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PubMed2002

Effect of constitutive- and inducible-cyclooxygenase in the carbachol-induced pituitary-adrenocortical response during social stress.

Acetylcholine potently stimulates the hypothalamic-pituitary-adrenal (HPA) axis. Cholinergic receptor agonist carbachol, given intraperitoneally (i.p.) or into the lateral cerebral ventricle (i.c.v.) to non-anesthetized rats acts via multiple pathways to stimulate the HPA axis. The present study sought to determine 1) the functional selectivity of carbachol for cholinergic muscarinic and/or nicotinic receptors involved in the stimulation of HPA axis; 2) the involvement of prostaglandins (PGs) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the carbachol-induced ACTH and corticosterone secretion in non-stressed rats and animals exposed to social crowding stress for 7 days (24 per a cage for 6). Carbachol was given i.c.v. or i.p. and cholinergic receptor antagonists or cyclooxygenase isoenzyme antagonists were given by the same routes 15 min earlier. One hour after the last injection trunk blood was taken for ACTH and corticosterone determinations. Atropine (0.1 microg i.c.v.), a cholinergic receptor antagonist, totally abolished the carbachol (2 microg i.c.v.)-induced ACTH and corticosterone secretion and mecamylamine (20 microg i.c.v.), a selective nicotinic receptor antagonist, did not affect this secretion. This finding indicates that carbachol functions as a selective central cholinergic muscarinic receptor agonist for the HPA axis stimulation. Crowding stress significantly diminished the carbachol (0.2 mg/kg i.p.)-induced plasma ACTH and corticosterone levels measured 1 hr after administration. Pretreatment with indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase inhibitor, significantly diminished the ACTH and corticosterone responses to carbachol (0.2 mg/kg i.p.) in control rats and moderately decreased these responses in stressed rats. Piroxicam (0.2 and 2.0 mg/kg i.p.), a COX-1 inhibitor, considerably impaired the carbachol-induced ACTH and corticosterone responses in control rats and markedly diminished these responses in stressed rats. A selective COX-2 blocker, compound NS-398 (0.2 and 2.0 mg/kg i.p.), substantially decreased the carbachol-induced hormones secretion in control rats but did not markedly alter this secretion in stressed rats. These results indicate that in the carbachol-induced HPA axis activation PGs generated by COX-1 are considerably and to a much greater extent involved than PGs generated by COX-2. Social stress markedly diminishes the mediation of PGs generated by COX-1 but PGs synthesized by COX-2 do not substantially participate in the carbachol-induced HPA response.

Adrenal Cortex↗

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↗

PubMed1994

Effect of carbachol in the absence and presence of phenylephrine on Rb+ efflux and tension in rabbit left atria.

The muscarinic agonist carbachol produced a concentration-dependent increase in 86Rb+ efflux and decrease in tension in isolated, electrically stimulated rabbit left atria. However, the lowest concentration of carbachol tested produced only a very small increase in 86Rb+ efflux, while it caused a relatively greater decrease in tension. 4-Aminopyridine and pertussis toxin attenuated the carbachol-stimulated 86Rb+ efflux and negative inotropic effect. However, 4-aminopyridine had a greater inhibitory effect on carbachol-stimulated 86Rb+ efflux than on carbachol-induced decreases in tension. Pre-treatment of rabbits with pertussis toxin completely abolished the increase in 86Rb+ efflux and decrease in tension produced by carbachol in the presence of the alpha-adrenoceptor agonist phenylephrine. 4-Aminopyridine attenuated the negative inotropic response to carbachol in the presence of phenylephrine, but had less effect on the carbachol-induced increase in 86Rb+ efflux under these conditions. These results suggest that carbachol-induced increases in K+ efflux may contribute at least in part to the negative inotropic responses to carbachol in the presence and absence of phenylephrine. However, this may not be sufficient to explain the direct negative inotropic response of left atria to carbachol.

4-Aminopyridine↗

PubMed1996

Carbachol induces inward current in neostriatal neurons through M1-like muscarinic receptors.

The effects of carbachol on rat neostriatal neurons were examined in the slice and the freshly dissociated neuron preparations using intracellular and whole-cell voltage-clamp recording methods. Superfusion of carbachol (30 microM) produced a depolarization concomitant with an increase in the rate of spontaneous action potentials. This depolarization was associated with an increase in the input resistance. The carbachol-induced membrane depolarization was blocked by pirenzepine (1 microM), a selective M1 muscarinic receptor antagonist. In other experiments, we observed that carbachol induced a transient inward current on the freshly dissociated neostriatal neuron at a holding potential of -60 mV in a concentration-dependent manner underlying the whole-cell voltage-clamp mode. The inward current caused by carbachol was not reduced by tetrodotoxin (1 microM), calcium-free recording solution or Cd2+ (100 microM). However, it was blocked by Ba2+ (100 microM). In addition, the carbachol-induced inward current reversed polarity at about the potassium equilibrium potential. The whole-cell membrane inward current in response to voltage-clamp step from -90 to -140 mV was reduced by 30 microM carbachol. With stronger hyperpolarization beyond the potassium equilibrium potential, carbachol produced a progressively greater reduction in membrane current. This inhibitory effect was also abolished by Ba2+ (100 microM). A concentration of 30 microM carbachol-induced inward current could be reversibly antagonized by the M1 muscarinic receptor antagonist pirenzepine (0.1-1 microM), with an estimated IC50 of 0.3 microM. However, other muscarinic receptor subtype (M2 or M3) antagonists could also block the carbachol-induced inward current. The rank order of antagonist potency was: pirenzepine (M1 antagonist) > 4-diphenylacetoxy-N,N-methyl-piperidine methiodide (M3/M1 antagonist) > gallamine (M2 antagonist). Based on these pharmacological data, we concluded that carbachol can act at M1-like muscarinic receptors to reduce the membrane K+ conductances and excite the neostriatal neurons.

Animals↗

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↗

PubMed1989

Role of calcium in mediating action of carbachol in T84 cells.

To examine the role of calcium in mediating carbachol's action in secretory epithelia, we simultaneously measured intracellular free [Ca] [( Ca]i) and transepithelial chloride transport 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. Chloride transport was measured as the short-circuit current (Isc) with a voltage clamp. Monolayers were pretreated with cyclic AMP to augment the response of Isc to carbachol, a procedure that did not qualitatively change the response of the monolayer to carbachol. The carbachol-induced changes in Isc appeared to be dependent on the increase in [Ca]i. First, carbachol caused both Isc and [Ca]'i to increase in parallel. Isc increased from 32 +/- 5 to 70 +/- 9 microA and then declined to 57 +/- 16 microA while [Ca]'i increased from 72 +/- 14 to 156 +/- 22 nM and then declined to 133 +/- 45 nM. Second, the carbachol-induced increases in Isc and [Ca]'i were correlated. The greater the hormone-stimulated rise in [Ca]'i, the higher the increase in Isc. Third, carbachol's stimulation of Isc was blunted by preventing the calcium spike with the cellular calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Although the carbachol-induced increase in [Ca]'i appeared necessary for the increase in Isc, it was not clear if carbachol's action was solely the result of an increase in [Ca]'i. Increasing [Ca]'i with ionomycin, although causing Isc and [Ca]'i to increase in parallel, failed to increase Isc to the levels observed with carbachol. These experiments suggest that although the carbachol-induced increase in Isc is dependent on the increase in [Ca]i, the hormone may activate a second process that increases the sensitivity of the calcium-activated transport process to changes in [Ca]i.

Calcium↗

PubMed1989

Carbachol in the presence of guanosine 5'-O-(3-thiotriphosphate) stimulates the breakdown of exogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol by rat brain membranes.

The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTP gamma S (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 microM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 microM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTP gamma S was greatest on a percentage basis at the lowest free Ca2+ concentrations. Above 1 microM free Ca2+, the stimulatory effect was lost, whereas 10 microM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 microM. The best stimulation of PI breakdown due to carbachol plus GTP gamma S was seen with 0.3 microM free Ca2+ and 100 microM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 microM GTP gamma S alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 microM GTP gamma S. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTP gamma S in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.

Adenosine Triphosphate↗

PubMed1982

Effects of carbachol, cholecystokinin, and insulin on protein phosphorylation in isolated pancreatic acini.

The role of protein phosphorylation in the regulation of pancreatic function by carbachol, cholecystokinin-octapeptide (CCK8), and insulin was investigated using isolated pancreatic acini of the mouse. Carbachol and CCK8 increased the phosphorylation of a Mr = 32,500 particulate protein and Mr = 16,000 and 23,000 soluble proteins. These agents also caused the dephosphorylation of Mr = 21,000 and 20,500 soluble proteins. Alterations in phosphorylation produced by carbachol were dose-dependent (maximal at 1-3 microM) and consistent with the dose-response relationship for carbachol-induced amylase secretion. Maximal dephosphorylation of the Mr = 21,000 and 20,500 proteins occurred within 1 min of addition of carbachol and was concurrent with the first detectable stimulation of amylase cyclase by carbachol. Moreover, atropine inhibition of carbachol-induced amylase secretion was concurrent with reversal of dephosphorylation of these two proteins. The calcium ionophore A23187, which mimicks the actions of carbachol and CCK8 on the pancreas, also mimicked the effects of these agents on protein phosphorylation, suggesting that pancreatic protein phosphorylation is regulated by calcium. Insulin, which enhances many of the actions of carbachol and CCK8 on the pancreas, alone increased the phosphorylation of the Mr = 16,000, 23,000, and 32,500 proteins and enhanced in an additive manner the effects of carbachol and CCK8 on the latter two proteins. By contrast, phosphorylation of the Mr = 21,000 or 20,500 proteins was not altered by insulin either in the absence or presence of carbachol or CCK8. In conclusion, the results suggest that alterations in phosphorylation of specific acinar cell proteins may provide a mechanism by which hormones and neurotransmitters interact in the regulation of pancreatic function.

Animals↗

PubMed1995

Corticosteroid-mediated modulation of carbachol responsiveness in CA1 pyramidal neurons: a voltage clamp analysis.

Pyramidal neurons in the rat hippocampal CA1 area contain mineralocorticoid (MRs) and glucocorticoid (GRs) receptors for corticosterone. Previous current clamp experiments showed that depolarizations evoked by carbachol (1-3 microM) depend on relative MR/GR occupation: carbachol responses are small with predominant MR-activation and larger when both receptor types are occupied. Multiple K-conductances underlie the carbachol-induced depolarization. In the present study we used the single electrode voltage clamp technique to examine which K-conductances modulated by carbachol are sensitive to corticosteroid treatment in vitro. We observed that 1 microM carbachol significantly reduced the IK,Leak while the IM was hardly affected; carbachol effects on the IK,Leak were significantly reduced under conditions of predominant MR activation compared to simultaneous activation of MRs and GRs. With a higher (10 microM) carbachol dose, steroid modulation of the IK,Leak showed a similar tendency. The amplitude of the IM was largely reduced by 10 microM carbachol but appeared to be not affected by steroid treatment. We conclude that the previously described suppression of the carbachol-induced depolarization with predominant activation MRs is caused by an attenuation of the carbachol action on the IK,Leak.

Adrenal Cortex Hormones↗

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↗

PubMed1996

Asymmetric signal transduction in polarized ileal Na(+)-absorbing cells: carbachol activates brush-border but not basolateral-membrane PIP2-PLC and translocates PLC-gamma 1 only to the brush border.

In ileal Na+ absorptive cells, carbachol inhibits NaCl absorption and its component brush-border Na+/H+ exchanger, acting via basolateral membrane (BLM) receptors. This carbachol effect involves brush-border but not BLM protein kinase C. In the present work we describe another asymmetric aspect of signal transduction in these epithelial cells, this time involving phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC). Thirty seconds and 1 min after carbachol treatment, brush-border PIP2-specific PLC activity increased, returning to control levels by 2.5 min. Involvement of brush-border tyrosine kinase(s) in this effect was suggested by inhibition of the carbachol effect on NaCl absorption by the tyrosine kinase inhibitor genistein, added to the mucosal but not the serosal surface. Luminal genistein pretreatment also prevented the carbachol-induced increase in brush-border PLC activity. In contrast, carbachol exposure did not change the BLM PIP2-specific PLC activity. Western analysis and immunoprecipitation demonstrated that PLC-gamma 1 is present in the brush border and that carbachol increases the PLC-gamma 1 amount in the brush border. Both the brush border and BLM contain PLC-beta 3 and a small amount of PLC-delta 1 but no PLC-beta 1, whereas BLM lacks detectable PLC-gamma 1. No change in PLC-beta 3 or PLC-delta 1 amount in the brush border occurred with carbachol exposure. No change in tyrosine phosphorylation of brush-border PLC-gamma 1 occurred with carbachol treatment. The Ca2+ ionophore A23187 did not alter PIP2-specific PLC activity in either the brush border or the BLM. These studies demonstrate that carbachol but not Ca2+ ionophore effects on brush-border NaCl absorption are associated with increases in brush-border but not BLM PIP2-specific PLC activity and in the amount of brush-border PLC-gamma 1, and involve tyrosine phosphorylation. This asymmetric aspect of epithelial signal transduction, together with the previous demonstration of localization of high-sensitivity IP3 stores to the apical membrane area in intestinal epithelial cells, shows that different aspects of signal transduction occur at the apical and basolateral membranes in epithelial and requires studies in both domains to define mechanisms of intracellular signalling.

Animals↗

PubMed1997

Ileal microvillar protein villin is tyrosine-phosphorylated and associates with PLC-gamma1. Role of cytoskeletal rearrangement in the carbachol-induced inhibition of ileal NaCl absorption.

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-gamma1 (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-gamma1. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-gamma1 and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-gamma1. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-gamma1 association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.

Actins↗

PubMed2004

Isoflurane reduces the carbachol-evoked Ca2+ influx in neuronal cells.

BACKGROUND: The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca transient increase ([Ca]cyt) was eliminated by K or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca]cyt transient involved Ca influx through the plasma membrane. METHODS: Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La (100 microm), of the L-type Ca channel blocker nitrendipine (10 microm), and of the N-type Ca channel blocker omega-conotoxin GVIA (0.1 microm) on isoflurane modulation of the carbachol-evoked [Ca]cyt transient. [Ca]cyt was detected with fura-2 and experiments were carried out at 37 degrees C. RESULTS: Isoflurane reduced the peak and area of the carbachol-evoked [Ca]cyt transient in the presence but not in the absence of extracellular Ca. La had a similar effect as the removal of extracellular Ca. Omega-conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. CONCLUSIONS: The current data are consistent with previous observations in that the carbachol-evoked [Ca]cyt transient involves both Ca release from intracellular Ca stores and Ca entry through the plasma membrane. It was found that isoflurane attenuates the carbachol-evoked Ca entry. The isoflurane sensitive Ca entry involves a cationic channel different from the L- or N- type voltage-dependent Ca channels. These results indicate that isoflurane attenuates the carbachol-evoked [Ca]cyt transient at a site at the plasma membrane that is distal to the muscarinic receptor.

Anesthetics, Inhalation↗

PubMed1985

Effects of locally and systemically administered cholinoceptor antagonists on the secretory response of human eccrine sweat glands to carbachol.

Eleven healthy male volunteers participated in a study comparing the effects of locally and systemically administered cholinoceptor antagonists on the secretory response of sweat glands to intradermally injected carbachol chloride. Atropine sulphate administered locally into the skin antagonised the response to carbachol: the dose-response curve for carbachol was shifted to the right without any depression of the maximum of the curve. The nicotinic receptor antagonists hexamethonium bromide and (+)-tubocurarine chloride, however, had little effect on the response to carbachol. Atropine sulphate, administered systemically by intramuscular injection, caused a non-surmountable antagonism of the response to carbachol: the maximum of the dose-response curve was depressed with little change in the value of ED50. Atropine methonitrate (a mixed muscarinic/nicotinic receptor antagonist), and hexamethonium bromide (a nicotinic receptor antagonist), both with poor access to the central nervous system, were injected intramuscularly: both caused non-surmountable antagonism of the response to carbachol. It is concluded that the response to carbachol is mediated by muscarinic rather than nicotinic receptors. The effect of atropine sulphate on the response to carbachol depends on the route of administration: while locally applied atropine sulphate appears to act as a competitive antagonist, systemically applied atropine sulphate, like atropine methonitrate and hexamethonium bromide, appears to act in a non-competitive manner. It is suggested that the systemically administered cholinoceptor antagonists reduce the response to carbachol by interacting with cholinoceptors in sympathetic ganglia: such an interaction would reduce the impulse flow in sudomotor fibres resulting in decreased sensitivity of the sweat glands to carbachol.

Adult↗

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↗

PubMed1971

Actions of acetylcholine and carbachol on the chick biventer cervicis muscle.

1. The possibility that carbachol may act on the chick biventer cervicis muscle by releasing acetylcholine was investigated.2. Log dose-response curves for acetylcholine and carbachol were constructed in the presence of triethylcholine, tubocurarine and physostigmine, in both innervated and denervated chick biventer cervicis muscles.3. The log dose-response curve for acetylcholine was shallower than that for carbachol and was moved to the left by triethylcholine, indicating potentiation. In the presence of physostigmine, the curve was as steep as that for carbachol, and like that for carbachol was moved to the right by triethylcholine, indicating antagonism. In the presence of tubocurrine, the log dose-response curves for both acetylcholine and carbachol were moved to the right.4. The log dose-response curves for both acetylcholine and carbachol were moved to the left by chronic denervation, indicating increased sensitivity to these drugs. Both curves were moved similar distances to the right by excess magnesium ion concentration. No tachyphylaxis developed to repeated administration of carbachol in the presence of triethylcholine or tubocurarine, either in innervated or denervated muscles.5. It was concluded that the main action of triethylcholine in the chick biventer cervicis muscle is a tubocurarine-like action, which, in the presence of acetylcholine, is masked by a weak anticholinesterase action. It was also concluded that the diminished response to carbachol in the presence of triethylcholine is due to the postjunctional blocking action of triethylcholine, and that carbachol owes none of its activity to an ability to release acetylcholine from the nerve terminals.

Acetylcholine↗

PubMed1988

Short-term desensitization of guinea-pig taenia caecum induced by carbachol occurs at intracellular Ca stores and that by histamine at H1-receptors.

1. In Ca-free solution, the contractile response of guinea-pig taenia caecum to 10(-4) M carbachol was mediated through muscarinic receptors and was reduced time-dependently by desensitization with 10(-4) M carbachol, but not 10(-4) M histamine. On the other hand, the response to 10(-4) M histamine was shown to be mediated through H1-receptors and to be reduced time-dependently by desensitization with either 10(-4) M histamine or 10(-4) M carbachol. 2. The maximal K+ contraction was not changed by desensitization with carbachol or histamine. Thus, contractile proteins and voltage-dependent Ca channels maintain their normal functions. 3. To study the coupling of Ca channel activity in cell surface membrane to receptor activation, the contractile responses elicited by carbachol and histamine added simultaneously with Ca to Ca-free solution were measured. The response elicited by carbachol plus Ca was not changed by desensitization with carbachol, while that elicited by histamine plus Ca was reduced by desensitization with histamine. These results show that desensitization by carbachol occurs at a post-receptor site, whereas that induced by histamine occurs at H1-receptors. 4. After desensitization with carbachol, but not histamine, the contractile response to 5 x 10(-2) M caffeine in Ca-free solution was significantly reduced. 5. These results show that short-term desensitization of guinea-pig taenia caecum by carbachol is heterologous and occurs at intracellular Ca stores, while that induced by histamine is homologous and occurs at histamine H1-receptors.

Animals↗

PubMed1989

Sodium-dependent membrane current induced by carbachol in single guinea-pig ventricular myocytes.

1. In the presence of either barium (0.2 mM) or caesium (20 mM), carbachol (3-300 microM) depolarized isolated guinea-pig ventricular myocytes. Carbachol induced an inward current under voltage clamp at a holding potential equal to the resting potential (-75 mV). 2. Acetylcholine and oxotremorine also evoked an inward current but were less effective than carbachol. Atropine (0.3 microM) prevented the depolarization and inward current induced by carbachol and acetylcholine but not by oxotremorine. Moreover, oxotremorine, but not carbachol, induced an inward current in the absence of extracellular sodium. 3. Carbachol increased membrane chord conductance when it induced an inward current. These effects were recorded under experimental conditions that suppressed the voltage- and time-dependent sodium current (tetrodotoxin) and calcium current (cadmium), the inwardly rectifying potassium current, iK1 (caesium, barium and tetraethylammonium) and the current generated by the sodium-potassium pump (zero external potassium). 4. Under these same experimental conditions, the steady-state I-V relationship in the presence of carbachol was subtracted from that in its absence. The apparent reversal potential (Erev) was 25 mV with extracellular Na+ ([ Na+]o) at 143 mM and intracellular Na+ ([Na+]i) at 11 mM. Replacement of [Na+]o by N-methyl-D-glucamine was associated with a shift of the apparent Erev to more negative voltages by approximately 61 mV per tenfold change of [Na+]o. 5. Isoprenaline induced an inward current in ventricular myocytes that depended upon sodium entry, required the accumulation of cyclic AMP and which was partially suppressed by acetylcholine (Egan, Noble, Noble, Powell, Twist & Yamaoka, 1988). In contrast to the current evoked by beta-adrenoceptor agonist, the current induced by muscarinic agonist was smaller and sustained. Moreover, the carbachol-induced current was not suppressed by prior addition of isoprenaline. 6. The findings are consistent with the mechanism that carbachol activates a plasma membrane ion channel that admits sodium and thereby increases intracellular sodium activity. The estimated increase of intracellular sodium activity from electrophysiological data agrees quantitatively with that obtained from measurements with sodium-sensitive microelectrodes (Korth & Kühlkamp, 1985). 7. The ability of carbachol to increase sodium influx may be the first step in a series of reactions that eventually alters sodium-calcium exchange and could account for catecholamine-independent stimulation of developed force in mammalian ventricle.

Acetylcholine↗