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At least 19 recordsLinked to original sources

PubMed1986

The effect of varying carbachol concentration on the slope of Schild plots of selective beta-adrenoceptor antagonists in the carbachol-contracted guinea-pig trachea.

The effect of varying the level of smooth muscle tone induced by carbachol on the Schild analysis of atenolol (beta 1-selective) and ICI 118,551 (beta 2-selective) with salbutamol as agonist, on the guinea-pig tracheal preparation has been examined. When 10(-6) M carbachol was used to induce near-maximal smooth muscle tone, Schild plot slopes for atenolol and ICI 118,551 were less than 1. Slopes of Schild plots for both drugs were equivalent to 1 when 10(-7) M carbachol was used to produce approximately half-maximal smooth muscle tone. Depletion of neuronal noradrenaline by prior treatment with reserpine had no effect on the Schild analysis. Salbutamol produced maximal relaxation and was more potent when tone was induced with 10(-7) M carbachol, but was less effective at 10(-6) M carbachol. Pretreatment with reserpine increased the potency of salbutamol at each concentration of carbachol. The results suggest that either the level of smooth muscle tone or an unknown effect associated with a high level of smooth muscle tone induced by carbachol may contribute to low slope values of Schild plots of selective beta-adrenoceptor antagonists in the carbachol-contracted guinea-pig trachea. The carbachol-contracted guinea-pig trachea can be used to determine theoretically valid pA2 values for selective beta-adrenoceptor antagonists as long as substantially less than a maximal level of smooth muscle tone is induced by carbachol.

Adrenergic beta-Antagonists↗

PubMed2003

Ethanol blocks both basic fibroblast growth factor- and carbachol-mediated neuroepithelial cell expansion with differential effects on carbachol-activated signaling pathways.

We have expanded neuroepithelial cells dissociated from the embryonic rat telencephalon in serum-free defined medium containing basic fibroblast growth factor (bFGF) in order to generate a model neuroepithelium to study the interaction of ethanol with both growth factor- and transmitter-stimulated proliferation. Ethanol blocked proliferation stimulated by bFGF and by carbachol, an agonist at muscarinic acetylcholine receptors, in a dose-dependent manner. In addition, ethanol attenuated autonomous expansion of neuroepithelial cells occurring following withdrawal of bFGF. The latter effect was associated with an increase in the number of apoptotic cells identified by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling labeling. We studied the effects of ethanol on carbachol-stimulated signaling pathways critical to its proliferative effects. Ethanol significantly reduced carbachol-stimulated Ca(2+) signaling, as well as Erk1/Erk2, Akt and cyclic AMP-response element-binding phosphorylations in a dose-dependent manner. Comparison of the potency of ethanol in attenuating carbachol-stimulated proliferation and signal transduction showed that mitogen-activated protein kinase phosphorylation was less sensitive to ethanol than the other parameters. The results indicate that ethanol's suppression of proliferation induced by carbachol in this model neuroepithelium likely involves multiple signaling pathways. These effects in vitro may help to explain the devastating effects of prenatal ethanol exposure in vivo, which contribute to the fetal alcohol syndrome.

Acetylcholine↗

PubMed1985

A novel cyclic GMP-lowering agent, LY83583, blocks carbachol-induced cyclic GMP elevation in rabbit atrial strips without blocking the negative inotropic effects of carbachol.

A novel cyclic GMP-lowering agent, LY83583(6-anilino-5,8-quinolinedione), was used to investigate the possibility that increases in myocardial cyclic GMP levels are responsible for the negative inotropic effects of cholinergic agonists. Concentrations of carbachol from 0.3 to 3 microM elevated cyclic GMP levels in electrically paced rabbit atrial strips by 75 to 200% and decreased contractile force in the strips by 30 to 60%. Pretreatment of the muscles for 10 min with 10 microM LY83583 significantly lowered resting cyclic GMP levels and completely blocked the elevation of cyclic GMP by these concentrations of carbachol. However, the negative inotropic effects of carbachol were not blocked by the LY83583. These results indicate that the negative inotropic effects of carbachol in rabbit atrium are not mediated by increases in tissue levels of cyclic GMP.

Aminoquinolines↗

PubMedDate not supplied

An analysis of the carbachol-induced emotional-defensive response in cats: dependence of the response on different solvents of carbachol.

The effect of intrahypothalamic injections of carbachol (25.1 nmole into each hemisphere) dissolved in three different solvents (artificial cerebrospinal fluid, physiological saline and bidistilled water) was investigated. The purpose of this study was to find out whether the qualitative and/or quantitative character of the carbachol-induced emotional-defensive behavior depended on the sort of the solvent used. The analysis was performed on the basis of behavioral and electroencephalographic changes. Apart from routine EEG recordings, integrated measurements of the amplitude of alpha, beta, delta, and theta waves recorded rom the posterior hypothalamus, the midbrain central gray matter and from the dorsal hippocampus were performed as well as quantitative measurements of the characteristic growling. Intrahypothalamic injections of carbachol, independently of the solvent used, evoked similar vegetative, behavioral and electroencephalographic changes (in the routine recordings). The number of growls and the total duration of growling did not differ significantly either. Some statistically significant changes were found in the integrated amplitudes of EEG waves. They concerned, however, only alpha and theta rhythms and appeared in the midbrain central gray matter. Moreover, they did not affect the time course and the quantitative and qualitative features of the evoked emotional-defensive response. All used solvents are equivalent and may be applied interchangeably in experiments of this type.

Animals↗

PubMed1988

Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration.

Nominally K-sensitive microelectrodes were used to measure carbachol (CCh) in order to study the dependence of muscarinic effects on CCh concentration and exposure time in guinea pig hippocampal slices. Interference presumably originating from tissue choline-compounds was neutralized by pre-equilibration of the slices with 500 microM choline and calibration of the CCh-sensitive microelectrodes in the presence of the same choline-concentration. Muscarinic depolarization and reduction of the afterhyperpolarization (AHP) following a train of action potentials by bath applied CCh were monitored in granule cells and CA3 pyramidal neurons by intracellular recording. A fast bath application mode of CCh was designed, by which CCh tissue concentration reached a peak after 2-3 min and was washed out with a half time of about 8 min. After application of 30 nmol CCh in this way, the AHP was reduced according to the variation of CCh concentration over time. Neurons depolarized with some delay after the reduction of the AHP and started to repolarize 1 min before the peak of tissue CCh concentration (0.6 microM) was reached. Pirenzepine (1-10 microM) blocked only the depolarization, while atropine (1-10 microM) blocked both the depolarization and the reduction of the AHP. When superfusing with CCh containing saline, 80% of the final concentration was reached in the bath after 12 min, but in the tissue only after 45 min. The slow increase of tissue CCh concentration was concurrent with the slow decrease of the AHP. No effect on the membrane potential was observed. Atropine, but not pirenzepine, blocked the reduction of the AHP. Superfusion with a high CCh concentration (100-300 microM) containing saline depolarized neurons and reduced the AHP. Then pirenzepine repolarized neurons, whereas atropine both repolarized the cells and restored the AHP. It is concluded tha the muscarinic depolarization depends not only on the CCh concentration, but also on the rate of rise of CCh, while the reduction of the AHP depends solely on the concentration. This result is discussed in terms of the possibility that the depolarization is mediated by a short term desensitizing M1 muscarinic receptor subtype and the reduction of the AHP is mediated by a M2 muscarinic receptor subtype.

Animals↗

PubMed1998

Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion.

We have examined the role of tyrosine phosphorylation in regulation of calcium-dependent chloride secretion across T84 colonic epithelial cells. The calcium-mediated agonist carbachol (CCh, 100 microM) stimulated a time-dependent increase in tyrosine phosphorylation of a range of proteins (with molecular masses ranging up to 180 kDa) in T84 cells. The tyrosine kinase inhibitor, genistein (5 microM), significantly potentiated chloride secretory responses to CCh, indicating a role for CCh-stimulated tyrosine phosphorylation in negative regulation of CCh-stimulated secretory responses. Further studies revealed that CCh stimulated an increase in both phosphorylation and activity of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein kinase. Chloride secretory responses to CCh were also potentiated by the mitogen-activated protein kinase inhibitor, PD98059 (20 microM). Phosphorylation of ERK in response to CCh was mimicked by the protein kinase C (PKC) activator, phorbol myristate acetate (100 nM), but was not altered by the PKC inhibitor GF 109203X (1 microM). ERK phosphorylation was also induced by epidermal growth factor (EGF) (100 ng/ml). Immunoprecipitation/Western blot studies revealed that CCh stimulated tyrosine phosphorylation of the EGF receptor (EGFr) and increased co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr. An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 microM), reversed CCh-stimulated phosphorylation of both EGFr and ERK. Tyrphostin AG1478 also potentiated chloride secretory responses to CCh. We conclude that CCh activates ERK in T84 cells via a mechanism involving transactivation of the EGFr, and that this pathway constitutes an inhibitory signaling pathway by which chloride secretory responses to CCh may be negatively regulated.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Carbachol regulation of rabbit ileal brush border Na+-H+ exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent.

The epithelial brush border membrane (BBM) Na(+)-H(+) exchanger 3 (NHE3) is the major transport protein responsible for ileal electroneutral Na(+) absorption. We have previously shown that ileal BBM NHE3 activity is rapidly inhibited by carbachol, an agonist that mimics cholinergic activation in digestion. In this study, we investigated the mechanisms involved in this NHE3 inhibition. Carbachol decreased the amount of ileal Na(+) absorptive cell BBM NHE3 within 10 min of exposure. Based on OptiPrep gradient centrifugation, carbachol increased the amount of NHE3 in early endosomes and decreased the amount of NHE3 in BBM, consistent with effects on NHE3 trafficking. The decrease in BBM NHE3 occurred in the detergent-soluble BBM fraction with no change in the amount of NHE3 in the BBM detergent-resistant membranes. The size of BBM NHE3 complexes increased in carbachol-exposed ileum, as studied with sucrose gradient centrifugation. The NHE3 complex size increased in the total BBM, but did not change in the detergent-soluble fraction. This suggests that carbachol treatment enhanced the association of proteins with NHE3 complexes specifically in the detergent-resistant fraction of ileal BBM. NHERF2, alpha-actinin-4 and protein kinase C were among those NHE3-associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol-mediated inhibition since: (1) c-Src was rapidly activated in the detergent-resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na(+) absorption was completely abolished by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, the carbachol-induced increase in the size of NHE3-containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent-soluble fraction but not the detergent-resistant membrane; and (3) the molecular level, at which NHE3 complex-associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co-IP of NHERF2 with alpha-actinin-4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol-induced and Src-mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity.

Actinin↗

PubMed1996

Activation of the protein phosphatase calcineurin during carbachol exposure decreases the extent of recovery from end-plate desensitization.

1. Our previous studies demonstrated that protein kinase C (PKC) activity is required for acetylcholine (ACh) sensitivity to recover fully at snake twitch fiber end plates after prolonged exposure to carbachol. In the present studies, we have investigated whether protein phosphatase(s), activated during carbachol exposure, dephosphorylated critical membrane proteins, which required rephosphorylation by PKC to maintain end-plate sensitivity. End-plate sensitivity was assessed from measurements of miniature end-plate currents (MEPCs) and carbachol-activated currents (EPCCARBS). Conductance of ACh-activated channels was determined from patch-clamp recordings of single-channel currents. 2. Pretreatment of snake muscle preparations with the protein kinase inhibitor staurosporine (0.5 microM), followed by a 10-min exposure to 540 microM carbachol, reduced mean MEPC amplitudes to values 30-40% less than those recorded before carbachol exposure. Conversely, at control end plates exposed to carbachol, the mean MEPC amplitude was reduced by only approximately 5% compared with precarbachol values. This staurosporine-induced decrease in ACh sensitivity could be prevented by pretreatment with the protein phosphatase 2B (calcineurin) inhibitor deltamethrin (0.5 microM), whereas okadaic acid (5 microM) and calyculin A (0.5 microM), inhibitors of protein phosphatases 1 and 2A, had no effect. 3. After a 10-min exposure to 540 microM carbachol, EPCCARB amplitudes (produced by local superfusion with 20 microM carbachol) were significantly smaller at staurosporine-treated end plates than at control end plates. In contrast, the EPCCARB amplitude recorded from end plates pretreated with both deltamethrin and staurosporine was not significantly different from that recorded at control end plates. 4. Substitution of 10 mM Mn2+ for external Ca2+ during the exposure to 540 microM carbachol prevented the decrease in MEPC amplitude recovery at staurosporine-treated end plates. These results suggested that the alteration in sensitivity at staurosporine-treated end plates was calcium dependent. 5. At control end plates, a single population of ACh-activated channels (45-50 pS) is observed both before and after a 10-min exposure to 540 microM carbachol. Conversely, at staurosporine-treated end plates, after exposure to carbachol, a second population of small-conductance (25-30 pS) ACh-activated channels is present in addition to the predominant 45- to 50-pS ACh-activated channels. In preparations pretreated with both deltamethrin and staurosporine, after carbachol exposure, there was a significant decrease in the frequency of small-conductance ACh-activated channels. Deltamethrin treatment alone produced no small-conductance channels before or after a 10-min exposure to 540 microM carbachol. Also, no small-conductance ACh-gated channels were recorded at PKC-inhibited end plates after carbachol exposure either with pretreatment with 10 microM cyclosporin A (another inhibitor of calcineurin) or with the substitution of 10 mM Mn2+ for Ca2+ during the 10-min agonist exposure. 6. We propose that during prolonged exposure to the nicotinic agonist carbachol, calcium influx through ACh-gated channels elevates the level of ionized calcium at the inner surface of the post-junctional membrane and that this local rise in intracellular calcium activates the calcium-dependent phosphatase calcineurin. Dephosphorylation of some key membrane protein by calcineurin leads to a decrease in the extent of recovery from desensitization. Under normal conditions, this process is effectively reversed by PKC activity and end-plate sensitivity recovers fully. However, when PKC is inhibited, the extent of recovery of end-plate sensitivity is decreased, and associated with this decrease is the presence of small-conductance ACh-activated channels not normally recorded at snake twitch fiber end plates.

Animals↗

PubMed1986

Effects of verapamil on the response of the guinea-pig tracheal muscle to carbachol.

The effects of verapamil on the contraction of the guinea-pig tracheal smooth muscle induced by calcium (Ca2+) or barium (Ba2+) were investigated in three different conditions: (a) in excess K solution, (b) in the presence of carbachol, and (c) in excess K solution containing carbachol. In order to clarify the contractions, the effects of removal and readdition of the divalent cations were also investigated. In Ca2+-loaded tissues, application of carbachol in Ca-free medium produced a transient contraction, the magnitude of which decreased the longer the duration of exposure to Ca2+-free solution. In Ca2+-depleted, Ba2+-loaded tissues, application of carbachol in a Ba2+- and Ca2+-free medium produced a transient contraction the magnitude of which decreased the longer the duration of exposure to the Ba2+- and Ca2+-free solution. After exposure to Ca2+-free solution for 40 min, the sensitivity of the tissue to Ca2+ was greater in the presence of 30 microM carbachol (ED50 = 0.06 mM) than in the presence of 40 mM K+ (ED50 = 0.3 mM). The Ca2+-sensitivity in the presence of 30 microM carbachol plus K+ (40 mM) was not different from that in the presence of 30 microM carbachol alone. In Ca2+-free solution, the sensitivity of the tissue to Ba2+ in the presence of 40 mM K+ (ED50 = 1.4 mM) was not different from that observed in the presence of 30 microM carbachol (ED50 = 1.3 mM). 6 After exposure to Ca2+-free solution, verapamil produced a parallel rightward shift in the concentration-response curves to added Ca2+ and Ba2+ in the presence of either 40 mM K+, 30 microM carbachol or 40mM K+ plus 30 microM carbachol. 7 The pA2 values of verapamil against Ca2+ responses in the presence of 40 mM K+, 30 microM carbachol and 40 mM K+ plus 30 microM carbachol were 7.0, 6.5 and 6.5, respectively. The pA2 values of verapamil against Ba2+ responses under these conditions were 7.1, 7.0 and 7.1, respectively. 8 It is concluded that the sustained contraction produced by carbachol requires the influx of Ca2+ and that Ba2+ can substitute for Ca2+ in this process. Furthermore, the ionic channels which admit Ca2+ may be modified by carbachol to different degrees depending on the presence of Ca2+ or Ba2+. Such changes alter the affinity of the channel to verapamil.

Animals↗

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↗

PubMed1995

Characteristics of the analgesic effects and drug interactions of intrathecal carbachol in rats.

BACKGROUND: Intrathecal carbachol produces consistent analgesia in animals without appreciable adverse effects. Little is known about the ability of this drug to provide analgesia as stimulus intensity is increased. Likewise, there are few data regarding interactions between carbachol and other intrathecal analgesics. METHODS: Using two different noxious radiant heat intensities, one applied to each hind limb, analgesic effects of 1, 3, 10, and 30 micrograms intrathecal carbachol on paw withdrawal latencies were measured. Similar testing was done for intrathecal morphine and clonidine. ED50 fractions (1/2, 1/4, 1/8, 1/16) of drug combinations of carbachol-morphine and carbachol-clonidine were administered, responses to the low intensity stimulus were recorded, and the ED50 of each combination was established and isobolographic analysis of the drug interactions was carried out. RESULTS: The 30-micrograms dose of carbachol was associated with transient agitation, salivation, and hind limb weakness. No other adverse effects were noted. The ED50 (95% confidence interval) of intrathecal carbachol was 2.34 micrograms (1.34-4.04) for low intensity stimulation and 12.64 micrograms (4.18-38.25) for high intensity. There was no significant difference between high- and low-intensity ED50 values for intrathecal morphine and clonidine. The analgesic effect of the carbachol-morphine and carbachol-clonidine combinations were significantly greater than the calculated additive effects. The ED50 for the carbachol-morphine combination was 12% of the expected additive value and the ED50 for the carbachol-clonidine combination was 30% of the expected additive value. CONCLUSIONS: Intrathecal carbachol provides analgesia to noxious thermal stimulation of the hind paw in rats. It is relatively less effective at providing analgesia than intrathecal morphine or clonidine when stimulus intensity is raised. Intrathecal carbachol is synergistic when combined with intrathecal morphine or clonidine.

Analgesics, Non-Narcotic↗

PubMed1972

The depolarizing action of acetylcholine or carbachol in intestinal smooth muscle.

1. The membrane potential of the longitudinal muscle of the guinea-pig ileum was recorded intracellularly with glass micro-electrodes.2. Acetylcholine or carbachol depolarized the membrane. The depolarization produced by 1.4 x 10(-6)M carbachol was only 3.6 mV less than that produced by 5.5 x 10(-5)M.3. When the change in size of the electrotonic potential was used to estimate the increase in membrane conductance produced by different concentrations of carbachol, the increase in conductance was about tenfold at 1.4 x 10(-6)M and about 100-fold at 5.5 x 10(-5)M. There was a significant (P < 0.025) regression of the change in size of the electrotonic potential on the logarithm of the concentration of carbachol over this dose range. This and other observations suggest that it is not the availability of receptors which curtails the depolarization produced by concentrations of carbachol in excess of 1.4 x 10(-6)M.4. Reducing the external sodium concentration shifted the level of peak depolarization produced by carbachol negatively, and increasing the external sodium concentration shifted it positively.5. Reducing the external chloride from 134 to 13 mM had no significant effect on the level of peak depolarization produced by carbachol. Reducing the external chloride to 7 mM shifted the level of peak depolarization 3.1 mV in a positive direction.6. Increasing the external potassium concentration had little effect on the level of peak depolarization produced by carbachol, and decreasing the external potassium shifted the level of peak depolarization positively.7. It was possible to account for the observed relationship between membrane potential and membrane conductance if the assumption was made that carbachol opens additional ion channels in the membrane which have an equilibrium potential of about -9 mV. It is suggested therefore that the depolarizing action of carbachol on this smooth muscle is limited by its equilibrium potential, and that the equilibrium potential and the membrane potential in the presence of large concentrations of carbachol (e.g. 5.5 x 10(-5)M) are very close. Hence changes in the level of peak depolarization produced by varying the ionic composition of the external solution probably reflect changes in the equilibrium potential fairly closely.8. A simple model in which carbachol opens conductances to sodium, potassium and chloride ions and in which these conductances are independent of external ion concentrations (and voltage) could account for the effects on the equilibrium potential of varying [Na(+)](0) and [Cl(-)](0) but not [K(+)](0).9. It is suggested that carbachol increases the conductance to sodium, potassium and possibly other cations, and that, if it increases chloride conductance, then the increase produced is small relative to the increase in conductance to cations.

Acetylcholine↗

PubMed1993

Suppression of hypoglossal motoneurons during the carbachol-induced atonia of REM sleep is not caused by fast synaptic inhibition.

The depression of upper airway motor activity that develops during the rapid eye movement (REM) stage of sleep is a major factor allowing upper airway obstructions to occur in patients with sleep apnea syndrome. Microinjections of carbachol, a cholinergic agonist, into the dorsal pontine tegmentum of chronically instrumented cats produce REM sleep. In acutely decerebrate cats, carbachol induces postural atonia, eye movements and a depression of the motor output to respiratory pump and upper airway muscles. In lumbar motoneurons, the depression of activity is due to a glycinergic inhibition that has the same characteristics during natural REM sleep in chronic cats and carbachol-induced atonia in decerebrate cats (Neurophysiology, 57 (1987) 1118-1129). The mechanisms that lead to the suppression of upper airway motoneuronal activity during REM sleep are unknown. In this study, we assessed whether the depression of hypoglossal (XII) nerve activity induced by pontine carbachol injections is caused by inhibitory amino acids acting within the XII nucleus. In decerebrate, paralyzed and artificially ventilated cats, we recorded the activities of both XII nerves (genioglossal branches), one phrenic and a cervical motor branch (to monitor postural activity). Postural atonia and respiratory depression were induced by pontine carbachol injections. The inhibitory amino acid receptor antagonists, strychnine (glycine receptors) or bicuculline (GABAA receptors), were injected (100-250 nl; 1.0-2.5 mM) into one XII nucleus (the other served as control) in an attempt to reduce or abolish the depression subsequently induced by pontine carbachol. Prior to the carbachol injections, both antagonists caused similar elevations of XII nerve activity on the treated side (30-40%). However, following carbachol, the XII nerve activity on the treated side was depressed to about 25% of the (pre-antagonist and pre-carbachol) control level, whereas the depression on the untreated side was slightly greater, to 10-15% of the control. Additional injections of antagonists during the carbachol-induced depression produced no further increase in nerve activity. This minor effect of the antagonists on the carbachol-induced depression of XII nerve activity was in contrast to the marked disinhibitory effects that both antagonists had on the XII nerve response to electrical stimulation of the lingual nerve. The latter was used as a control for the ability of strychnine and bicuculline to exert disinhibitory effects within the XII nucleus. Thus, there is little, if any, contribution of these inhibitory amino acids to the depression of XII motoneurons during the carbachol-induced, REM sleep-like postural and respiratory depression; mechanisms other than fast synaptic inhibition must be involved.

Animals↗

PubMed1998

Interaction between 3 alpha-hydroxy-5 alpha-pregnan-20-one and carbachol in the control of neuronal excitability in hippocampal slices of female rats in defined phases of the oestrus.

The effects of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and carbachol on CA1 and dentate gyrus action potentials were studied in hippocampus slices in premature, follicular and luteal phase rats. A 0.5 nL droplet of allopregnanolone (12.5 mumol L-1), carbachol (5 mumol L-1) or a mixed solution of 12.5 mumol L-1 allopregnanolone and 5 mumol L-1 carbachol was applied locally onto the stratum oriens-pyramidale or granular layer. The amplitude of CA1 population spike (POPSP) was reduced by allopregnanolone (-38 +/- 3%) and carbachol (-21 +/- 4%) in the luteal phase slices. The mixture of allopregnanolone and carbachol doubled this inhibition (-77 +/- 6%). The inhibition caused by allopregnanolone and the mixture of allopregnanolone and carbachol in CA1 was significantly larger in the luteal phase than in the follicular phase (P = 0.02 and 0.0002). In the granular layer of the dentate gyrus, these inhibitions showed no significant difference between the phases. Neither in CA1 nor in the dentate gyrus did the carbachol inhibition differ between the phases. Perfusion with 5-10 mumol L-1 carbachol caused an increasing inhibition of the POPSP during the first few minutes. Thereafter the inhibition gradually diminished and was replaced by a facilitation. The local allopregnanolone inhibition was enhanced by simultaneous carbachol perfusion. Picrotoxin (100 mumol L-1) substantially reduced the allopregnanolone but not the carbachol inhibition. Atropine (10 mumol L-1) blocked the carbachol response, but not the allopregnanolone inhibition. Perfusion with a mixed solution of picrotoxin and atropine reduced, but did not block, the inhibition caused by local application of allopregnanolone or by the mixture of allopregnanolone and carbachol. Our data suggest that neuroprogestine modulators of the GABAA-receptor-mediated inhibition may play a significant role in the control of the cholinergic excitation in the hippocampus.

Action Potentials↗

PubMed1999

Carbachol-induced sustained tonic contraction of rat detrusor muscle.

OBJECTIVE: To investigate the underlying contractile mechanism of the sustained tonic contraction (SuTC) induced by repetitive carbachol application in rat detrusor muscles. MATERIALS AND METHODS: Longitudinal muscle strips with no mucosa were obtained from the anterior wall of the urinary bladder in 12-week-old Sprague-Dawley rats. Carbachol (5 micromol/L) was applied repetitively to induce SuTC. The carbachol-induced SuTC was assessed in the presence of various Ca2+-channel blockers and drugs affecting intracellular Ca2+ concentration. RESULTS: The first application of carbachol elicited a large phasic contraction followed by a tonic contraction (TC); the carbachol-induced contraction was completely reversed by washing out the solution. However, the initial phasic contraction was not reproduced after a second or further application of carbachol. There was consistently only a SuTC with no phasic contraction. The amplitude of the SuTC was 85% of the TC induced by the first carbachol application. The application of atropine (1 micromol/L) to the bath completely blocked SuTC. The carbachol-induced SuTC was insensitive to nicardipine (5 micromol/L) and extracellular polyvalent cations (1 mmol/L, La3+, Co2+, Cd2+, Ni2+ ). Moreover, a similar SuTC was induced even after the complete elimination of extracellular Ca2+ by adding 2 mmol/L EGTA to the Ca2+-free Tyrode solution. To exclude intracellular Ca2+ sources related to the sarcoplasmic reticulum (SR), the effects of SR Ca2+ pump inhibitors, cyclopiazonic acid (CPA, 10 micromol/L) and thapsigargin (0.5 micromol/L) were tested. The carbachol-induced SuTC was insensitive to pretreatment with CPA and/or thapsigargin. To deplete the ryanodine-sensitive Ca2+ pool, muscle strips were repetitively stimulated with caffeine (10 mmol/L) in the presence of 10 micromol/L ryanodine, which did not affect the carbachol-induced SuTC. CONCLUSIONS: Although the characteristics of the carbachol-induced SuTC have not been defined, these results show that a significant proportion of the carbachol-induced contraction in rats is contributed by the SuTC, which is present even in the complete absence of external Ca2+. The SuTC was not affected by limiting the contributions of internal Ca2+ sources. This suggests that the SuTC in rat bladders is unrelated to known Ca2+ mobilization mechanisms.

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↗

PubMed1998

Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells.

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.

Animals↗