     

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells

The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChRs) on apoptosis in human skin fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with pilocarpine in the presence or absence of specific antagonists. Pilocarpine stimulates apoptosis, total inositol phosphates (InsP) accumulation and nitric oxide synthase (NOS) activity. All these effects were inhibited by atropine, mustard hydrochloride (4‐DAMP) and pirenzepine, indicating that M₁ and M₃ mAChRs are implicated in pilocarpine action. Pilocarpine apoptotic action is accompanied by caspase‐3 and JNK activation. The intracellular pathway leading to pilocarpine‐induced biological effects involved phospholipase C, calcium/calmodulin and extracellular calcium as U‐73122, W‐7, verapamil, BAPTA and BAPTA‐AM blocked pilocarpine effects. L ‐NMMA, a NOS inhibitor, had no effect, indicating that the enzyme does not participate in the apoptosis phenomenon. These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on the apoptotic human skin fibroblast process. J. Cell. Physiol. 222: 640–647, 2010. © 2009 Wiley‐Liss, Inc.     

乙酰胆碱对小鼠胰岛B细胞电活动作用的分析

用细胞内电位记录和细胞外微电泳技术,研究乙酰胆碱对小鼠胰岛Ｂ细胞电活动的作用,微电泳ＡＣｈ使Ｂ细胞胞膜去极化５－１０ｍＶ和锋电位电位发放数增加１１－１７／３０ｓ。这种效应具有葡萄糖依赖性,并被阿托品完全阻断,而哌仓西平可阻抑ＡＣｈ效应的７０％。ＡＣｈ的膜去极化作用不依赖于细胞外Ｃａ＾２＋,而可被河豚毒阻断;ＡＣｈ增加锋电位数的效应依赖于细胞外Ｃａ＾２＋,但不被异捕定阻断。结果表明：ＡＣｈ增强Ｂ细胞     

Influence of acetylcholine agonists and antagonists on the swelling of etiolated wheat (Triticum aestivum L.) mesophyll protoplasts

Etiolated wheat (Triticum aestivum L.) mesophyll protoplasts swell within 30 min in darkness after a red light (R) pulse or addition of acetylcholine (ACh), if 0.5 mM CaCl₂ is present in the medium. In addition, ACh is also able to induce swelling in the presence of both 0.1 mM KCl or NaCl. Besides ACh, only carbamylcholine out of the choline derivatives tested was active in induction of swelling in the presence of K⁺ or Na⁺. The K⁺/Na⁺-dependent ACh-induced protoplast swelling was nullified by a ‘calmodulin inhibitor’, but not by Ca²⁺-channel blockers, Li⁺ or VO ₄ ^3- . The antagonists atropine (of muscarine-sensitive ACh receptors, mAChRs) andd-tubocurarine (of nicotine-sensitive ACh receptors, nAChRs) nullified the Ca²⁺ — and the K⁺/Na⁺-dependent protoplast swelling responses, respectively, while having no effect on the Ca²⁺-dependent R-induced swelling response. Moreover, muscarine and nicotine mimicked ACh in the Ca²⁺- and K⁺/Na⁺-dependent swelling responses respectively. Just as is the case in animal cells, the proposed mAChRs appear to be associated with a phosphatidylinositol-dependent pathway, whereas the proposed nAChRs are phosphatidylinositol independent. Similarity between the action of ACh via the proposed mChRs and R via phytochrome in protoplast swelling indicates they share in common signal-transduction pathway. We dedicate this paper to Hans Mohr on the occasion of his 60th birthday We thank the Department of Molecular Biology of the Agricultural University, Wageningen for the use of the photomicroscope and Dr. G. Fassina, Department of Pharmacology, University of Padua, Italy for the gift of nifedipine. These studies were supported by The Foundation for Fundamental Biological Research (BION) which is subsidized by The Netherlands Organization for the Advancement of Research (NWO). A.T. was also supported by: a Research Fellowship from the Agricultural University, Wageningen; a Visitors Fellowship from NWO, the Netherlands; RP II 12.15 from Ministry of Education, Poland.     

Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux

Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.     

Cholinergic transmission from mechanosensory afferents to an identified nonspiking interneuron in the crayfish Procambarus clarkii girard

Pharmacological properties of excitatory synaptic transmission from mechanosensory afferents to an identifiable nonspiking interneuron of crayfish were studied by drug perfusion experiments using acetylcholine (ACh) agonists and antagonists. Application of carbachol, a general agonist of ACh, caused sustained depolarization of the interneuron and a decrease in the peak amplitude of its excitatory synaptic response to sensory stimulation on the soma side. Similar depolarization was observed during application of carbachol under the low-Ca²⁺, high-Mg²⁺ condition. The peak amplitude was also reduced by application of nicotine and tetramethylammonium, both of which also caused sustained depolarization of the inter-neuron. By contrast, perfusion of muscarinic agonists, muscarine, oxotremorine and pilocarpine, reduced the peak amplitude without affecting the membrane potential of the interneuron. Perfusion of nicotonic antagonists of ACh, d-tubocurarine and hexamethonium, caused reduction of the peak amplitude without any change in the membrane potential. A muscarinic antagonist atropine was also effective in blocking the synaptic transmission but at higher concentration than d-tubocurarine. The results suggest that the ACh receptors on the nonspiking interneuron belong to a previously characterized class of crustacean cholinergic receptors resembling the nicotinic subtype of vertebrates.     

Actions of acetylcholine on the salivary gland cells of the pond snail, Planorbis corneus

Intracellular recordings have been made from salivary gland cells of the pond snail Planorbis corneus. Gland cells produced a dose-dependent biphasic response to the bath application of acetylcholine (ACh), an initial depolarization being followed by a hyperpolarization. Nicotine and the nicotinic agonist tetramethylammonium had an excitatory action on the gland cells. The muscarinic agonists acetyl-beta-methyl choline and arecoline were also stimulants, but muscarine, bethanechol and pilocarpine produced no response from gland cells at 10(-3) M. A number of cholinergic antagonists, including atropine, hexamethonium and curare, effectively blocked the response to ACh. The depolarizing phase of the ACh response resulted from an increased membrane permeability to Na+ ions, though the participation of other ionic species cannot be ruled out. The hyperpolarizing phase of the ACh response was produced by the activity of an electrogenic Na+/K+ pump.     

Multiple muscarinic receptor subtypes in the canine pulmonary circulation.

The vascular response to the muscarinic receptor agonist acetylcholine (ACh) in the presence of selected antagonists was examined in the isolated blood-perfused canine left lower lung lobe under conditions of normal (resting) and elevated vascular tone. At normal vascular tone, ACh (1-5 mumol) produced a dose-dependent increase in pulmonary arterial pressure (Ppa), total pulmonary vascular resistance (PVR), and downstream resistance (Rds) without altering upstream resistance (Rus). Pirenzepine (50 and 100 nM), the prototype M1-selective antagonist, and gallamine, an M2-selective antagonist, as well as atropine (50 nM) and secoverine (100 nM), nonselective antagonists, attenuated (P less than 0.05) the ACh-induced increase in Ppa and Rds. With elevated vascular tone induced by serotonin infusion, ACh produced a dose-dependent increase in Ppa in 19 of 25 lobes, although Rus decreased while Rds increased in all lobes. At high vascular tone, pirenzepine or gallamine attenuated the ACh-induced increase in Rds, whereas Rus was not affected. Secoverine and atropine antagonized ACh-induced increases in both Rds and Rus. The pA2 values (i.e., the negative log antagonist concentration requiring a doubling of ACh dose for an equivalent increase in Rds) for gallamine, pirenzepine, secoverine, and atropine were 6.1 +/- 0.1, 7.4 +/- 0.1, 8.3 +/- 0.2, and 10.2 +/- 0.3, respectively. These results suggest that 1) ACh increases PVR in the dog by constricting the venous segments (downstream) of the pulmonary circulation via activation of pulmonary vascular muscarinic receptors under conditions of both normal and elevated vascular tone, 2) both M1- and non-M1-muscarinic receptor subtypes appear to participate in mediating the ACh-induced increase in Rds, and 3) ACh moderately relaxes the upstream (arterial) vessels, especially under conditions of elevated tone.     

Contribution of nicotinic receptors to the function of synapses in the central nervous system: The action of choline as a selective agonist of α7 receptors

The α7-nicotinic receptor (nAChR)-selective agonist choline and nAChR-subtype-selective antagonists led to the discovery that activation of both α7 and α4β2 nAChRs located in CA1 interneurons in slices taken from the rat hippocampus facilitates the tetrodotoxin (TTX)-sensitive release of γ-aminobutyric acid (GABA). Experiments carried out in cultured hippocampal neurons not only confirmed that preterminal α7 and α4β2 nAChRs modulate the TTX-sensitive release of GABA, but also demonstrated that evoked release of GABA is reduced by rapid exposure of the neurons to acetylcholine (ACh, 10 μM-1 mM) in the presence of the muscarinic receptor antagonist atropine (1 μM). This effect of ACh, which is fully reversible and concentration-dependent, is partially blocked by superfusion of the cultured neurons with external solution containing either the α7-nAChR-selective antagonist methyllycaconitine (MLA, 1 nM) or the α4β2-nAChR-selective antagonist dihydro-β-erythroidine (DHβE, 100 nM). A complete blockade of ACh-induced reduction of evoked release of GABA was achieved only when the neurons were perfused with external solution containing both MLA and DHβE, suggesting that activation of both α7 and α4β2 nAChRs modulates the evoked release of GABA from hippocampal neurons. Such mechanisms may account for the apparent involvement of nAChRs in the psychological effects of tobacco smoking, in brain disorders (e.g., schizophrenia and epilepsy), and in physiological processes, including cognition and nociception.     

Catecholamine secretion by hamster adrenal cells.

Abstract— Suspensions of isolated adrenal cells were prepared by digesting hamster adrenal glands with collagenase, and the secretion of catecholamine from these cells was studied. Acetylcholine (ACh) produces a dose-dependent increase in catecholamine secretion; half-maximal secretion is produced by 3 μm -ACh, and maximal secretion by 100 μm -ACh. The cholinergic receptor in these cells appears to be nicotinic, since catecholamine secretion is stimulated by the nicotinic agonists nicotine and dimeth-ylphenylpiperaziniurn, but not by the muscarinic agonists pilocarpine or oxotremorine. ACh-induced catecholamine secretion is inhibited by hexamethonium, tubocurarine, and atropine, but is not inhibited by α-bungarotoxin. ACh-induced catecholamine secretion is dependent upon the presence of extracellular Ca²⁺, and appears to occur by exocytosis, since the release of catecholamine is accompanied by the release of dopamine β-monooxygenase, but not of lactate dehydrogenase. These biochemical studies complement the morphological evidence for exocytosis in hamster adrenal glands, and indicate that catecholamine secretion from hamster chromaffin cells is similar to that from chromaffin cells of other species.     

Acetylcholine receptors of thoracic dorsal midline neurones in the cockroach, Periplaneta Americana

The actions of acetylcholine and cholinergic ligands have been studied using dorsal midline neurones from the rnetathoracic ganglion of the cockroach Periplaneta americana.Both nicotine and oxotremorine depolarized dorsal midline neuronal cell bodies.Dose-response curves for nicotine and oxotremorine saturated at different levels. Nicotine-induced depolarizations were completely or partially blocked by mecamylamine, d-tubocurarine, strychnine, and bicuculline, but were insensitive to alpha-bungarotoxin(100 nM), atropine (100 micronM),Scopolamine (10 micronM), and pirenzepine (50 micronM). Following pretreatment with collagenase, the dorsal midline neurones were sensitive to high doses of alpha-bungarotoxin (3 micronM). Oxotremorine-induced depolarizations were blocked by scopolamine (10 micronM) atropine (100 micronM), and pirenzepine (50 micronM) and were insensitive to mecamylamine (10 micronM) and d-tubocurarine (100 micronM). The results indicate the coexistence of at least two distinct acetylcholine receptors on dorsal midline neuronal cell bodies in the cockroach metathoracic ganglion.     

Direct evidence that release-stimulating alpha7* nicotinic cholinergic receptors are localized on human and rat brain glutamatergic axon terminals

The existence on glutamatergic nerve endings of nicotinic acetylcholine receptors (nAChRs) mediating enhancement of glutamate release has often been suggested but not demonstrated directly. Here, we study the effects of nAChR agonists on [3 H]-d-aspartate ([3 H]-d-ASP) release from synaptosomes superfused in conditions known to prevent indirect effects. Nicotinic receptor agonists, while unable to modify the basal [3 H]-d-ASP release from human neocortex or rat striatal synaptosomes, enhanced the Ca2+ -dependent exocytotic release evoked by K+ (12 mm) depolarization. Their rank order of potency were anatoxin-a > epibatidine > nicotine > ACh (+ atropine). The anatoxin-a effect, both in human and rat synaptosomes, was antagonized by mecamylamine, alpha-bungarotoxin or methyllycaconitine. The basal release of [3 H]ACh from human cortical synaptosomes was increased by (-)-nicotine (EC50 = 1.16 +/- 0.33 microm) or by ACh plus atropine (EC50 = 2.0 +/- 0.04 microm). The effect of ACh plus atropine was insensitive to alpha-bungarotoxin, methyllycaconitine or alpha-conotoxin MII, whereas it was totally antagonized by mecamylamine or dihydro-beta-erythroidine. To conclude, glutamatergic axon terminals in human neocortex and in rat striatum possess alpha7* nicotinic heteroreceptors mediating enhancement of glutamate release. Release-enhancing cholinergic autoreceptors in human neocortex are nAChRs with a pharmacological profile compatible with the alpha4beta2 subunit combination.     

Muscarinic pharmacology of the inhibition of adenylate cyclase in N18TG2 neuroblastoma cells

This study characterizes the muscarinic cholinergic receptors associated with the inhibition of adenylate cyclase on N18TG2 neuroblastoma cell membranes. Agonists could be divided into two classes: oxotremorine, acetylcholine, carbachol and arecoline exerted the most efficacious and potent inhibition, while McN-A343, bethanechol and AHR-602 were partial agonists. Both quinuclidinyl benzilate and atropine maximally antagonized the inhibitory effect of McN-A343, carbachol and oxotremorine. Pirenzepine was almost as potent as atropine in reversing the inhibitory effect of McN-A343, but was 300 times less potent than atropine or quinuclidinyl benzilate in antagonizing the effects of either carbachol or oxotremorine. Gallamine was ineffective as an antagonist at concentrations up to 1 mM. These results suggest that the receptors that modulate this inhibition are of the M₂ type, since they were activated by carbachol, acetylcholine and oxotremorine, but much less by McN-A343 and AHR-602 (both M₁ selective agonists). The full agonists were blocked by atropine and quinuclidinyl benzilate but not by low concentrations of pirenzepine (M₁ selective antagonist).