Negative Chronotropic Effect Of Carbachol Research Articles

Modulation of the M2 Muscarinic Acetylcholine Receptor Activity with Monoclonal Anti-M2 Receptor Antibody Fragments

Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.

Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells.

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after approximately 4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.

Interaction of edrophonium with muscarinic acetylcholine M2 and M3 receptors.

It has been reported that edrophonium can antagonize the negative chronotropic effect of carbachol. This study was undertaken to evaluate in detail the interaction of edrophonium with muscarinic Mz and M3 receptors. A functional study was conducted to evaluate the effects of edrophonium on the concentration-response curves for the negative chronotropic effect and the bronchoconstricting effect of carbachol in spontaneously beating right atria and tracheas of guinea pigs. An electrophysiologic study was conducted to compare the effects of edrophonium on carbachol-, guanosine triphosphate (GTP)gama S-, and adenosine-induced outward K+ currents in guinea pig atrial cells by whole cell voltage clamp technique. A radioligand binding study was conducted to examine the effects of edrophonium on specific [3HIN-methylscopolamine (NMS) binding to guinea pig atrial (M2) and submandibular gland (M3) membrane preparations, and on atropine-induced dissociation of [3H]NMS. Edrophonium shifted rightward the concentration-response curves for the negative chronotropic and bronchoconstricting effects of carbachol in a competitive manner. The pA2 values for cardiac and tracheal muscarinic receptors were 4.61 and 4.03, respectively. Edrophonium abolished the carbachol-induced outward current without affecting the GTPgamma S- and adenosine-induced currents in the atrial cells. Edrophonium inhibited [3H]NMS binding to M2 and M3 receptors in a concentration-dependent manner. The pseudo-Hill coefficient values and apparent dissociation constants of edrophonium for M2 and M3 receptors were 1.02 and 1.07 and 21 and 34 microM, respectively. Edrophonium also changed dissociation constant values of [3H]NMS without affecting its maximum binding capacities. Edrophonium binds to muscarinic M2 and M3 receptors nonselectively, and acts as a competitive antagonist.

Inactivation of GiαProteins Increases Arrhythmogenic Effects ofβ-Adrenergic Stimulation in the Heart

Chronic treatment of rats with carbachol downregulates M-cholinoceptors and inhibitory, pertussis toxin (PTX)-sensitive G proteinalpha-subunits (Gialpha) and sensitizes the heart to arrhythmogenic effects of isoprenaline (ISO), suggesting a causal relationship. To test this hypothesis by a more direct and quantitative approach, nine groups of rats were treated for 24 h with increasing doses of PTX (1.25-200 microg/kg i.v.). Inactivation of cardiac Gialpha was determined biochemically by 32P-ADP-ribosylationin vitro and functionally by measuring contractile effects of carbachol. Effects of ISO were studied in spontaneously beating right atria (RA) and isolated papillary muscles (PM; paced at 1 Hz). PTX increased heart rate in conscious animals (ECG) with a bell-shaped dose-dependency (maximal increase 120 beats/min at 7.5 microg/kg). PTX dose-dependently inactivated 25-85% of total cardiac Gialpha, which linearly correlated with a loss of the direct negative chronotropic effect of carbachol in atria, but not with a loss of its indirect negative inotropic effect in PM. The latter was resistant up to PTX 20 microg/kg (=70% inactivation). The decrease in Gialpha closely correlated with an increased efficacy of ISO to induce spontaneous contractile activity (automaticity) in PM. At 3 micromol/l ISO, all PM from PTX 200 microg/kg beat spontaneously compared to 10% in control. In contrast, pretreatment with PTX only modestly and not clearly dose-dependently increased the inotropic potency of ISO (PTX 100 microg/kg: EC50 28v 81 nmol/l in control) and did not affect the chronotropic effect of ISO. The disparity of the functional consequences of PTX treatment suggest that under physiological conditions, Gialpha serve mainly to suppress arrhythmogenic, but not or to a minor extent, positive chronotropic or inotropic effects of beta-adrenoceptor activation.

901-63 Role of Nitric Oxide in the Autonomic Modulation of Atrioventricular Nodal Conduction in Dogs

Nitric oxide (NO), synthesized from L-arginine, is involved in the modulation of autonomic neurotransmission. Recent in vitro and in vivo studies have shown that the negative chronotropic effect of carbachol or vagal stimulation was partially blocked by an L-arginine analog, NG-monomethyl-L-arginine (L-NMMA), that competetively inhibits NO synthase (NOS). The purpose of the present study was to assess in an open chest canine model whether the L-arginine-NO pathway may also playa role in the autonomic modulation of AV nodal conduction, and if so, whether this is prejunctional or postjunctional. To test for prejunctional actions, we assessed the dromotropic response to cervical vagal stimulation (VS: 2 Hz, 4.0 V. 4 ms) and VS during ansae subclaviae stimulation (SS: 2 Hz, 3.0 V. 4 ms, n = 5). To test for postjunctional actions, we determined the dromotropic response to intravenous isoproterenol infusion (5 mcg/min) before and during vagal stimulation (VS ± ISO, n = 5). VS, SS and ISO were repeated during selective infusion of L-NMMA retrogradely into the AV node artery (AVNA) and following reversal of NOS inhibition with L-arginine (100 mg/kg). Shown is the change in AV nodal conduction time (AH, msec) produced by VS before and during SS or isoproterenol at control state (saline infusion into AVNA, 1 ml!min), during L-NMMA infusion (4 mg/min) into AVNA, and after IV injection of L-arginine (100 mg/kg).Empty CellVSVS/SSVS/ISOControl139 ± 29120 ± 2795 ± 23L-NMMA*118 ± 21101 ± 2981 ± 31L-arginine†136 ± 27117 ± 2491 ± 26*p < 0.01 vs baseline†p < 0.01 vs L-NMMA p < 0.01 vs baseline p < 0.01 vs L-NMMA The negative dromotropic effect of VS. VS during SS and ISO is attenuated by L-NMMA and restored toward baseline by L-arginine, suggesting that NO plays a partial role at both prejunctional and postjunctional sites in the vagal modulation of AV nodal conduction.

Nitric oxide synthases and cardiovascular signaling

Nitric oxide (NO) synthesized from l-arginine is a ubiquitous intercellular chemical messenger involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The recent isolation of molecular clones for NO synthases has permitted the characterization of several distinct enzyme isoforms and has allowed us to identify a family of related genes. NO synthesized in vascular endothelial cells appears to play an important role in the control of vascular tone and platelet aggregation, apparently through the activation of guanylate cyclase activity in target tissues mediated by NO. The rote of the NO signaling pathway in the direct modulation of cardiac function is less well characterized. We have found that inhibitors of NO synthase can modulate the response of neonatal or adult rat ventricular myocytes exposed to muscarinic or adrenergic agonists. The effects of carbachol on the inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes are blocked by l- N-monomethylarginine, an l-arginine analog that inhibits NO synthase, and by methylene blue, an inhibitor of NO; these agents have no effect on the basal beating rate of these cells. The negative chronotropic effect of carbachol is also mimicked by analogs of cyclic guanosine monophosphate (cGMP), a second messenger implicated in mediating the action of NO in other cell types. Production of NO can be detected directly in carbachol-stimulated neonatal myocytes using a reporter cell bioassay. In studies of adult cardiac myocyte contractility, the NO synthase inhibitor l-nitro-arginine was found to increase the inotropic effect of the β-adrenergic agonist isoproterenol on adult rat ventricular myocytes but had no effect on basal contractility. Thus, the physiologic response of isolated neonatal and adult ventricular myocytes to both muscarinic cholinergic and β-adrenergic stimulation is mediated, at least in part, by products of an endogenous NO synthase.

Control of cardiac muscle cell function by an endogenous nitric oxide signaling system.

Nitric oxide (NO) synthesized from L-arginine is a ubiquitous intracellular chemical messenger and is involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The role of the NO-signaling pathway in the direct modulation of cardiac function is less well characterized. In this report, the effects of inhibitors of NO synthase (NOS) were examined in isolated neonatal and adult rat ventricular myocytes exposed to either muscarinic or adrenergic agonists. Carbachol (10 microM) caused a 91% inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes. N omega-monomethyl-L-arginine, an L-arginine analog that inhibits NOS, and methylene blue, an inhibitor of NO, blocked the negative chronotropic effect of carbachol but had no effect on the basal beating rate of these cells. The inhibition by N omega-monomethyl-L-arginine of the negative chronotropic effect of carbachol was reversed by adding excess L-arginine. The negative chronotropic effect of carbachol was also mimicked by analogs of cGMP, a second messenger implicated in mediating the action of NO in other cell types. Production of NO could be detected directly in carbachol-stimulated neonatal myocytes by using a reporter cell bioassay. The regulation of adrenergic responsiveness by the NO signaling system was also documented in studies of adult cardiac myocyte contractility. The NOS inhibitor N omega-nitro-L-arginine significantly increased the inotropic effect of the beta-adrenergic agonist isoproterenol on electrically stimulated adult rat ventricular myocytes, whereas this inhibitor had no effect on basal contractility. Inhibition of NO production by N omega-monomethyl-L-arginine in these cells, as measured by reporter cell bioassay, was also reversible with excess L-arginine. Thus, the physiologic response of isolated neonatal and adult ventricular myocytes to both muscarinic cholinergic and beta-adrenergic stimulation is mediated, at least in part, by products of an endogenous NOS.

Pertussis Toxin-Sensitive G-Protein Activation Does Not Influence the Response to BAY K 8644 in Embryonic Chick Myocytes

Pretreatment of embryonic chick cardiac myocytes with pertussis toxin (1-2.5 micrograms ml-1 for 22 h) abolished the negative chronotropic effects of carbachol but not the positive inotropic effects of Bay K 8644. Neither guanosine 5'-O-3-thiotriphosphate (GTP gamma S 500 microM intracellularly), nor pertussis toxin (0.5-1 microgram ml-1 for 22 h) modified the agonist effects of Bay K 8644 on calcium channel currents recorded under whole-cell voltage clamp. These findings indicate that pertussis-sensitive guanine nucleotide binding (G)-proteins does not modulate the effects of calcium channel activators in embryonic chick cardiac myocytes.

Discrimination by N-ethylmaleimide between the chronotropic and inotropic response to muscarinic receptor stimulation in rat atrium.

N-ethylmaleimide (NEM) rapidly blocked the negative chronotropic effect of carbachol on rat right atrium. In contrast, NEM did not reduce the negative inotropic response to muscarinic (M) receptor stimulation. Carbachol inhibited the specific binding of [3H]-N-methylscopolamine [( 3H]-NMS) to membranes of rat atria as reflected by a shallow inhibition curve. Both guanosine triphosphate (GTP) and NEM shifted the [3H]-NMS inhibition curves of carbachol to the right. Pretreatment of the atrial membranes with NEM abolished the GTP-induced rightward shift. However, when instead of the membranes the intact atria were pre-incubated with NEM, no interaction between NEM and GTP in the membranal preparation was observed. The results indicate that NEM sharply discriminated between the inotropic and chronotropic effects to M-receptor stimulation in rat atria. The inhibitory effect of NEM on the M-receptor-mediated negative chronotropic effect in rat atrium cannot be explained by an interaction of the sulfhydryl reagent with GTP-binding proteins, like Ni or No.