A comparison of affinity constants for muscarine-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29 degrees C and in ileum at 29 degrees C and 37 degrees C.
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1 The affinity of 17 compounds for muscarine-sensitive acetylcholine receptors in atrial pacemaker cells and ileum of the guinea-pig has been measured at 29 degrees C in Ringer-Locke solution. Measurements were also made at 37 degrees C with 7 of them. 2 Some of the compounds had much higher affinity for the receptors in the ileum than for those in the atria. For the most selective compound, 4-diphenylacetoxy-N-methylpiperidine methiodide, the difference was approximately 20-fold. The receptors in the atria are therefore different the structure from those in the ileum. 3 The effect of temperature on affinity are not the same for all the compounds, tested indicating different enthalpies and entropies of adsorption and accounting for some of the difficulty experienced in predicting the affinity of new compounds. (+info)
Effects of calcium-antagonistic drugs on the stimulation by carbamoylcholine and histamine of phosphatidylinositol turnover in longitudinal smooth muscle of guinea-pig ileum.
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A number of drugs classed as calcium antagonists, spasmolytics, non-specific receptor antagonists or receptor antagonists with multiple sites of action were tested to determine whether they prevent the stimulation of phosphatidylinositol turnover caused in various tissues by the activation of receptors which increase cell-surface Ca2+ permeability. The experiments were done with fragments of longitudinal smooth muscle from guinea-pig ileum; these were incubated in vitro with 32Pi and either 100 muM-carbamoylcholine or 100 muM-histamine, in the presence of antagonistic drugs at concentrations at least sufficient to cause complete blockade of smooth-muscle contraction. The phosphatidylinositol response to carbamoylcholine was not changed by cinchocaine, papaverine, nifedipine, dibenamine, amethocaine, cinnarizine, lidoflazine, methoxyverapamil, prenylamine or two antimuscarinic alkane-bis-ammonium compounds, and the response to histamine was unaffected by the first four drugs. In contrast, phenoxybenzamine prevented the increase in phosphatidylinositol labelling caused by either carbamoylcholine or histamine. The insensitivity of the phosphatidylinositol response to most of the drugs provides further experimental support for the conclusion that the receptor-stimulated phosphatidylinositol breakdown which initiates the increase in phosphatidylinositol turnover is not caused by an increase in intracellular Ca2+. The simplest interpretation of the available information appears to be that phosphatidylinositol breakdown plays a role in the coupling between the receptor-agonist interaction and the opening of cell-surface Ca2+ gates [Michell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147]. If this is correct, then phenoxybenzamine must exert its inhibitory effects on phosphatidylinositol breakdown early in this sequence of events, but the drugs must act at a stage later than phosphatidylinositol breakdown. The unexpected difference in the effects of dibenamine and phenoxybenzamine, which are chemically very similar, may provide a useful experimental tool with which to explore the way in which activated receptors provoke the opening of cell-surface Ca2+ gates. (+info)
Cholinergic modulation of neostriatal output: a functional antagonism between different types of muscarinic receptors.
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It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (RN) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic RN increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M1,4 muscarinic receptor antagonist pirenzepine but not by M2 or M3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing RN and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M1- and M2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits. (+info)
Mechanisms of verapamil inhibition of action potential firing in rat intracardiac ganglion neurons.
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The effects of verapamil and related phenylalkylamines on neuronal excitability were investigated in isolated neurons of rat intracardiac ganglia using whole-cell perforated patch-clamp recording. Verapamil (>/=10 microM) inhibits tonic firing observed in response to depolarizing current pulses at 22 degrees C. The inhibition of discharge activity is not due to block of voltage-dependent Ca2+ channels because firing is not affected by 100 microM Cd2+. The K+ channel inhibitors charybdotoxin (100 nM), 4-aminopyridine (0.5 mM), apamin (30-100 nM), and tetraethylammonium ions (1 mM) also have no effect on firing behavior at 22 degrees C. Verapamil does not antagonize the acetylcholine-induced inhibition of the muscarine-sensitive K+ current (M-current) in rat intracardiac neurons. Verapamil inhibits the delayed outwardly rectifying K+ current with an IC50 value of 11 microM, which is approximately 7-fold more potent than its inhibition of high voltage-activated Ca2+ channel currents. These data suggest that verapamil inhibits tonic firing in rat intracardiac neurons primarily via inhibition of delayed outwardly rectifying K+ current. Verapamil inhibition of action potential firing in intracardiac neurons may contribute, in part, to verapamil-induced tachycardia. (+info)
Sequestration of G-protein beta gamma subunits by different G-protein alpha subunits blocks voltage-dependent modulation of Ca2+ channels in rat sympathetic neurons.
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The membrane-delimited and voltage-dependent inhibition of N-type Ca2+ channels is mediated by Gbeta gamma subunits. Previously, exogenous excess GDP-bound GalphaoA has been shown to dramatically attenuate the norepinephrine (NE)-mediated Ca2+ current inhibition by sequestration of Gbeta gamma subunits in rat superior cervical ganglion (SCG) neurons. In the present study, we determined whether the attenuation of NE-mediated modulation is specific to GalphaoA or shared by a number of closely related (Galphatr, GalphaoB, Galphai1, Galphai2, Galphai3, Galphaz) or unrelated (Galphas, Galphaq, Galpha11, Galpha16, Galpha12, Galpha13) Galpha subunits. Individual Galpha subunits from different subfamilies were transiently overexpressed in SCG neurons by intranuclear injection of mammalian expression vectors encoding the desired protein. Strikingly, all Galpha subunits except Galphaz nearly blocked basal facilitation and NE-mediated modulation. Likewise, VIP-mediated Ca2+ current inhibition, which is mediated by cholera toxin-sensitive G-protein, was also completely suppressed by a number of Galpha subunits overexpressed in neurons. Galphas expression produced either enhancement or attenuation of the VIP-mediated modulation-an effect that seemed to depend on the expression level. The onset of the nonhydrolyzable GTP analog, guanylylimidodiphosphate-mediated facilitation was significantly delayed by overexpression of different GDP-bound Galpha subunits. Taken together, these data suggest that a wide variety of Galpha subunits are capable of forming heterotrimers with endogenous Gbeta gamma subunits mediating voltage-dependent Ca2+ channel inhibition. In conclusion, coupling specificity in signal transduction is unlikely to arise as a result of restricted Galpha/Gbeta gamma interaction. (+info)
Muscarinic receptor activity induces an afterdepolarization in a subpopulation of hippocampal CA1 interneurons.
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Cholinergic input to the hippocampus may be involved in important behavioral functions and the pathophysiology of neurodegenerative diseases. Muscarinic receptor activity in interneurons of the hippocampus may play a role in these actions. In this study, we investigated the effects of muscarinic receptor activity on the excitability of different subtypes of interneurons in rat hippocampal CA1. Most interneurons displayed an afterhyperpolarizing potential (AHP) after depolarization by injected current or synaptic stimulation. In the presence of a muscarinic agonist, the AHP of a subset of these interneurons was replaced by an afterdepolarization (ADP), often of sufficient magnitude to evoke action potentials in the absence of further stimulation. The ADP was insensitive to cadmium and low extracellular calcium. It was blocked by low extracellular sodium but not by tetrodotoxin or low concentrations of amiloride. Muscarinic ADPs were sometimes observed in isolation but were often accompanied by depolarizing, hyperpolarizing, or biphasic changes in the membrane potential. Interneurons with muscarinic ADPs were found in all strata of CA1 and did not fall into a single morphological classification. The potential functions of the prolonged action potential output of interneurons produced by the ADP could include changes in hippocampal circuit properties and facilitation of the release of peptide cotransmitters in these interneurons. (+info)
Nitric oxide acts independently of cGMP to modulate capacitative Ca(2+) entry in mouse parotid acini.
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Carbachol- and thapsigargin-induced changes in cGMP accumulation were highly dependent on extracellular Ca(2+) in mouse parotid acini. Inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) resulted in complete inhibition of agonist-induced cGMP levels. NOS inhibitors reduced agonist-induced Ca(2+) release and capacitative Ca(2+) entry, whereas the inhibition of sGC had no effect. The effects of NOS inhibition were not reversed by 8-bromo-cGMP. The NO donor GEA-3162 increased cGMP levels blocked by the inhibition of sGC. GEA-3162-induced increases in Ca(2+) release from ryanodine-sensitive stores and enhanced capacitative Ca(2+) entry, both of which were unaffected by inhibitors of sGC but reduced by NOS inhibitors. Results support a role for NO, independent of cGMP, in agonist-mediated Ca(2+) release and Ca(2+) entry. Data suggest that agonist-induced Ca(2+) influx activates a Ca(2+)-dependent NOS, leading to the production of NO and the release of Ca(2+) from ryanodine-sensitive stores, providing a feedback loop by which store-depleted Ca(2+) channels are activated. (+info)
Differential inflammatory modulation of canine ileal longitudinal and circular muscle cells.
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The aim of this study was to identify the subtypes of muscarinic receptors that mediate in vivo and in vitro canine ileal longitudinal muscle contractions and whether their role is modulated by inflammation. Previous studies have reported that circular muscle contractions are suppressed in ileal inflammation induced by mucosal exposure to ethanol and acetic acid. We found that inflammation had no significant effect on in vivo and in vitro spontaneous or muscarinic receptor-mediated contractions of the longitudinal muscle. The longitudinal muscle contractions were mediated primarily by the M(3) receptor subtype. However, the IC(50) of the M(2) receptor antagonist methoctramine was only 10 times greater than that of the M(3) receptor antagonist 4-DAMP in the longitudinal muscle, whereas it was 224 times greater in the circular muscle. M(2) receptor-coupled decrease of intracellular cAMP occurred in the longitudinal but not in the circular muscle from the normal ileum. Inflammation did not alter this coupling in the longitudinal muscle but established it in the circular muscle. In conclusion, M(2) receptors may play a greater role in the mediation of longitudinal muscle contractions than circular muscle contractions. Inflammation does not alter the contractility or the relative role of muscarinic receptor subtypes in longitudinal muscle cells. However, it modulates the M(2) receptor coupling to adenylate cyclase in the circular muscle. (+info)