cGMP-mediated inhibition of cardiac L-type Ca2+ current by a monoclonal antibody against the M2 ACh receptor

J. H. M. Nascimento1, L. Sallé2, J. Hoebeke3, J. Argibay2, and N. Peineau2

2 Laboratoire de Physiologie des Cellules Cardiaques et Vasculaires, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6542, Faculté des Sciences, Parc de Grandmont, 37200 Tours; 3 Centre National de la Recherche Scientifique Unité Prope de Recherche 9021, Institute de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France; and 1 Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil


    ABSTRACT
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ABSTRACT
INTRODUCTION
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The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M2 receptor were studied on the L-type Ca2+ currents (ICa,L) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated ICa,L but did not significantly affect basal ICa,L. Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated ICa,L were not modified in presence of B8E5. Inhibition of ICa,L by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase {1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one}. These results indicate that the antibody B8E5 inhibits the beta -adrenergic-stimulated ICa,L through activation of the M2 muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.

M2 muscarinic receptor; guanosine 3',5'-cyclic monophosphate-dependent protein kinase pathway; autoantibodies; guinea pig ventricular myocytes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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CIRCULATING AUTOANTIBODIES against G protein-coupled cardiovascular receptors such as beta -adrenergic and M2 muscarinic receptors have been demonstrated in patients with idiopathic dilated cardiomyopathy (36, 70), malignant hypertension (15), chronic Chagas' heart disease (3, 4, 10, 24, 57, 58), and also in mice experimentally infected with Trypanosoma cruzi (43). Functional studies have demonstrated that these autoantibodies are able not only to bind to target receptors in the myocardium, but also to induce receptor-mediated biological responses, as partial agonists (24, 46). In chronic chagasic patients, an association was found between the presence of autoantibodies against muscarinic receptors and cardiac dysfunction, pointing to a pathogenic role of such antibodies in the development of chagasic cardiomyopathy (24, 46).

It has been reported that the antigenic targets for these autoantibodies are located in the second extracellular loop of beta -adrenergic and M2 muscarinic receptors (14, 16, 39). In a recent study, Elies et al. (11) characterized a monoclonal antibody (B8E5) that recognizes the sequence VRTVE, corresponding to the NH2-terminal part of the second extracellular loop of the human M2 muscarinic receptor. The agonist-like activity of this antibody was demonstrated by its ability to decrease the beating rate of neonatal rat cardiomyocytes in culture (11).

In the present study, we examined the functional effects of the monoclonal antibody B8E5 on L-type Ca2+ channels after beta -adrenergic prestimulation. We show that B8E5 inhibits the beta -adrenergic responsiveness of L-type Ca2+ current (ICa,L) via a cGMP-dependent pathway.


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Isolation of single cells. All experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Guinea pigs were killed by cervical dislocation. The hearts were quickly excised, and single ventricular myocytes were isolated using collagenase and pronase digestion as described elsewhere (34). Isolated cells were placed in a 100-µl chamber on the stage of an inverted microscope (Nikon Diaphot). The chamber was continuously perfused at a rate of 880 µl/min with the standard Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 11 glucose, 0.33 NaH2PO4, and 10 HEPES (pH adjusted to 7.3 with NaOH).

Electrophysiology. Whole cell voltage-clamp experiments were conducted using a patch-clamp amplifier (Axopatch 200A; Axon Instruments). The patch pipettes had resistances of 2-5 MOmega when filled with the internal solution. After achieving whole cell configuration, capacitance and series resistance were compensated. The series resistance was 10.9 ± 0.4 MOmega (183 cells) and was compensated by 60-80%. The mean capacitance of the cells was 53.4 ± 1.6 pF (183 cells).

Cells were internally dialyzed with pipette solution, which contained (in mM) 110 CsCl, 30 tetraethylammonium (TEA)-Cl, 5 MgATP, 0.1 GTP, 10 HEPES, and 10 EGTA (pH adjusted to 7.1 with CsOH). To eliminate ion currents other than ICa,L once whole cell recording was achieved, the bath solution was switched to an extracellular solution (TEA-Cs solution) where Na+ were substituted by TEA and K+ were replaced by Cs+ containing (in mM) 140 TEA-Cl, 6 CsCl, 1 MgCl2, 2 CaCl2, 11 glucose, and 10 HEPES (pH adjusted to 7.3 with TEA-OH).

Experiments were conducted at room temperature (24-26°C). Currents were low-pass filtered at 2 kHz and acquired with an analog-to-digital converter (Digidata 1200; Axon Instruments) and pCLAMP 6.02 software (Axon Instruments), at a sampling rate of 5 kHz. ICa,L was elicited every 6 s by voltage-clamp steps from a holding potential of -80 to 0 mV for 500 ms. ICa,L amplitude was determined as the difference between peak inward current and the current at the end of the depolarizing pulse. The effects of the anti-M2 antibody B8E5 on ICa,L are presented as percentage variations from the amplitude of the isoproterenol (ISO)-stimulated ICa,L (or other activator-stimulated ICa,L).

Drugs. ISO, IBMX, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) were purchased from Sigma. Except for forskolin, which was dissolved in alcohol at a final concentration of 0.1%, and ODQ in DMSO, all other drugs were dissolved in aqueous solutions.

Antibody purification and properties. Monoclonal antibody B8E5 is of IgG2a isotype and recognizes the pentapeptide VRTVE corresponding to sequence 168-172 of the human M2 muscarinic receptor (48). The antibody was purified from ascitic fluids by adsorption on Protein A (Hi-Trap Protein A; Pharmacia, Uppsala, Sweden) in PBS (150 mM NaCl, 10 mM phosphate, pH 7.4) and subsequent elution with 0.1 M citrate buffer at pH 3.6. The purity of the antibody was assessed by electrophoresis on SDS polyacrylamide gels and its active concentration defined by surface plasmon resonance in a BIAcore instrument as described by Christensen (8). The active concentration corresponded to 20-30% of the concentration of antibodies as measured by absorbance at 280 nm.

Statistical analysis. Results are expressed as means ± SE (n = number of cells). Statistical significance was evaluated by the two-tailed Student's t-test or ANOVA. In all cases, P < 0.05 was considered statistically significant.


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Effect of B8E5 on nonstimulated and ISO-stimulated ICa. We first tested the effects of B8E5 on basal (nonstimulated) ICa,L of guinea pig ventricular myocytes. Like it has been reported for ACh, a specific muscarinic agonist, this antibody did not change the amplitude of basal ICa,L significantly. After 6 min of superfusion with 12 nM B8E5, the mean variation of basal ICa,L was 0.79 ± 6.03% (8 cells). Therefore, to study the effect of B8E5 on ICa,L, guinea pig ventricular myocytes were prestimulated with ISO. At a concentration of 0.1 µM, ISO increased the peak amplitude of ICa,L by 192 ± 29% (7 cells), consistent with the concentration-dependent stimulation of ICa,L by ISO previously reported for guinea pig cells (65). The superfusion of the monoclonal antibody in the presence of ISO showed a significant inhibition of ISO-stimulated ICa,L. B8E5 at 12 nM reduced the ISO-stimulated ICa,L by 42.8 ± 6.6% (7 cells). A typical time course for the reduction of ICa,L induced by B8E5 is illustrated in Fig. 1A. The effect of B8E5 on ISO-stimulated ICa,L reached a steady level in 2-4 min after the onset of superfusion, and it was irreversible under the time limits used for measurement. Figure 1B shows the current traces recorded at the time points denoted by the letters a, b, and c in Fig. 1A. Peak ICa,L amplitude, normalized relative to cell capacitance (pA/pF) as a function of current-voltage (I-V) relationships, before and after exposure to ISO or ISO plus B8E5 is plotted in Fig. 1C. A comparison of the I-V curves shows that ISO, at 0.1 µM, evoked an increase of ICa,L amplitude at every voltage with respect to the control conditions and, as reported in the literature, shifted the peak ICa,L to more negative potentials (40). Figure 1C also shows a decrease of ICa,L amplitude in presence of ISO with B8E5, relative to ISO-stimulated ICa,L, but with no significant shift in peak current potential. Figure 1D compares the inhibitory effect of carbachol (CCh), a muscarinic agonist, and B8E5 on the ISO-stimulated ICa,L. CCh at 1 µM reduced the stimulatory effect of ISO on ICa,L by 26.97 ± 8.07% (n = 6). In nine other experiments, a higher concentration of CCh (10 µM) inhibited the ISO-stimulated ICa,L by 60.62 ± 9.07%. Atropine, a nonspecific muscarinic antagonist, significantly attenuated the inhibitory effects of CCh and B8E5 on ISO-stimulated ICa,L. In the presence of 10 µM atropine, 10 µM CCh reduced ICa,L by only 18.6 ± 10.5% (n = 4). Application of 12 nM B8E5, in the presence of 10 µM atropine, induced no significant effect on ISO-stimulated ICa,L. The mean ICa,L variation was -3.6 ± 2.0% in seven cells.


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Fig. 1.   Effect of the anti-M2 muscarinic receptor antibody B8E5 on isoproterenol (ISO)-stimulated L-type Ca2+ current (ICa,L) in guinea pig ventricular myocytes. A: time course of changes in ICa,L current amplitude elicited by 0.1 µM ISO and the application of 12 nM B8E5. B: individual current traces recorded under different conditions: control (a), ISO (b), and ISO + B8E5 (c) at the times indicated in A. C: current-voltage relationship of ICa,L in control condition, in the presence of 0.1 µM ISO, and in the presence of 0.1 µM ISO + 12 nM B8E5. Symbols and bars represent means ± SE of 58, 19, and 14 different ventricular myocytes, respectively. D: percent inhibition of ISO-stimulated ICa,L induced by carbachol (CCh) or B8E5, in the absence or presence of 10 µM atropine (Atro).

Effect of B8E5 on electrophysiological parameters of ISO-stimulated ICa,L. To investigate the effect of B8E5 on the voltage dependence of the Ca2+ channel, activation and inactivation curves for ICa,L were determined. The ICa,L activation curve was constructed from the I-V relationship by dividing ICa,L by the driving force. The apparent reversal potential of ICa,L (ECa) was estimated by linear regression through the data points in the descending part of the I-V curve where ICa,L is fully activated (+20 to +40 mV). The amplitude of ICa,L at each potential was normalized to the predicted maximal current at that voltage estimated from the fully activated I-V relationship. Figure 2A shows the normalized estimated steady-state activation curves in control, 0.1 µM ISO, and in presence of both ISO and 12 nM B8E5. The experimental points were fitted to a Boltzmann function to determine the half-activation voltage (V1/2) and the slope factor (k). Under control conditions, V1/2 was -11.0 ± 0.5 mV and k was 5.2 ± 0.1 mV (n = 58). In the presence of ISO, V1/2 was -26.7 ± 2.9 mV and k was 4.5 ± 0.2 mV (n = 15). Under both ISO and B8E5, V1/2 was -28.0 ± 1.1 mV and k was 4.8 ± 0.3 mV (n = 14). Thus ISO induces a modification of the voltage dependence of activation of the Ca2+ channel characterized by a negative shift with no change in the slope as previously described (40). When B8E5 was superfused together with ISO, no statistical difference was observed either in V1/2 or k. Similar results were obtained when we looked at the steady-state inactivation curves, obtained by using the classical double-pulse protocol for a voltage range of -80 to +40 mV from a holding potential of -80 mV and test pulse of 0 mV. The results were fitted, at the interval from -50 to 0 mV, with a Boltzmann function to determine the parameters of steady-state inactivation. The inactivation plots in control, 0.1 µM ISO, and with 12 nM B8E5 are shown in Fig. 2B. Under control conditions, V1/2 was -30.3 ± 0.8 mV and k was 4.8 ± 0.1 mV (n = 38). In the presence of ISO, V1/2 was -38.8 ± 1.3 mV and k was 5.9 ± 0.2 mV (n = 17). When B8E5 was superfused in the presence of ISO, V1/2 was -40.2 ± 0.7 mV with a k of 5.8 ± 0.36 mV (n = 14). The results are comparable with those obtained for the activation curve, a negative shift for the half-inactivation voltage without change in the slope between the control condition and in the presence of ISO, whereas the presence of the antibody induces no statistical difference in the two parameters compared with ISO alone.


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Fig. 2.   Voltage dependence of activation and inactivation parameters of ICa,L. A: voltage activation curve of ICa,L obtained from the current-voltage relationship. ISO shifted the voltage of half-activation compared with the control curve, whereas the application of the anti-M2 antibody B8E5 with ISO does not change the activation parameters relative to the ISO curve. B: steady-state voltage-dependent inactivation curves, under the same conditions as in A, show similar results. Inactivation was measured by stepping to 0 mV after applying 1,000-ms conditioning pulses to potentials between -80 and +40 mV. Holding potential, -80 mV. C: effect of B8E5 on ICa,L recovery from inactivation. The time course for recovery of ISO-stimulated ICa,L was not modified by B8E5.

Another possible source of ICa,L decrease by antibody action could be a modification of the time constant for recovery from inactivation of Ca2+ channels, such that even if depolarizing pulses were applied every 6 s, a slowing of the recovery process could induce a decrease in ICa,L amplitude. To test this possibility, the time course of recovery from inactivation was determined at a holding potential of -80 mV under the different experimental conditions. The recovery from inactivation could be fitted by a single exponential, with a time constant (tau ) in control conditions of 101.7 ± 6.7 ms (n = 18). Figure 2C shows a significant slowing of the time of recovery in the presence of 0.1 µM ISO (tau  = 159.4 ± 12.0 ms, n = 12), but no difference was detected by adding 12 nM B8E5 (tau  = 162.3 ± 16.4 ms, n = 13). In any case, recovery was complete in ~2 s.

Participation of the cAMP-dependent pathway in the effect of B8E5. It is known that ISO increases ICa,L via a phosphorylation mechanism involving the beta -adrenergic receptor and a cAMP-dependent pathway and that this effect can be antagonized by the activation of the M2 muscarinic receptor. The mechanism for this muscarinic inhibition is based on the reduction of intracellular cAMP concentration by a number of distinct reactions (32). Our aim was to evaluate whether the reduction of ICa,L induced by the antibody is mediated by cAMP reduction. Furthermore we investigated which of the two principal mechanisms known to reduce cAMP, either the inhibition of adenylyl cyclase via the stimulation of Gi protein or the stimulation of cGMP-dependent phosphodiesterase (PDE), was being activated.

Forskolin is a potent activator of adenylyl cyclase (29). Superfusing the cells with 10 µM forskolin increased the amplitude of ICa,L by 185.6 ± 39.9% (n = 6), as illustrated in Fig. 3A. In accordance with the results observed in the presence of ISO, inhibition of this effect was obtained by the addition of B8E5. In six cells tested, 12 nM B8E5 reduced the forskolin-activated ICa,L by 25.6 ± 6.3% (Fig. 3D).


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Fig. 3.   Action of the anti-M2 antibody B8E5 on ICa,L in the presence of activators of protein kinase A-dependent phosphorylation. A: application of 10 µM forskolin (Fsk) induced an increase of ICa,L amplitude that was partially reversed by application of the B8E5 antibody. B: blockage of cAMP degradation by 10 µM IBMX provoked ICa,L enhancement, which was inhibited by 12 nM B8E5. Arrowhead indicates the start of B8E5 application. C: the same result was obtained when 10 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) was included in the internal pipette solution. The first point in the curve was recorded at ~6 min after patch rupture. D: summary of the effects of B8E5 (12 nM) on ICa,L in the presence of ISO, 8-BrcAMP dialysis, forskolin, or IBMX. B8E5-induced inhibition is expressed as percentage reduction over the amplitude of stimulated ICa,L. Values are means ± SE, with number of experiments in parentheses. Gaps in B and C are points missing during the determination of current-voltage relationships.

The participation of a PDE in ICa,L inhibition by B8E5 was excluded when, in the presence of the nonspecific PDE inhibitor IBMX (61), it was still possible to decrease the ICa,L amplitude with B8E5. IBMX (10 µM) increased ICa,L by 142.9 ± 24.7% (n = 5). In four myocytes, the percent inhibition of IBMX-stimulated ICa,L by B8E5 was 43.2 ± 5.7% (Fig. 3, B and D). These data suggest that ICa,L amplitude reduction by B8E5 is mediated not by a reduction of cAMP levels, but by another mechanism. To reinforce this hypothesis we used 8-BrcAMP, a cAMP analog more resistant to hydrolysis by PDEs than cAMP (49). The external application or internal dialysis of 10 µM 8-BrcAMP led to an increase in ICa,L that was reversed by the application of 12 nM B8E5 (38.4 ± 6.9%, n = 4; Fig. 3, C and D).

Participation of the cGMP-dependent pathway in the effect of B8E5. Because the previous results suggest that the cAMP pathway does not participate in the reduction of ICa,L by B8E5, an alternative mechanism was tested. It was shown that muscarinic stimulation would increase cGMP levels in cardiac myocytes (29, 51). Indeed, in guinea pig ventricular myocytes, cGMP inhibits cAMP-activated ICa,L, but the mechanism seems to be independent of cGMP-stimulated PDEs (35). Therefore, we decided to investigate the participation of a cGMP-dependent protein kinase (cGMP-PK)-mediated pathway, by using 8-BrcGMP, a potent membrane-permeable stimulator of cGMP-PK (9). As shown in Fig. 4A, superfusion of 100 µM 8-BrcGMP in the presence of 0.1 µM ISO significantly reduced the peak amplitude of ISO-stimulated ICa,L. In four cells, 0.1 µM ISO increased ICa,L by 130.7 ± 15.6%, whereas the addition of 100 µM 8-BrcGMP in the presence of ISO decreased ICa,L by 42.33 ± 5.5%. The finding that 8-BrcGMP reduces ISO-stimulated ICa,L, in a manner similar to B8E5, lends support to the view that B8E5 may be acting through a muscarinic receptor-mediated elevation of cGMP levels. To further test this hypothesis, we used ODQ, a selective inhibitor of nitric oxide (NO)-sensitive guanylyl cyclase (5, 23). The superfusion of 10 µM ODQ in the presence of B8E5 and ISO after the prestimulation of ICa,L by 0.1 µM ISO blocked the inhibitory effect of B8E5. At steady state, the peak reduction of ISO-stimulated ICa,L was 5.6 ± 5.9% (n = 6). A typical experiment is shown in Fig. 4B.


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Fig. 4.   A: effect of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on ISO-stimulated ICa,L. Time course of changes in ICa,L amplitude recorded during the superfusion of 0.1 µM ISO and 100 µM 8-BrcGMP + 0.1 µM ISO. B: 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) blocks the effect of the anti-M2 muscarinic receptor antibody B8E5 on ISO-stimulated ICa,L in ventricular guinea pig myocytes. Time course of changes in ICa,L amplitudes in presence of ISO and in presence of ISO+ ODQ+ B8E5 is indicated by the bars above the graph.


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The present study demonstrates that a monoclonal antibody raised against the second extracellular loop of the muscarinic receptor can decrease the beta -adrenergic responsiveness of ICa,L in guinea pig ventricular myocytes by acting as an allosteric agonist through activation of the muscarinic receptor. We also show that the decrease of ICa,L through the M2 muscarinic receptor does not pass by the classical cAMP-dependent pathway and that an additional cAMP-independent mechanism exists to reverse the increase in ICa,L amplitude induced by beta -adrenergic stimulation. From our results and other reports (25, 59), it is reasonable to assume a participation of cGMP in the ICa,L reduction evoked by anti-M2 receptor antibodies.

The effect passes through the M2 muscarinic receptor. The immunochemical and pharmacological properties of anti-M2 muscarinic receptor antibodies found in idiopathic dilated cardiomyopathy (17) or Chagas' disease (10, 25) can be mimicked by the polyclonal rabbit antibodies directed against the second extracellular loop of the target receptor (20). Moreover, these antibodies have a functional muscarinic activity, as shown by the induction of a negative inotropism and/or chronotropism in vivo (71) as well as in vitro on rat myocytes (18, 19) or rat papillary muscle (54). Polyclonal antibodies are, however, poor tools to unravel activating mechanisms because polyclonality can induce pleiotropy of response. The availability of a monoclonal anti-M2 muscarinic receptor antibody with agonist-like effects on neonatal rat cardiomyocytes in vitro (11) makes these mechanistic studies possible. Indeed, this monoclonal antibody (B8E5) was shown to exert its effect only through its target receptor (11). We confirmed this result by complete inhibition of the effects of B8E5 with the muscarinic antagonist atropine.

How is ICa, L decreased? The M2 receptor is the predominant subtype of muscarinic receptors present in the heart of mammalian species (6, 7, 12). Additionally, some species could coexpress the M1 receptor (21, 52). Activation of M2 and M4 receptor couples via a pertussis toxin-sensitive G protein (Gi/Go) to inhibition of adenylyl cyclase and inhibits increases in intracellular cAMP, which leads to a reduction in the previously enhanced ICa,L. M1, M3, and M5 receptors couple preferentially via Gq/11 to phospholipase C, with subsequent formation of inositol phosphates and diacylglycerol (for recent reviews, see Refs. 6, 7, 12). It is largely accepted that the M2 muscarinic inhibitory effect on ICa,L occurs through the inhibition of adenylyl cyclase, in particular through the activation of Gi protein (29, 37). The decrease of adenylyl cyclase activity leads to a reduction of cAMP production and protein kinase A (PKA) activity. In the present study, this process does not seem to mediate the decrease of ICa,L amplitude evoked by the antibodies. The reduction was still observed when the level of cAMP was sustained using a nonhydrolyzable cAMP analog. Inhibition of ICa,L by ACh without changes in cAMP levels or PKA activity has been previously reported (for review, see Ref. 41). ACh can activate the production of cGMP (29), and it has been demonstrated that, in cardiac cells, ACh can, via activation of NO synthase (NOS), increase the cGMP level (for review see Ref. 55). Interestingly, the autoantibodies against M2 receptors, present in sera of patients with Chagas' disease, also seem to act by the NO signaling pathway (59). However, some reports have shown that ACh could increase cGMP without increasing NO (22) and also in the presence of NOS inhibitors (45, 56). Studies on NOS-3 knockout mice were inconclusive, since contradictory findings on the ACh receptor-mediated ICa,L modulation were observed (28, 67). Recently, however, it was shown that NO could exert a negative effect on ICa,L by a cGMP pathway (1). Other reports also indicate an obligatory participation of NO production in the signal transduction cascade involved in the M2 muscarinic acetylcholine receptor-mediated inhibitory responses of the heart (2, 13, 27).

In amphibian cells, cGMP activates a cGMP-stimulated PDE that hydrolyzes cAMP and thereby decreases ICa,L (30). But even if the anti-M2 muscarinic receptor antibodies from chagasic patients have been reported to increase cGMP levels via NO (59, 60), the participation of PDEs in the inhibition observed here is excluded for two reasons. First, the preponderant PDE in guinea pig ventricular cardiomyocytes is the cGMP-inhibited PDE (PDE3), so cGMP production would induce an increase of cAMP and consequently an enhancement of ICa,L amplitude (47, 53). Second, the decrease of ICa,L by B8E5 was observed in the presence of a nonselective PDE inhibitor (IBMX), which suggests the presence of another target for the action of cGMP. An alternative mechanism that could be proposed is the decrease of ICa,L via activation of cGMP-PK. Levi et al. (35) were the first to describe the participation of cGMP-PK, in guinea pig cardiomyocytes. These results have been confirmed in guinea pig (44, 47, 53, 68), rat (42, 62), chick (26, 64), and rabbit cardiac cells (63). This hypothesis is supported by the observations that a potent stimulator of cGMP-PK, the cGMP-analog 8-BrcGMP, mimicked the effect of B8E5 on ISO-stimulated ICa,L and that the selective inhibitor of NO-sensitive guanylyl cyclase (ODQ) prevents the B8E5 decrease of ICa,L.

Why do the antibodies seem to activate only the cGMP pathway and not the Gi pathway? This could be due to the different active conformations induced with B8E5 compared with a normal muscarinic agonist, CCh. Indeed, the antibody B8E5 acts by functional dimerization of the muscarinic receptor, since monovalent Fab fragments of this antibody lack a negative chronotropic effect, but their activity can be restored after cross-linking of the fragments with an anti-mouse IgG (11). Functional dimerization of the receptors has already been postulated for the muscarinic receptor by Maggio et al. (38) and for beta 2-adrenergic receptor by Hebert et al. (31) and is actually proposed as an important functional property of G protein-coupled receptors (50). Supposing the multiple activating states of G protein-coupled receptors, postulated by Kenakin (33), the monoclonal antibody could induce an active dimeric conformation leading to Gq activation but not Gi/o activation. This hypothesis seems to contradict the results of Valenzuela et al. (66), who have shown that the accentuated antagonism induced by CCh was absent in Goalpha knockout mouse ventricular myocyte, indicating an obligate participation of Go for the muscarinic regulation of ICa,L. In fact, our results confirm that the antibody does not act as full agonist-like ACh or CCh but induces a specific dimeric conformation that could associate with Gq protein, although other transduction mechanisms cannot be excluded. This is also confirmed by the fact that, in contrast to CCh, the antibodies do not lead to desensitization of the receptor (69).

Clinical implications. Autoantibodies against M2 receptors are associated with some of the pathological symptoms present in cardiomyopathies such as chronic Chagas' disease (24, 25, 46). Although targeted to different epitopes at the second extracellular loop of the M2 receptor, these antibodies share with the monoclonal antibody the agonist-like activity and seem to act by inducing a dimeric conformation that allows the activation of an alternate transduction pathway, which is probably mediated by Gq. This could lead to potentially deleterious consequences for heart function, as has been shown in rabbits that were immunized with a peptide encompassing the epitope recognized by the monoclonal antibody (19). Therefore, the monoclonal antibody B8E5 could be of value in further studies of molecular mechanisms of receptor activation by such autoantibodies and could point to potential therapeutic interventions that could eliminate antibody-induced pathological symptoms.


    ACKNOWLEDGEMENTS

We thank Dr. Rozenn Elies for the gift of the monoclonal antibody. We also thank Dr. Jean-Yves Le Guennec for helpful criticism and Dr. Ian Findlay for reading the manuscript.


    FOOTNOTES

This work was supported by le Conseil Régional du Centre. J. H. M. Nascimento was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Amparo à Pesquisa do Estado do Rio de Janiero. Johan Hoebeke was supported by European Grant BMH4-CT95-1008.

Address for reprint requests and other correspondence: J. H. M. Nascimento, Instituto de Biofisica Carlos Chagas Filho, CCS Bloco G UFRJ, 21949-900. Rio de Janeiro, Brazil (E-mail: jhmn{at}biof.ufrj.br).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4 August 2000; accepted in final form 8 June 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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