High Concentrations Of Carbachol Research Articles

Store-operated Ca2+ entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity, Ca2+ and force

In the myometrium SR Ca2+ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca2+ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca2+-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca2+ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca2+]i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca2+ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca2+ and depolarization continued a point was reached when electrical and Ca2+ spikes and phasic contractions ceased, and a maintained, tonic force and Ca2+ was produced. Lanthanum, a non-selective blocker of store-operated Ca2+ entry, but not the L-type Ca2+ channel blocker nifedipine (1–10μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca2+ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca2+ release is coupled to marked Ca2+ entry, via store operated Ca2+ channels, leading to depolarization and enhanced electrical and mechanical activity.

Enhanced nicotinic receptor mediated relaxations in gastroesophageal muscle fibers from Barrett's esophagus patients

Increased nicotinic receptor mediated relaxation in the gastroesophageal antireflux barrier may be involved in the pathophysiology of reflux. This study is designed to determine whether the defects we previously identified in gastroesophageal reflux disease patients in- vivo are due to abnormalities of the gastric sling, gastric clasp, or lower esophageal circular (LEC) muscle fibers. Muscle strips from whole stomachs and esophagi were obtained from 16 normal donors and 15 donors with histologically proven Barrett's esophagus. Contractile and relaxant responses of gastric sling, gastric clasp, or LEC fibers were determined to increasing concentrations of carbachol and to nicotine after inducing maximal contraction to bethanechol. Muscarinic receptor density was measured using subtype selective immunoprecipitation. Barrett's esophagus gastric sling and LEC fibers have decreased carbachol-induced contractions. Barrett's esophagus sling fibers have decreased M2 -muscarinic receptors and LEC fibers have decreased M3 receptors. Relaxations of all three fiber types are greater in Barrett's esophagus specimens to both high carbachol concentrations and to nicotine following bethanechol precontraction. The maximal response to bethanechol is greater in Barrett esophagus sling and LEC fibers. The increased contractile response to bethanechol in Barrett's specimens indicates that the defect is likely not due to the smooth muscle itself. The enhanced nicotinic receptor mediated response may be involved in greater relaxation of the muscles within the high-pressure zone of the gastroesophageal junction during transient lower esophageal sphincter relaxations and during deglutitive inhibition and may be involved in the pathophysiology of gastroesophageal reflux disease.

(−)-Carvone: Antispasmodic effect and mode of action

(−)-Carvone is a monoterpene ketone found in spearmint (Mentha spicata var. crispa) essential oil that is widely used as an odor and flavor additive. An intestinal antispasmodic effect was recently reported for (−)-carvone, and it has been shown to be more potent than its (+)-antipode. The mechanism of (−)-carvone action in the intestines has not been investigated. To gain a better understanding of the (−)-carvone antispasmodic effect, we investigated its pharmacological effects in the guinea pig ileum. Terminal portions of the ileum were mounted for isotonic contraction recordings. The effect of (−)-carvone was compared with that of the classical calcium channel blocker (CCB) verapamil. In isolated ileal smooth muscle, (−)-carvone did not produce direct contractile or relaxation responses and did not modify electrically elicited contractions or low K+-evoked contractions. The submaximal contractions induced by histamine (p<0.001), BaCl2 (p<0.05), and carbachol (p<0.01) were significantly reduced by (−)-carvone. The contractile response elicited by high concentrations of carbachol was reduced but not abolished by (−)-carvone. No additive action was detected with co-incubation of (−)-carvone and verapamil on carbachol-induced contraction. (−)-Carvone reduced the contraction induced by high K+ and was almost 100 times more potent than verapamil. Thus, (−)-carvone showed a typical and potent CCB-like action. Many effects described for both (−)-carvone and spearmint oil can be explained as a CCB-like mode of action.

Food allergy alters jejunal circular muscle contractility and induces local inflammatory cytokine expression in a mouse model

BackgroundWe hypothesized that food allergy causes a state of non-specific jejunal dysmotility. This was tested in a mouse model.MethodsBalb/c mice were epicutaneously sensitized with ovalbumin and challenged with 10 intragastric ovalbumin administrations every second day. Smooth muscle contractility of isolated circular jejunal sections was studied in organ bath with increasing concentrations of carbamylcholine chloride (carbachol). Smooth muscle layer thickness and mast cell protease-1 (MMCP-1) positive cell density were assayed histologically. Serum MMCP-1 and immunoglobulins were quantified by ELISA, and mRNA expressions of IFN-γ, IL-4, IL-6 and TGFβ-1 from jejunal and ileal tissue segments were analyzed with quantitative real-time PCR.ResultsOvalbumin-specific serum IgE correlated with jejunal MMCP-1+ cell density. In the allergic mice, higher concentrations of carbachol were required to reach submaximal muscular stimulation, particularly in preparations derived from mice with diarrhoea. Decreased sensitivity to carbachol was associated with increased expression of IL-4 and IL-6 mRNA in jejunum. Smooth muscle layer thickness, as well as mRNA of IFN-γ and TGF-β1 remained unchanged.ConclusionIn this mouse model of food allergy, we demonstrated a decreased response to a muscarinic agonist, and increased levels of proinflammatory IL-6 and Th2-related IL-4, but not Th1-related IFN-γ mRNAs in jejunum. IgE levels in serum correlated with the number of jejunal MMCP-1+ cells, and predicted diarrhoea. Overall, these changes may reflect a protective mechanism of the gut in food allergy.

Potentiation of carbachol-induced detrusor smooth muscle contractions by β-adrenoceptor activation

In strips of rabbit bladder free of urothelium, the β-adrenoceptor agonist, isoproterenol, significantly reduced basal detrusor smooth muscle tone and inhibited contractions produced by low concentrations of the muscarinic receptor agonist, carbachol. During a carbachol concentration–response curve, instead of inhibiting, isoproterenol strengthened contractions produced by high carbachol concentrations. Thus, the carbachol concentration–response curve was shifted by isoproterenol from a shallow, graded relationship, to a steep, switch-like relationship. The tyrosine kinase inhibitor, genistein, inhibited carbachol-induced contractions only in the presence of isoproterenol. Contraction produced by a single high carbachol concentration (1 µM) displayed 1 fast and 1 slow peak. In the presence of isoproterenol, the slow peak was not strengthened, but was delayed, and U-0126 (mitogen-activated protein kinase kinase inhibitor) selectively inhibited this delay concomitantly with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. Isoproterenol reduced ERK phosphorylation only in the absence of carbachol. These data support the concept that, by inhibiting weak contractions, potentiating strong contractions, and producing a more switch-like concentration–response curve, β-adrenoceptor stimulation enhanced the effectiveness of muscarinic receptor-induced detrusor smooth muscle contraction. Moreover, β-adrenoceptor stimulation changed the cellular mechanism by which carbachol produced contraction. The potential significance of multi-receptor and multi-cell crosstalk is discussed.

Relationship between carbachol hyperstimulation-induced pancreatic intracelluar trypsinogen and NF-κB activation in rats in vitro

The relationship between intracellular trypsinogen activation and NF-kappa B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachol) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc) and NF-kappa B inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The activity of NF-kappa B was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-kappa B in pancreatic acinar cells treated with high concentrations of carbachol (10(-3) mol/L) in vitro was significantly decreased as compared with control group (P<0.01). The addition of 10(-2) mol/L PDTC resulted in a significant decrease of NF-kappa B activities in pancreatic acinar cells after treated with high concentrations of carbachol (10(-3) mol/L) in vitro, but the intracellular trypsinogen activity was not obviously inhibited (P>0.05). It was concluded that intracellular trypsinogen activation is likely involved in the regulation of high concentrations of carbachol-induced NF-kappa B activation in pancreatic acinar cells in vitro. NF-kappa B activation is likely not necessary for high concentrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Inhibitory effects of the alpha-2 adrenergic agonists clonidine and dexmedetomidine on enhanced airway tone in ovalbumin-sensitized guinea pigs

The alpha-2 adrenergic agonists clonidine and dexmedetomidine are used as an antihypertensive and a sedative, respectively. The aim of this study was to determine the effects of these agonists on ovalbumin-sensitized airway tone in guinea pigs. The animals were divided into two groups: control and sensitized. The sensitized group received ovalbumin intraperitoneally and was boosted by exposure to aerosolized ovalbumin. The effects of the alpha-2 agonists were investigated by measuring (1) total lung resistance and (2) smooth muscle tension using a tracheal ring preparation. In the control group, acetylcholine significantly increased total lung resistance in a dose-dependent manner. In the sensitized animals, total lung resistance was significantly higher (by 95%) at 6 mug kg-1 acetylcholine than that in the control group. Both clonidine and dexmedetomidine had a slight but significant inhibitory effect on the response curve of lung resistance at higher concentrations of carbachol, a potent muscarinic receptor agonist. Similar to the data obtained in the control group, both clonidine and dexmedetomidine significantly decreased total lung resistance and the inhibitory effects of these alpha-2 agonists on lung resistance were significantly distinguishable. Similar direct inhibitory effects of the alpha-2 agonists on carbachol-induced muscle contraction were observed in both the control and sensitized groups, the inhibitory effects in the sensitized group being significantly greater. Both clonidine and dexmedetomidine can relax the airway even in the hyper-reactive state.

Relationship between carbachol hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen or NF-κB activation in ratsin vitro

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-kappaB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10(-3) mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P < 0.01) following the treatment with a high concentration of carbachol (10(-3) mol/L) in vitro. The addition of 10(-2) mol/L PDTC didn't result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol (10(-3) mol/L) in vitro (P > 0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

Excitatory effect of M1 muscarinic acetylcholine receptor on automaticity of mouse heart

We have investigated the effects of relatively high concentration of carbachol (CCh), an agonist of muscarinic acetylcholine receptor (mAChR), on cardiac automaticity in mouse heart. Action potentials from automatically beating right atria of mice were measured with conventional microelectrodes. When atria were treated with 100 microM CCh, atrial beating was immediately arrested and diastolic membrane potential (DMP) was depolarized. After exposure of the atria to CCh for approximately 4 min, action potentials were regenerated. The regenerated action potentials had lower frequency and shorter duration when compared with the control. When atria were pre-exposed to pirenzepine (1 microM), an M1 mAChR antagonist, there was complete inhibition of CCh-induced depolarization of DMP and regeneration of action potentials. Pre-exposure to AFDX-116 (11 ({2-[(diethylamino)-methyl]-1 -piperidyl}acetyl)-5,11 -dihydro-6H-pyridol[2,3-b][1,4] benzodiazepine-6-one base, 1 microM), an M2 mAChR antagonist, failed to block CCh-induced arrest of the beating. However, prolonged exposure to CCh elicited gradual depolarization of DMP and slight acceleration in beating rate. Our data indicate that high concentration of CCh depolarizes membrane potential and recovers right atrial automaticity via M1 mAChR, providing functional evidence for the role of M1 mAChR in the atrial myocytes.

Effects of increased intracellular cAMP on carbachol-stimulated zymogen activation, secretion, and injury in the pancreatic acinar cell.

A characteristic of acute pancreatitis is the premature activation and retention of enzymes within the pancreatic acinar cell. Because ligands linked to cAMP production may prevent some forms of pancreatitis, we evaluated the effects of increased intracellular cAMP in the rat pancreatic acinar cell. Specifically, this study examined the effects of the cholinergic agonist carbachol and agents that increase cAMP [secretin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)] on zymogen activation (trypsin and chymotrypsin), enzyme secretion, and cellular injury in isolated pancreatic acini. Although cAMP agonists affected the responses to physiological concentrations of carbachol (1 microM), their most prominent effects were observed with supraphysiological concentrations (1 mM). When secretin was added to 1 mM carbachol, there was a slight increase in zymogen activation, but no change in the secretion of amylase or chymotrypsin. Furthermore, coaddition of secretin increased parameters of cell injury (trypan blue exclusion, lactic dehydrogenase release, and morphological markers) compared with carbachol (1 mM) alone. Although directly increasing cellular cAMP by 8-Br-cAMP caused much greater zymogen activation than carbachol (1 mM) alone or with secretin, 8-Br-cAMP cotreatment reduced all parameters of injury to the level of unstimulated acini. Furthermore, 8-Br-cAMP dramatically enhanced the secretion of amylase and chymotrypsin from the acinar cell. This study demonstrates that increasing acinar cell cAMP can overcome the inhibition of enzyme secretion caused by high concentrations of carbachol and eliminate acinar cell injury.

Labeled carbon dioxide (C18O2): an indicator gas for phase II in expirograms.

Carbon dioxide labeled with 18O (C18O2) was used as a tracer gas for single-breath measurements in six anesthetized, mechanically ventilated beagle dogs. C18O2 is taken up quasi-instantaneously in the gas-exchanging region of the lungs but much less so in the conducting airways. Its use allows a clear separation of phase II in an expirogram even from diseased individuals and excludes the influence of alveolar concentration differences. Phase II of a C18O2 expirogram mathematically corresponds to the cumulative distribution of bronchial pathways to be traversed completely in the course of exhalation. The derivative of this cumulative distribution with respect to respired volume was submitted to a power moment analysis to characterize volumetric mean (position), standard deviation (broadness), and skewness (asymmetry) of phase II. Position is an estimate of dead space volume, whereas broadness and skewness are measures of the range and asymmetry of functional airway pathway lengths. The effects of changing ventilatory patterns and of changes in airway size (via carbachol-induced bronchoconstriction) were studied. Increasing inspiratory or expiratory flow rates or tidal volume had only minor influence on position and shape of phase II. With the introduction of a postinspiratory breath hold, phase II was continually shifted toward the airway opening (maximum 45% at 16 s) and became steeper by up to 16%, whereas skewness showed a biphasic response with a moderate decrease at short breath holding and a significant increase at longer breath holds. Stepwise bronchoconstriction decreased position up to 45 +/- 2% and broadness of phase II up to 43 +/- 4%, whereas skewness was increased up to twofold at high-carbachol concentrations. Under all circumstances, position of phase II by power moment analysis and dead space volume by the Fowler technique agreed closely in our healthy dogs. Overall, power moment analysis provides a more comprehensive view on phase II of single-breath expirograms than conventional dead space volume determinations and may be useful for respiratory physiology studies as well as for the study of diseased lungs.

Activation of nitric oxide synthase through muscarinic receptors in rat parotid gland

Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) activity was found coupled to muscarinic receptor activation as well as to nitric oxide-mediated amylase secretion elicited by carbachol. Parotid glands presented a predominant M 3 and a minor muscarinic M 1 acetylcholine receptor population, though carbachol stimulated NOS activity only through muscarinic M 3 receptors as revealed in the presence of 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP) and pirenzepine. Amylase secretion induced by carbachol appeared to be partly mediated by nitric oxide and nitric oxide-induced signaling since N-nitro- l-arginine methyl ester ( l-NAME) inhibited the effect as well as did methylene blue. A negative regulation of NOS by protein kinase C activation in the presence of a high concentration of carbachol was seen in parotid glands and this inhibition was paralleled by amylase secretion.

On the mechanisms of cholinergic control of the sinoatrial node discharge.

It has been proposed that cholinergic agonists inhibit the sinoatrial node discharge by shifting the activation range of the hyperpolarization-activated inward current If to more negative values or by increasing potassium conductance. In the former instance, cesium will potentiate the cholinergic inhibition by blocking any residual If; in the latter instance, Cs+ and Ba2+ will antagonize the inhibitory action by blocking K+ channels. The changes in discharge induced by high and low concentrations of carbachol were studied using an electrophysiologic technique in isolated guinea pig sinoatrial node perfused in vitro in the absence and presence of different concentrations of Cs+ and Ba2+. In Tyrode solution, high carbachol concentrations (0.5-2 microM) slowed the sinoatrial node by hyperpolarizing the membrane and by reducing the amplitude of diastolic depolarization; and stopped the sinoatrial node by preventing the attainment of threshold potential. Adding Cs+ (10 mM) to carbachol increased the rate in slowly discharging sinoatrial node and induced spontaneous discharge in quiescent sinoatrial node. In high [K+]o (approximately 12 mM), carbachol slowed or stopped the slow responses and adding Cs+ accelerated or induced discharge. Both in Tyode and in high [K+]o, in the presence of Cs+, carbachol did stop the sinoatrial node. In the presence of carbachol, Ba2+ (0.1 mM) accelerated or induced discharge, as Cs+ did. Atropine (1 microM) prevented both the slowing or suppression by carbachol and the acceleration of sinoatrial node by Cs+ in the presence of carbachol. Low carbachol concentrations (0.05-0.1 microM) decreased the rate to a similar extent in the absence and the presence of a low concentration of Cs+ (2 mM, which blocks If but not K+ channels), but markedly less in the presence of 0.5-0.75 mM Ba2+ (which block K+ channels but not If). We conclude that cholinergic agonists slow or stop the sinoatrial node by a shifting the membrane potential toward the more negative subsidiary pacemaker range and away from the threshold. The results with Cs+ and Ba2+ indicate that both high and low concentrations of carbachol decrease sinoatrial node discharge by activating the I(K,ACh) channels rather than by decreasing If.

CAMP-dependent potentiation of the Ca 2+-activated release of the anionic fluorescent dye, calcein, from rat parotid acinar cells

A recent study indicates that elevation of [Ca 2+] i enhances the release of calcein, an anionic fluorescent dye, from isolated exocrine acinar cells, so cytoplasmic calcein is useful for monitoring the secretion of organic anions. In this study, we investigated the effect of cAMP on the calcein release evoked by elevation of [Ca 2+] i. Isoproterenol, forskolin and dibutyryl cyclic AMP (dbcAMP) did not induce the release of calcein from isolated parotid acinar cells, but they potentiated the carbachol-induced release of calcein. Although cytoplasmic calcein is released through an increase in [Ca 2+] i, isoproterenol potentiated the carbachol-induced release of calcein without affecting the increase in [Ca 2+] i evoked by a high concentration of carbachol (10 −6 M). Charybdotoxin, a K + channel blocker, inhibited both the carbachol-induced release and the potentiation by isoproterenol. However, the calcein permeation pathways mediating the carbachol-induced release and the isoproterenol-potentiated release exhibited distinct sensitivities to anion channel blockers. Our results indicate that the calcein release induced by carbachol is potentiated through an increase in intracellular levels of cAMP. Although both the Ca 2+-activated release and the cAMP-potentiated release may be coupled to Ca 2+-activated K + efflux, increases in both [Ca 2+] i and [cAMP] i may activate the calcein conduction pathway which is not activated by an increase in [Ca 2+] i alone.

High carbachol increases the electrically induced [Ca 2+] i transient in the single isolated ventricular myocyte of rats

In order to investigate the mechanisms responsible for the inotropic effects of muscarinic acetylcholine receptor stimulation by high concentrations of muscarinic receptor agonists, we studied the effects of carbachol at 30–300 μM on the electrically induced [Ca 2+] i transient of rat isolated ventricular myocytes. Carbachol at this dose range increased the amplitude and duration of the electrically induced [Ca 2+] i transient time and dose dependently. It also increased the resting fluorescence ratio and time to 80% decline of amplitude from the peak. At 100–300 μM the increase in [Ca 2+] i transient was followed by a cluster of Ca 2+ oscillations in 50–83% of the cells studied. The effects were blocked by atropine, but not pertussis toxin. Depletion of Ca 2+ from sarcoplasmic reticulum by ryanodine, which itself reduced the amplitude of the [Ca 2+] i transient and increased resting fluorescence, abolished the effect of carbachol on the [Ca 2+] i transient without affecting its effect on resting fluorescence ratio. The caffeine-induced [Ca 2+] i transient was unaffected by prior addition of carbachol in a Ca 2+ free and low Na + solution. Inhibition of Ca 2+ influx by the L-type Ca 2+ channel blocker, verapamil, which itself reduced the amplitude of the [Ca 2+] i transient without affecting the resting fluorescence ratio, attenuated the augmentation of the amplitude of the [Ca 2+] i transient elicited by carbachol. Ni 2+, a non-specific Ca 2+ channel blocker and an inhibitor of Na +Ca 2+ exchange, abolished the effects of carbachol on both [Ca 2+] i transient and resting fluorescence ratio. Low external Na +, which increased the resting fluorescence ratio due to its inhibitory effect on Na +Ca 2+ exchange, also abolished the effects of carbachol. The results indicate that the inotropic effect of muscarinic acetylcholine receptor stimulation by high concentrations of a muscarinic receptor agonist may be due to an increase in the electrically induced [Ca 2+] i transient in ventricular myocytes via a process which is not pertussis toxin sensitive. The increase in the electrically induced [Ca 2+] i transient may result from increases in Na +Ca 2+ exchange and influx of Ca 2+ via voltage-gated Ca 2+ channels, and mobilization of Ca 2+ from the intracellular store. The mobilization of Ca 2+ from the intracellular store is a secondary event. The study has provided evidence for the first time that muscarinic acetylcholine receptor stimulation by high concentrations of carbachol increases Ca 2+ influx via the Ca 2+ channel and mobilization of Ca 2+ from its intracellular store. The study has also demonstrated for the first time the occurrence of Ca 2+ oscillations induced by high concentrations of carbachol.

Molecular and functional identification of m1 muscarinic acetylcholine receptors in rat ventricular myocytes.

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol ( > 10 mumol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mumol/L) increased the basal Ca2+ level from 96 +/- 7 to 118 +/- 8 nmol/L and the peak Ca2+ transient level from 519 +/- 32 to 640 +/- 36 nmol/L (mean +/- SEM P < .05 for both, n = 8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m1 mAChR-selective antagonist. In contrast, the m2 mAChR-selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m1 mAChRs are expressed on adult rat ventricular myocytes in addition to m2 mAChRs. The results further suggest that m1 mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.

Carbachol but Not Acetylcholine Inhibits Contraction by the Protein Kinase C-Dependent and -Independent Pathways in the Smooth Muscle of Guinea Pig Taenia Caeci

In the intestinal smooth muscle of guinea pig taenia caeci, acetylcholine and carbachol induced a transient contraction followed by a sustained contraction. The magnitudes of the transient and sustained contractions were similar when muscle was stimulated with acetylcholine (0.1 μM – 1 mM) or a lower concentration (0.1 μM) of carbachol. However, higher concentrations of carbachol (1–100 μM) induced significantly smaller sustained contraction than the transient contraction. In the 45 mM KCl-stimulated strips, addition of 100 μM carbachol induced a transient increase followed by a sustained decrease in the contractile tension. In contrast, acetylcholine (0.1 μM – 1 mM) showed only weak inhibitory effects on the high K+-induced contraction either in the absence or presence of a cholinesterase inhibitor, 0.5 αM diisopropylfluorophosphate. The same concentration of diisopropylfluorophosphate shifted the concentration-response curve for acetylcholine to lower concentrations. In the muscles pretreated with 3 μM phorbol 12-myristate 13-acetate for 24 hr to desensitize protein kinase C, sustained contractions induced by higher concentrations of carbachol (1-100 μM) were significantly greater than those in the strips without the treatment with phorbol ester. However, the transient contraction and the contraction induced by a lower concentration (0.1 μM) of carbachol were not changed by the treatment with phorbol ester. Pretreatment with phorbol ester attenuated the inhibitory effect of carbachol on the high K+-induced contraction. These results suggest that the inhibitory effects of carbachol is composed of two phases: protein kinase C-independent transient inhibition and protein kinase C-dependent sustained inhibition.

Determinants of airway smooth muscle shortening in excised canine lobes.

There is marked heterogeneity of airway narrowing in intraparenchymal airways in response to bronchoconstricting stimuli. We hypothesized that this heterogeneity results from variations in the structure of the airway wall. Freshly excised dog lung lobes were inflated to transpulmonary pressures (PL) of between 5 and 15 cmH2O, and an aerosol containing a high concentration of carbachol was administered. The lobes were fixed and processed for light-microscopic examination and morphometric analysis of membranous airway dimensions. The relationships of smooth muscle shortening to PL and airway dimensions were analyzed using multiple linear regression. The results show that airway smooth muscle shortening was greater at lower PL and in airways with larger internal perimeter and a greater number of folds per internal perimeter and that it was less in airways with greater inner wall area. We conclude that the magnitude and variability of airway smooth muscle shortening and airway narrowing in response to maximal constricting stimuli are influenced by mechanical factors related to airway wall geometry.

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

The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP3) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140-280% of baseline at 10(-3) M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30-50% of the maximal stimulation) obtained at low carbachol concentrations (< 10(-5) M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP3 accumulation induced by 10(-7) M (endothelium-dependent submaximal effect) and 10(-4) M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP3 production by 10(-4) M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC50): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H- dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11- dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M1 receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

G-protein control of voltage dependence as well as gating of muscarinic metabotropic channels in guinea-pig ileum.

1. Voltage-dependent properties of muscarinic receptor cationic current activated by carbachol in single smooth muscle cells have been studied using patch-clamp recording techniques. Cells were obtained by enzymic digestion from the longitudinal muscle layer of guinea-pig small intestine. 2. The inward cationic current showed a pronounced U-shaped current-voltage relationship (inward current negative). The relationship of cationic conductance to voltage could be described by a Boltzman distribution which was shifted 36 mV in the negative direction on the voltage axis by increasing fractional receptor occupancy (by increasing agonist concentration from 3 to 300 microM), and in the positive direction by desensitization during prolonged application of agonist. Cationic channels opened by low and high concentrations of carbachol at the same potential do not have identical properties. 3. Release of GTP within the cell, by flash photolysis of an inert caged precursor, had the same effect on the current-voltage relationship as increasing receptor occupancy by the agonist. Release of GDP beta S by flash photolysis had the opposite effect. 4. These various results could be explained if cationic channel opening upon receptor activation required binding of at least one alpha-GTP subunit, but the position of the activation curve on the voltage axis depended critically on the concentration of activated G-protein alpha-subunits in the cell.