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At least 19 recordsLinked to original sources
PubMed1991

The sources of calcium for carbachol-induced contraction in the circular smooth muscle of guinea-pig stomach.

1. The action of carbachol on the mechanical activity of circular muscle from guinea-pig upper stomach was studied. High concentrations of carbachol (e.g. 10(-4) M) produced a rapid phasic contraction followed by a smaller, sustained tonic contraction. Low concentrations (e.g. 10(-7) M) caused a contraction which did not generally show marked distinction between phasic and tonic components. 2. The response to 10(-7) M carbachol was very sensitive to 10(-5) M nifedipine as was the phasic response to 10(-4) M carbachol. The tonic contraction to the latter, however, was only slightly reduced by nifedipine. 3. The carbachol-induced contractions remaining in the presence of nifedipine were dose-related and very dependent on the presence of external calcium. 4. Carbachol, 10(-7) M, did not produce a contraction after 4 min exposure to calcium-free solution whereas 10(-4) M carbachol did and this was phasic in nature but much reduced relative to the control in normal Ca. 5. A phasic followed by a small tonic contraction to 10(-4) M carbachol was seen superimposed on the K contracture in tissues depolarized with 100 mM K, whereas only a small tonic response occurred for 10(-7) M carbachol. 6. In the absence of a functional carbachol-sensitive intracellular store, 10(-4) M carbachol was unable to trigger a contraction in calcium-free solution. However, when calcium was simultaneously readmitted with carbachol after exposure to calcium-free solution, a contraction occurred. 7. Carbachol, 10(-7) M, did not significantly increase inositol polyphosphate levels, whereas 10(-4) M carbachol did. The increase with 10-4M occurred rapidly peaking within 2min and was undetectable after 5 min, in the absence of lithium. 8. It is concluded that low concentrations of muscarinic agonist trigger a contraction predominantly through a nifedipine-sensitive route whereas higher concentrations further utilize intracellular calcium release and a receptor-operated extracellular calcium-dependent pathway. The former is probably associated with the phasic component and the latter with the tonic one.

Animals↗
PubMed1992

Protein kinase C does not participate in carbachol's secretory action in T84 cells.

We investigated the role of protein kinase C (PKC) in mediating carbachol's stimulation of transepithelial Cl- secretion in T84 cells. Direct PKC activation with phorbol 12-myristate 13-acetate (PMA) stimulated transepithelial Cl- transport (measured as the short-circuit current), demonstrating that PKC could interact with the secretory apparatus. Carbachol stimulated PKC activity, suggesting that the enzyme might participate in the hormone's action. Diacylglycerol metabolism inhibitors (DMIs), known to augment hormone-stimulated increases in diacylglycerol levels, potentiated the short-circuit current response to carbachol. The effect of DMIs was not due to amplification of carbachol-induced increases in PKC activity, however; PKC activity during carbachol stimulation was no higher in the presence of DMIs than in their absence. Augmentation of carbachol's action by DMIs appeared to be due to the direct activation of PKC which, like PMA, stimulated the Cl- conductance of the apical membrane (GCl). The effects of DMIs and carbachol on GCl were additive. Carbachol itself stimulated GCl but not by activating PKC; staurosporine did not blunt the effect of carbachol on GCl. Nor did staurosporine reduce the effect of carbachol on transepithelial Cl- secretion. These observations demonstrate that PKC does not participate in the secretory action of carbachol in T84 cells and suggest that direct PKC activation with DMIs and PMA stimulates an apical pool of PKC that is not accessible to carbachol applied to the basolateral membrane.

Alkaloids↗
PubMed1990

Carbachol desensitizes pancreatic enzyme secretion by downregulation of receptors.

First incubating guinea pig pancreatic acini with carbachol reduced the subsequent stimulation of amylase release caused by carbachol, cholecystokinin octapeptide (CCK-8), and bombesin but not that caused by vasoactive intestinal peptide, substance P, 8-bromoadenosine 3',5'-cyclic monophosphate, A23187, or 12-O-tetradecanoylphorbol-13-acetate. Carbachol also reduced the subsequent binding of N-[3H]methylscopolamine, 125I-CCK-8, and 125I-[Tyr4]bombesin. Pancreatic acini possess a high-affinity class of cholinergic receptors and a low-affinity cholinergic receptors appears to produce the reduction in carbachol-stimulated amylase release and binding of N-[3H]methylscopolamine. First incubating acini with carbachol caused a complete loss of high-affinity cholinergic receptors with no change in the number or affinity of low-affinity cholinergic receptors. Carbachol occupation of low-affinity cholinergic receptors appears to produce the reduction in CCK-8- and bombesin-stimulated amylase release and in binding of 125I-CCK-8 and 125I-[Tyr4]bombesin. Acini possess two classes of CCK receptors. One class has a high affinity for CCK-8; the other class has a low affinity for CCK-8. First incubating acini with carbachol caused a 60% decrease in the number of high-affinity CCK receptors with no change in the number of low-affinity receptors or the affinities of either class of receptors for CCK-8. Acini possess a single class of bombesin receptors, and first incubating acini with carbachol caused a 40% decrease in the number of bombesin receptors with no change in their affinity for bombesin. 12-O-tetradecanoyl phorbol-13-acetate reproduced the action of carbachol on binding of N-[3H]methylscopolamine and 125I-CCK-8 but not on binding of 125I-[Tyr4]bombesin, suggesting that carbachol activation of protein kinase C may in some way mediate the effect of carbachol on receptors for carbachol and those for CCK but not that on receptors for bombesin.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1976

An analysis of the mode of action of carbachol on the chick biventer cervicis nerve--muscle preparation.

The mechanism of contracture evoked by carbachol in the isolated chick biventer cervicis nerve--muscle preparation was studied. At concentrations lower than 11 muM, carbachol progressively induced contracture at a rate much slower than did acetylcholine. A spontaneous increase of the response to carbachol, but not to acetylcholine, was observed 2-4 hr after isolation of the muscle. By contrast, no change of the response occurred in the denervated muscle. Anticholinesterase treatment shifted the dose-response curve for carbachol markedly to the left as far as the contracture attained after 4-6 min incubation was concerned. The shift was much less marked for the dose-response curve plotted against the initial rate of response defined as the contracture obtained after 1 min incubation. No enhancement of response was detected by inhibition of acetylcholinesterase in the denervated muscle. beta-Bungarotoxin which blocked neuromuscular transmission within 30 min, caused a transient enhancement of the response to carbachol. After 2-3 hr treatment, however, the spontaneous increase of response to carbachol was counteracted by the toxin. No potentiation of the response by anticholinesterase agents was observed after toxin treatment. When added in the presence of physostigmine or echothiophate, beta-bungarotoxin reduced the response to carbachol to the control level in 2 hr. The response to carbachol in the muscle treated with alpha-bungarotoxin and washed subsequently was also not potentiated by anticholinesterase treatment. Repetitive nerve stimulation in the presence of hemicholinium-3 caused a neuromuscular blockade but did not appreciably antagonize the response to carbachol more than that to acetylcholine either in the absence or presence of physotigmine. When calcium ion in the medium was decreased from 2.7 to 0.54 mM, ther response to nerve stimulation was nearly completely inhibited, but the response to carbachol was not affected and could still be potentiated by anticholinesterase. It is concluded that carbachol has both direct and indirect effects on the chick biventer cervicis muscle. At a low concentration, particularly in the presence of an anticholinesterase and if sufficient time of incubation is allowed, the indirect effect caused by release of acetylcholine may become more prominent than the direct action on the post-synaptic receptor. Both the mechanism and the store of acetylcholine for this indirect action appear to be different from those for the nerve impulse.

Acetylcholine↗
PubMed1990

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol.

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1975

Pre- and postjunctional neuromuscular blockade by carbachol.

Carbachol, when applied to the bathing Ringer solution of frog sartorius muscles, caused depolarization of the endplate and a blockade of endplate potentials (EPP's), miniature EPP's (mepp's) and the iontophoretic acetylcholine potential. In muscles treated with an analog of hemicholinium-3, alpha, alpha'bis(dimethylammonium acetaldehyde diethylacetal)-p-p'-diacetylbiphenyl dibromide (DMAE), depolarization of the endplate by carbachol was blocked and the blockade by carbachol of the iontophoretic acetylcholine potential was prevented. These responses to carbachol were attributed to a postjunctional action that was antagonized by DMAE. In contrast, the blockade by carbachol of EPP's and mepp's was enhanced in DMAE-treated muscles at a time when carbachol-induced depolarization was blocked. This response to carbachol was attributed to a pre-junctional action. Carbachol either blocked transmitter release by a mechanism that was insensitive to DMAE or enhanced the prejunctional blocking actions of DMAE. Succinylcholine had actions similar to carbachol. DMAE prevented depolarization by succinylcholine but enhanced neuromuscular blockade by succinylcholine. SKF 525-A (beta-diethylaminoethyl diphenylpropylacetate hydrochloride), like DMAE, prevented depolarization but not transmission blockade caused by carbachol.

Acetylcholine↗
PubMed1991

Potential mechanisms involved in the negative coupling between serotonin 5-HT1A receptors and carbachol-stimulated phosphoinositide turnover in the rat hippocampus.

Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 microM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) greater than ipsapirone (20) greater than gepirone (120) greater than RU 24969 (140) greater than buspirone (560) greater than 1-(m-trifluoromethylphenyl)piperazine (1,500) greater than methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 microM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 microM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 microM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 microM) and arachidonic acid (100 microM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 microM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 microM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a gamma-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

GTP-binding protein involvement in membrane currents evoked by carbachol and histamine in guinea-pig ileal muscle.

1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea-pig ileum were used for recording membrane currents under whole-cell voltage clamp in response to carbachol (100 microM, unless otherwise stated) or histamine (100 microM) applied extracellularly. 2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca(2+)-activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents. 3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of -40 mV, which was considered to represent opening of cationic channels. The carbachol-induced inward current was much longer in duration and larger in size than the histamine-induced inward current. 4. Inclusion of GDP beta S (2 mM) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol. 5. When the holding potential was held at 0 mV with inclusion of GTP gamma S (0.1-1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break-through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTP gamma S had released Ca2+ stores. When the holding potential was held at -40 mV and GTP gamma S (0.1 or 0.2 mM) was present in the pipette, an inward current developed a few minutes after break-through. During the GTP gamma S-induced inward current, application of carbachol or histamine produced no further inward current. However, when 0.01 mM-GTP gamma S was included in the pipette solution, carbachol- and histamine-induced inward currents were potentiated. 6. Pretreated with 2-5 micrograms/ml pertussis toxin (PTX) did not change noticeably the outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Modulation of Na+, Cl- and HCO3- transport by carbachol in pig distal jejunum.

Acetylcholine and cholinomimetics such as carbachol are potent stimulants of epithelial Cl- secretion in the small and large intestines of several mammalian species. In this study, the effects of carbachol were characterized in vitro on active ion transport in sheets of submucosa-mucosa from the distal jejunum of swine. Carbachol (10 microM) produced an increase in serosa-positive short-circuit current (Isc) in this tissue after its serosal, but not luminal administration. The Na(+)-K(+)-Cl- transport blocker bumetanide (10 microM) produced a 50% decrease in the carbachol-induced Isc elevation after its serosal administration. Peak increases in Isc evoked by carbachol were significantly reduced by 60-85% in tissues bathed in media lacking Cl-, HCO3-, or both anions. The initial drug-induced increase in net charge transfer from serosa to lumen was dependent upon both HCO3- and Cl-, whereas sustained elevations in charge transfer were dependent upon extracellular Cl- only. Radiotracer flux analyses revealed that the drug decreased net Na+ absorption and increased Cl- secretion. In the absence of HCO3-, carbachol decreased Cl- absorption. The effects of carbachol on HCO3- transport were examined by pH-stat titration. The drug rapidly alkalinized the luminal medium immediately after its serosal administration. These results suggest that carbachol stimulates electrogenic anion secretion in the mucosa of the porcine distal jejunum. Furthermore, the ability of carbachol to inhibit spontaneous Na+ absorption is dependent upon extracellular HCO3-.

Animals↗
PubMed1992

Carbachol stimulates adenylate cyclase and phospholipase C and muscle contraction-relaxation in a reciprocal manner in dog iris sphincter smooth muscle.

In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulation of phospholipase C and muscle contraction or to the stimulation of adenylate cyclase and muscle relaxation, this was found to be dependent upon the concentration of the muscarinic agonist. In contrast to the dog, muscarinic receptors in iris sphincters from different mammalian species were found to be coupled to phospholipase C and contraction at all concentrations of carbachol investigated (1-100 microM). In the dog sphincter, lower concentrations (less than 5 microM) of carbachol stimulated myo-inositol 1,4,5-trisphosphate (IP3) production, inhibited cAMP formation and induced contraction, and higher concentrations (greater than 5 microM) enhanced cAMP formation, inhibited IP3 production and induced relaxation. The mechanisms for the stimulatory effects on cAMP formation through muscarinic receptors were investigated. Carbachol (25 microM) increased both basal and isoproterenol- and forskolin-stimulated cAMP levels. Atropine inhibited the carbachol-stimulated increase in cAMP levels in a dose-dependent manner with an IC50 of 9 nM. Intracellular Ca2+, derived from IP3-induced Ca2+ release and/or from muscarinic receptor-operated Ca2+ influx, and protein kinase C may mediate the muscarinic receptor-linked rise in intracellular cAMP. This conclusion is supported by the following findings. (1) At short time intervals (less than 1 min) carbachol (25 microM) increased IP3 production and contraction and this was followed (between 1 and 20 min) by cAMP formation and muscle relaxation. (2) Carbachol-stimulated IP3 production was detected at a concentration of the agonist 26-fold lower than that required for cAMP formation, and it was completely blocked by the phorbol ester, phorbol 12,13-dibutyrate (50 nM). (3) A Ca(2+)-calmodulin stimulated adenylate cyclase was demonstrated in membranes from dog iris sphincter but not in that from rabbit and bovine. (4) Trifluoperazine (0.1 microM), a calmodulin antagonist, inhibited the carbachol-stimulated cAMP accumulation. (5) The Ca2+ ionophore A23187 and the phorbol ester increased cAMP production in a dose-dependent manner. A23187 potentiated cAMP production induced by either carbachol or by the phorbol ester. (6) Muscarinic stimulation of cAMP production persisted even after the tissue was pretreated with the phorbol ester or staurosporine. (7) Nifedipine (0.01-0.5 microM), a Ca2+ channel antagonist, inhibited carbachol stimulation of cAMP production, suggesting the presence of a muscarinic receptor-operated Ca2+ influx pathway in this tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1992

Carbachol does not down-regulate substance P receptors in pancreatic acini.

In a previous study, we found that first incubating guinea pig pancreatic acini with carbachol caused desensitization of the enzyme secretory response to cholecystokinin-octapeptide (CCK-8), bombesin, and carbachol but not that to substance P. This carbachol-induced desensitization could be accounted for by carbachol-induced down-regulation of receptors for CCK-8, bombesin, and carbachol. Although carbachol did not desensitize the enzyme secretory response to substance P, an effect of carbachol on substance P receptors was not examined. In the present study, in dispersed acini from guinea pig pancreas, substance P caused a twofold increase in amylase secretion. Stimulation was half-maximal at 0.7 nM and was maximal at 10 nM. Analysis of the ability of substance P to inhibit binding of 125I-substance P to substance P receptors indicated that acini possess a single class of receptors for substance P (Kd = 0.8 +/- 0.1 nM; Bmax = 1,037 +/- 145 fmol/mg of DNA). There was a close correlation between the relative potency with which substance P stimulated amylase secretion (0.7 nM) and the potency for inhibiting binding of 125I-substance P (Kd = 0.8 nM). First incubating pancreatic acini with carbachol did not alter either substance P-stimulated enzyme secretion or binding of 125I-substance P to substance P receptors, whereas in the same experiments, carbachol reduced binding of 125I-CCK-8 to cholecystokinin receptors by 50% and decreased in CCK-8-stimulated enzyme secretion by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1992

Carbachol-induced decrease in thyroid cell adenylyl cyclase activity is independent of calcium and phosphodiesterase activation.

The mechanism of adenylyl cyclase desensitization by carbachol, an agent that stimulates polyphosphoinositide hydrolysis, was studied in thyroid cells. Incubation of cultured dog thyroid cells with 10 microM carbachol for 2-4 hr reduced the subsequent thyrotropic hormone (TSH) stimulation of adenylyl cyclase activity of membrane preparations by approximately 40%. This inhibition was reversed by atropine, occurred even in a Ca(2+)-free medium containing ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, and was not reproduced by the Ca2+ ionophore A23187. The carbachol effect was not prevented by simultaneous incubation of cells with either isobutylmethylxanthine, an inhibitor of phosphodiesterase, or H-7, an inhibitor of protein kinase. Pretreatment of cells with pertussis toxin to inactivate the Gi inhibitory protein also failed to affect the carbachol inhibition. Although carbachol did not reduce the basal or the TSH-stimulated cyclase activities when added to membranes directly during the assay, exposure of cells to carbachol for 2-4 hr resulted in long lasting inhibition of TSH-stimulated cyclase activity (for at least 24 hr); recovery was seen by 48 hr after its removal. Carbachol pretreatment had no effect on 125I-TSH binding to membranes but reduced the cyclase stimulation by not only TSH but also cholera toxin, guanosine 5'-O-(3-thio)triphosphate, and forskolin; it also significantly reduced the cholera toxin-mediated AD[32P]-ribosylation of Gs in membranes. These data indicate that carbachol-induced inhibition of adenylyl cyclase occurs beyond the level of TSH receptor binding and that Gs is a possible site of its action. Thus, in dog thyroid cells, carbachol, via muscarinic receptors, can reduce the adenylyl cyclase activity by a process that does not involve Ca2+ or activation of phosphodiesterase.

Adenylate Cyclase Toxin↗
PubMed1992

Carbachol potentiates isoprenaline-induced mucin secretion by rat submandibular gland.

We studied the effects of carbachol on isoprenaline-induced secretion of 3[H]glucosamine-labeled high molecular weight protein from in vitro fragments of rat submandibular gland. The concentration-response curve for isoprenaline was determined alone and in the presence of one of two concentrations of carbachol. Isobolic interaction indices were calculated from the resulting curves. The role of extracellular calcium on this interaction was assessed by examining the effect of increasing concentrations of [Ca2+]e on secretion induced by a fixed concentration of isoprenaline alone and in the presence of carbachol. Carbachol alone caused a small, but statistically significant, protein secretion. Carbachol markedly shifted the isoprenaline concentration-response curve to the left in a dose-dependent manner. In the absence of extracellular calcium, carbachol did not increase isoprenaline-induced secretion, but secretion increased as the [Ca2+]e was increased. The carbachol-induced increase in secretion reached a maximal level at approximately 2.0 mM [Ca2+]e. We conclude that carbachol's increase of isoprenaline-induced secretion of 3[H]glucosamine-labeled high molecular weight protein is not additive, but is true potentiation. This potentiation requires extracellular calcium, and may be maximal at physiological [Ca2+]e.

Animals↗
PubMed1991

Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells.

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.

Adenosine↗
PubMed1990

Activation of phospholipase C in rabbit brain membranes by carbachol in the presence of GTP gamma S; effects of biological detergents.

Rabbit brain cortical membranes incubated with carbachol in the presence of GTP gamma S show a marked increase in the degradation of exogenous phosphatidylinositol 4,5-bisphosphate. This activation of phospholipase C is dependent on the presence of deoxycholate and maximal at 0.8-1 mM deoxycholate. There is negligible activation by carbachol alone but in the presence of GTP gamma S a carbachol effect can be readily demonstrated. Optimal activation of phospholipase C by carbachol was seen at 10 to 100 nM free Ca2+. Washing cortical membranes with hypertonic buffer extracted 60% of the membrane protein yet the carbachol and GTP gamma S coupling remained intact. Incubation of the membranes with lysophosphatidylcholine, Nonidet P-40, sodium deoxycholate or digitonin at concentrations considerably less than those frequently used to solubilize membrane proteins abolished the carbachol response. Octyl glucoside and sodium cholate also uncoupled receptor regulation of phospholipase C but only at concentrations where solubilization of membrane proteins occurred. Prior exposure of membranes to carbachol did not prevent the uncoupling observed as a result of detergent treatment. Incubation of the membranes with carbachol and GTP gamma S did not appear to be accompanied by specific release of either active phospholipase C or inhibitors of phospholipase C activity.

Animals↗
PubMed1992

Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells.

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 microM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [32P]phosphoinositide breakdown, the first peak of carbachol (1 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2). The second peak was found to be related to [32P]PIP2 breakdown. Carbachol stimulated the release of [3H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [3H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [3H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-microM)-induced release of [3H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.

Alkaloids↗
PubMed1990

Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role.

To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K+ gradient (Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986. J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in [Ca]'i and gK. The carbachol-induced changes in gK appeared to be dependent on the increase in [Ca]i because stimulation of gK was significantly diminished when the hormone-induced increase in [Ca]'i was blunted, either by loading the cells with BAPTA or by reducing the extracellular [Ca]. The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state [Ca]'i. The changes in gK and [Ca]'i after stimulation with carbachol were correlated and ionomycin also increased gK and [Ca]'i in a parallel manner. The carbachol-induced delta gK per delta[Ca]'i, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gK.

Amphotericin B↗
PubMed1990

Effects of carbachol and (-)-N6-phenylisopropyladenosine on myocardial inositol phosphate content and force of contraction.

1. The effects of carbachol and the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA) on force of contraction and inositol lipid metabolism were studied in electrically driven left auricles and papillary muscles isolated from guinea-pig hearts. Both carbachol and PIA (0.01-10 microM) had concentration-dependent negative inotropic effects in auricles. In papillary muscles PIA had no inotropic effect. Carbachol also had no inotropic effect at low concentrations (0.01-1 microM) but at 10-100 microM it exerted a slight positive inotropic effect. 2. In auricles and papillary muscles both carbachol and PIA concentration-dependently increased inositol trisphosphate (IP3; significant at 1 microM). Accordingly phosphatidylinositol bisphosphate (PIP2), the precursor of IP3, was reduced. All effects of carbachol and PIA were antagonized by atropine (10 microM) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 20 microM) respectively, indicating receptor-mediated effects. 3. In auricles the negative inotropic effects of carbachol and PIA preceded the increase in IP3. 4. In papillary muscles the increase in IP3 preceded the slight positive inotropic effect of carbachol, indicating that the M-cholinoceptor-mediated increase in IP3 and force of contraction may be related. However, PIA showed a comparable increase in IP3 but no inotropic effect, indicating a dissociation between those parameters. 5. In conclusion, in previous studies a close relation between increases in IP3 and force of contraction has been shown after alpha 1-adrenoceptor stimulation. The present study with carbachol supports this view. However, the present data for PIA could not show such a close relationship, questioning the role of IP3 as an endogenous regulator of force of contraction.

Animals↗