Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors

The role of muscarinic receptors in the down‐regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10⁻⁶ M) and atropine (10⁻⁸ to 10⁻⁶ M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10⁻⁶ M) caused a slight depression in the amplitude of responses to ACh pressure‐applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA‐immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10⁻⁶ M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10⁻⁶ M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10⁻⁶ M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 420–431, 1999     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors.

The role of muscarinic receptors in the down-regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10(-6) M) and atropine (10(-8) to 10(-6) M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10(-6) M) caused a slight depression in the amplitude of responses to ACh pressure-applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA-immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10(-6) M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10(-6) M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10(-6) M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach,Periplaneta americana

Summary Intracellular microelectrode recording and ionophoretic application of carbamylcholine (CCh) were used to compare the cholinergic sensitivity of postsynaptic dendrites of an identified neurone with that of an identified presynaptic cholinergic axon.The axon of the lateral filiform hair sensory neurone (LFHSN) in the first-instar cockroachPeriplaneta americana was found to be as sensitive to CCh as the dendritic regions of giant interneurone 3 (GI 3). The CCh response of both neurones was unaffected by replacing Ca²⁺ with Mg²⁺, confirming that the ACh receptors are present on the neurones under test. The CCh response of both neurones was mimicked by ionophoretic application of nicotine. The responses were blocked by 10^–5 M mecamylamine and 10^–6 M d-tubocurarine and were not affected by muscarinic antagonists, suggesting that the ACh receptors present on GI 3 and LFHSN are predominantly nicotinic.The muscarinic agonist oxotremorine and the antagonists atropine and quinuclidinyl benzilate had no modulatory effect on LFHSN-GI 3 synaptic transmission.The latency of the LFHSN response to CCh was consistent with the hypothesis that ACh receptors are situated on the main axon/terminal within the neuropil of the ganglion. It has previously been shown that this region of the axon does not form output synapses (Blagburn et al. 1985a). This indirect evidence indicates that presynaptic or extrasynaptic ACh receptors are present in the membrane of a cholinergic axon.LFHSN was depolarized by synaptically-released ACh after normal or evoked spike bursts, suggesting that the nicotinic ACh receptors act as autoreceptors. However, it was not possible to obtain direct evidence to support the hypothesis that these receptors modulate ACh release.Abbreviations CCh carbamylcholine - GI giant interneurone - FHSN filiform hair sensory neurone - LFHSN lateral filiform hair sensory neurone - R _in input resistance - V depolarization - V _m resting potential     

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.     

Cholinergic transmission at mechanosensory afferents in the crayfish terminal abdominal ganglion

Electrical stimulation of mechanosensory afferents innervating hairs on the surface of the exopodite in crayfish Procambarus clarkii (Girard) elicited reciprocal activation of the antagonistic set of uropod motor neurones. The closer motor neurones were excited while the opener motor neurones were inhibited. This reciprocal pattern of activity in the uropod motor neurones was also produced by bath application of acetylcholine (ACh) and the cholinergic agonist, carbamylcholine (carbachol). The closing pattern of activity in the uropod motor neurones produced by sensory stimulation was completely eliminated by bath application of the ACh blocker, d-tubocurarine, though the spontaneous activity of the motor neurones was not affected significantly. Bath application of the acetylcholinesterase inhibitor, neostigmine, increased the amplitude and extended the time course of excitatory postsynaptic potentials (EPSPs) of ascending interneurones elicited by sensory stimulation. These results strongly suggest that synaptic transmission from mechanosensory afferents innervating hairs on the surface of the tailfan is cholinergic.Bath application of the cholinergic antagonists, dtubocurarine (vertebrate nicotinic antagonist) and atropine (muscarinic antagonist) reversibly reduced the amplitude of EPSPs in many identified ascending and spiking local interneurones during sensory stimulation. Bath application of the cholinergic agonists, nicotine (nicotinic agonist) and oxotremorine (muscarinic agonist) also reduced EPSP amplitude. Nicotine caused a rapid depolarization of membrane potential with, in some cases, spikes in the interneurones. In the presence of nicotine, interneurones showed almost no response to the sensory stimulation, probably owing to desensitization of postsynaptic receptors. On the other hand, no remarkable changes in membrane potential of interneurones were observed after oxotremorine application. These results suggest that ACh released from the mechanosensory afferents depolarizes interneurones by acting on receptors similar to vertebrate nicotinic receptors.Abbreviations ACh cetylcholine - mns motor neurones - asc int ascending interneurone     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

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.     

Effects of atropine on the release of newly synthesized acetylcholine from rat striatal slices at various concentrations of calcium ions

The release of acetylcholine (ACh) from brain tissue is known to be inhibited by muscarinic autoreceptors on cholinergic nerve terminals but the mechanism of the inhibition is not understood. Atropine brings about an increase of ACh release by removing the inhibitory action of autoreceptors. We investigated whether the effect of atropine on the release of [¹⁴C]ACh newly synthesized during incubations from [U-¹⁴C] glucose depends on the concentration of Ca²⁺ in the medium. In rat striatal slices incubated in the presence of an inhibitor of cholinesterases and of 30 mmol/l K⁺, significant increases in the release of [¹⁴C]ACh elicited by atropine were only observed during incubations with very low concentrations of Ca²⁺. This finding supports the view that the activation of presynaptic muscarinic autoreceptors in the brain affects the release of ACh by reducing the availability of Ca²⁺ that is required for transmitter liberation.     

Role of Acetylcholine Receptors and Dopamine Transporter in Regulation of Extracellular Dopamine in Rat Carotid Body Cultures Grown in Chronic Hypoxia or Nicotine

Abstract: Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for ～12 days in normoxia (Nox; 20% O₂), chronic hypoxia (CHox; 6% O₂), or chronic nicotine (CNic; 10 µM nicotine, 20% O₂), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (～15-min) exposure to hypoxia (5% O₂; ～6× basal), high extracellular K⁺ (30 mM; ～10× basal), nomifensine (1 µM; a selective DA uptake inhibitor; ～3× basal), and nicotine (100 µM; ～5× basal), but not methylcholine (300 µM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (～9× control), the stimulatory effect of high K⁺ (3–4× basal) and acute hypoxia (～2× basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (～9× control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (～2× basal), nicotine, nomifensine, and high K⁺ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 µM), a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-min), treatment with the muscarinic antagonist atropine (1 µM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.     

Age-Related Alterations In Pre-Synaptic and Receptor-Mediated Cholinergic Functions In Rat Brain

Fractional [³H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [³H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [³H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [³H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [³H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [³H]ACh release from striatum of both age groups is modulated mainly by M₃ muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-³H]inositol decreased with aging, carbachol-stimulated [³H]myo inositol-1-fosfat (IP₁) accumulation was found similar in striatal, cortical and hippocampal slices.     

Cholinergic Dilatation and Constriction of Feline Cerebral Blood Vessels Are Mediated by Stimulation of Phosphoinositide Metabolism via Two Different Muscarinic Receptor Subtypes

Abstract: The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP₃) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140–280% of baseline at 10^?3M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30–50% of the maximal stimulation) obtained at low carbachol concentrations (<10^?5M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP₃ accumulation induced by 10^?7M (endothelium-dependent submaximal effect) and 10^?4M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP₃ production by 10^?4M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC₅₀): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11-dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M₁ receptor subtype. In contrast, the submaximal stimulation of the PI metabolism elicited by 10^?7M carbachol in major arteries was blocked by the same antagonists with the following order of potency (in -log IC₅₀): 4-DAMP (8.38) > pirenzepine (7.25) > UH-AH 371 (6.25) > AF-DX 116 (5.72), which was reminiscent of an M₃ pharmacological profile. These findings indicate that stimulation of cerebrovascular muscarinic receptors is accompanied by PI hydrolysis via two distinct receptors, most probably the M₁ and M₃ subtypes that have been associated with constriction and dilatation, respectively, of cat cerebral arteries. Furthermore, these results provide strong evidence for an endothelial localization of the M₃ dilatatory receptors within the vessel wall.     

Modulatory action of acetylcholine on the Na-dependent action potentials in Kenyon cells isolated from the mushroom body of the cricket brain

Kenyon cells, intrinsic neurons of the insect mushroom body, have been assumed to be a site of conditioning stimulus (CS) and unconditioned stimulus (US) association in olfactory learning and memory. Acetylcholine (ACh) has been implicated to be a neurotransmitter mediating CS reception in Kenyon cells, causing rapid membrane depolarization via nicotinic ACh receptors. However, the long-term effects of ACh on the membrane excitability of Kenyon cells are not fully understood. In this study, we examined the effects of ACh on Na⁺ dependent action potentials (Na⁺ spikes) elicited by depolarizing current injection and on net membrane currents under the voltage clamp condition in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Current-clamp studies using amphotericin B perforated-patch recordings showed that freshly dispersed cricket Kenyon cells could produce repetitive Na⁺ spikes in response to prolonged depolarizing current injection. Bath application of ACh increased both the instantaneous frequency and the amplitudes of Na⁺ spikes. This excitatory action of ACh on Kenyon cells is attenuated by the pre-treatment of the cells with the muscarinic receptor antagonists, atropine and scopolamine, but not by the nicotinic receptor antagonist mecamylamine. Voltage-clamp studies further showed that bath application of ACh caused an increase in net inward currents that are sensitive to TTX, whereas outward currents were decreased by this treatment. These results indicate that in order to mediate CS, ACh may modulate the firing properties of Na⁺ spikes of Kenyon cells through muscarinic receptor activation, thus increasing Na conductance and decreasing K conductance.     

Cholinergic control of the mechanical properties of the catch connective tissue in the holothurian body wall

Effects of acetylcholine (ACh), ACh-agonists and antagonists were studied on the viscosity of the dermis of the sea cucumber Holothuria leucospilota. ACh and nicotinic agonists caused an early increase in viscosity and late decrease. Muscarinic agonists produced a viscosity decrease. The viscosity increase elicited by nicotine was inhibited by tubocurarine. The viscosity decrease caused by methacholine was suppressed by atropine. The mechanical properties of this connective tissue are very likely controlled by both nicotinic and muscarinic cholinoreceptors.     

Studies on the Characterization of the Subtype(S) of Muscarinic Receptor Involved in Prostacyclin Synthesis in Rabbit Cardiomyocytes

Abstract

The present study was conducted to localize and characterize the subtype(s) of muscarinic receptor involved in prostacyclin (PGI₂) production elicited by the cholinergic transmitter acetylcholine (ACh) in various cell types in the rabbit heart. ACh increased PGI₂ synthesis measured as 6-keto-PGF1α, in cultured coronary endothelial cells and freshly dissociated ventricular myocytes in a dose dependent manner but not in cultured coronary smooth muscle cells of rabbit heart. McN-A-343, a partially selective M₁ muscarinic ACh receptor (mAChR) agonist, did not alter 6-keto-PGF1α synthesis in these cell types. ACh induced 6-keto-PGF1α synthesis in coronary endothelial cells and ventricular myocytes was not altered by a low concentration (10^?8 M) of pirenzipine, an M₁ mAChR antagonist but was reduced by a higher concentration (10^?6 M). In coronary endothelial cells ACh induced 6-keto-PGF1α production was reduced by hexahydro-sila-difendial (HHSiD), an M₃ mAChR antagonist, and in ventricular myocytes by both 11-(2-[(di-ethylamino) methyl]-1-piperidinyl]acetyl-5,11-dihydro-6-H-pyrido-[2,3-b]-benzodiazepine-6 one] (AF-DX 116), an M₂ receptor antagonist, and HHSiD. The decrease by ACh of isoporterenol stimulated cAMP accumulation was minimized by AF-DX 116 but not by HHSiD or pirenzipine. Pertussis toxin treatment minimized ACh induced decrease in isoproterenol stimulated rise in cAMP and ATP release, but not ACh induced 6-keto-PGF1α synthesis. These data suggest that ACh stimulates prostacyclin production in coronary endothelial cells via M₃ mAChR and in ventricular myocytes M₂ and M₃ mAChR. Moreover, ACh induced decrease in cAMP, but not the increase in 6-keto-PGF1α production, is mediated by pertussis toxin sensitive Gαi proteins in these cells.     

Synaptic effects of nicotinic and muscarinic agonists in sympathetic ganglia of the frog

Superfusion of the isolated sympathetic ganglia of the frog with nicotinic agonists (suberyldicholine, tetramethylamonium, and dimethylphenylpiperazinium), as well as acetylcholine in the presence of atropine led to a brief depolarization of the neurons and blockade of synaptic transmission. The muscarinic agonists methylfurmethide (MFM) and methyldilvasen, cis^–, L(+), as well as acetylcholine elicited a stable depolarization which is not accompanied by disturbance in transmission. Oxotremorine at a concentration of 1·10^–5 M did not lead to the depolarization of the post-synaptic membrane, but at a concentration of 1·10^–6 M decreased the quantal EPSP content twofold, which indicates that the presynaptic receptors belong to the M₂ subtype. Inhibition of acetylcholinesterase significantly intensified the postsynaptic effect of MFM: a shift of the concentration-effect curve took place toward the side of lower MFM concentrations. It was shown that the post-synaptic muscarinic receptors of the ganglionic neurons possess varied sensitivity to the enantiomers of methyldilvasen and, consequently, are stereospecific. The identified functional properties of the cholinoreceptors of the ganglionic neurons explain the set of changes in synaptic transmission under conditions of the prolonged presence of a mediator in the synaptic cleft.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 227–234, March–April, 1988.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

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.     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.     

Cholinergic Receptors in the Human vas Deferens

Abstract

This study represents the first investigation demonstrating the contractile response to exogenous acetylcholine (ACh) in the isolated human vas deferens. Pharmacological characterization of cholinergic receptors was achieved using selective antagonists to define receptor subtypes. In the HVD the effect of exogenous ACh is revealed as a dose-dependent sudden increase in the basal tension of the vasa. The ACh receptors of the HVD were competitively antagonized by atropine (ATR) with a high pA₂ value (8.78). The main finding of this study is the presence of cholinergic receptors of the pharmacologically defined M₂-ACh subtype in the isolated HVD, according to the pA₂ values obtained with pirenzepine (PRZ) 7.39, AF-DX 116 (AF) 5.92 and 4-DAMP 5.65, M₁-ACh, _M2-ACh and M₃-ACh selective antagonists, respectively. Prazosin (PZ), a selective α₁-adrenergic antagonist, displayed a similar competitive antagonism for the contractile response evoked both by ACh (pA₂ = 8.69) and NE (pA₂ = 8.58) in the HVD. The antagonism exerted by PZ on the ACh-induced contractile response of the HVD, suggests that ACh probably acts at a presynaptic level stimulating the release of NE from an adrenergic neuron. According to these findings, the receptor involved in this action, located in the proximity of the nerve terminals, seems to be of the M₂-ACh subtype.