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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CIRCULATING
AUTOANTIBODIES against G protein-coupled cardiovascular receptors
such as -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
-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
-adrenergic prestimulation. We show that B8E5 inhibits the
-adrenergic responsiveness of L-type Ca2+ current
(ICa,L) via a cGMP-dependent pathway.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M 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 M
(183 cells) and was compensated by 60-80%. The mean
capacitance of the cells was 53.4 ± 1.6 pF (183 cells).
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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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.
|
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 -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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study demonstrates that a monoclonal antibody raised
against the second extracellular loop of the muscarinic receptor can
decrease the -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
-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 forClinical 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abi-Gerges, N,
Fischmeister R,
and
Méry P-F.
G protein-mediated inhibitory effect of a nitric oxide donor on the L-type Ca2+ current in rat ventricular myocytes.
J Physiol (Lond)
531:
117-130,
2001
2.
Balligand, J-L,
Kobzik L,
Han X,
Kaye DM,
Belhassen L,
O'Hara DS,
Kelly RA,
Smith TW,
and
Michel T.
Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes.
J Biol Chem
270:
14582-14586,
1995
3.
Borda, ES,
Cossio PM,
Vega M,
Araña RM,
and
Sterin-Borda L.
A circulating IgG in Chagas' disease which binds to -adrenoceptors of myocardium and modulates their activity.
Clin Exp Immunol
57:
679-686,
1984[ISI][Medline].
4.
Borda, ES,
and
Sterin-Borda L.
Antiadrenergic and muscarinic receptor antibodies in Chagas' cardiomyopathy.
Int J Cardiol
54:
149-156,
1996[ISI][Medline].
5.
Boulton, CL,
Southan E,
and
Garthwaite J.
Nitric oxide-dependent long-term potentiation is blocked by a specific inhibitor of soluble guanylyl cyclase.
Neuroscience
69:
699-703,
1995[ISI][Medline].
6.
Brodde, O-E,
and
Michel MC.
Adrenergic and muscarinic receptors in the human heart.
Pharmacol Rev
51:
651-689,
1999
7.
Caulfield, MP,
and
Birdsall NJM
International union of pharmacology. XVII. Classification of muscarinic acetylcholine receptors.
Pharmacol Rev
50:
279-290,
1998
8.
Christensen, LLH
Theoretical analysis of protein concentration determination using biosensor technology under conditions of partial mass transport limitation.
Anal Biochem
249:
153-164,
1997[ISI][Medline].
9.
Corbin, JD,
Ogreid D,
Miller JP,
Suva RH,
Jastorff B,
and
Doskeland SO.
Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase.
J Biol Chem
261:
1208-1214,
1986
10.
Elies, R,
Ferrari I,
Wallukat G,
Lebesgue D,
Chiale P,
Elizari M,
Rosenbaum M,
Hoebeke J,
and
Levin M.
Structural and functional analysis of the B cell epitopes recognized by anti-receptor autoantibodies in patients with Chagas' disease.
J Immunol
157:
4203-4211,
1996[Abstract].
11.
Elies, R,
Fu LXM,
Eftekhari P,
Wallukat G,
Schulze W,
Granier C,
Hjalmarson Å,
and
Hoebeke J.
Immunochemical and functional characterization of an agonist-like monoclonal antibody against M2 acetylcholine receptor.
Eur J Biochem
251:
659-666,
1998[Abstract].
12.
Felder, CC.
Muscarinic acetylcholine receptors: signal transduction through multiple effectors.
FASEB J
9:
619-625,
1995
13.
Feron, O,
Smith TW,
Michel T,
and
Kelly RA.
Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes.
J Biol Chem
272:
17744-17748,
1997
14.
Ferrari, I,
Levin MJ,
Wallukat G,
Elies R,
Lebesgue D,
Chiale P,
Elizari M,
Rosenbaum M,
and
Hoebeke J.
Molecular mimicry between the immunodominant ribosomal protein P0 of Trypanosoma cruzi and a functional epitope on the human 1-adrenergic receptor.
J Exp Med
182:
59-65,
1995[Abstract].
15.
Fu, LXM,
Herlitz H,
Wallukat G,
Hilme E,
Hedner T,
Hoebeke J,
and
Hjalmarson Å.
Functional autoimmune epitope on 1-adrenergic receptors in patients with malignant hypertension.
Lancet
344:
1660-1663,
1994[ISI][Medline].
16.
Fu, LXM,
Hoebeke J,
Magnusson Y,
Matsui S,
Matoba M,
Hedner T,
Herlitz H,
and
Hjalmarson Å.
Autoantibodies against cardiac G-protein coupled receptors in patients with cardiomyopathy but not with hypertension.
Clin Immunol Immunopathol
72:
15-20,
1994[ISI][Medline].
17.
Fu, MLX,
Magnusson Y,
Bergh CH,
Liljeqvist JA,
Waagstein F,
Hjalmarson A,
and
Hoebeke J.
Localization of a functional autoimmune epitope on the muscarinic acetylcholine-2 receptor in patients with idiopathic dilated cardiomyopathy.
J Clin Invest
91:
1964-1968,
1993[ISI][Medline].
18.
Fu, MLX,
Schulze W,
Wallukat G,
Hjalmarson A,
and
Hoebeke J.
Functional epitope analysis of the second extracellular loop of the human heart muscarinic acetylcholine receptor.
J Mol Cell Cardiol
27:
427-436,
1995[ISI][Medline].
19.
Fu, MLX,
Schulze W,
Wallukat G,
Hjalmarson A,
and
Hoebeke J.
A synthetic peptide corresponding to the second extracellular loop of the human M2 acetylcholine receptor induces pharmacological and morphological changes in cardiomyocytes by active immunization after 6 mo in rabbits.
Clin Immunol Immunopathol
78:
203-207,
1996[ISI][Medline].
20.
Fu, MLX,
Wallukat G,
Hjalmarson A,
and
Hoebeke J.
Agonist-like activity of anti-peptide antibodies directed against an autoimmune epitope on the heart muscarinic acetylcholine receptor.
Receptors Channels
2:
121-130,
1994[ISI][Medline].
21.
Gallo, MP,
Alloatti G,
Eva C,
Oberto A,
and
Levi RC.
M1 muscarinic receptors increase calcium current and phosphoinositide turnover in guinea-pig ventricular cardiocytes.
J Physiol (Lond)
471:
41-60,
1993[Abstract].
22.
Gallo, MP,
Ghigo D,
Bosia A,
Alloatti G,
Costamagna C,
Penna C,
and
Levi RC.
Modulation of guinea-pig cardiac L-type calcium current by nitric oxide synthase inhibitors.
J Physiol (Lond)
506:
639-651,
1998
23.
Garthwaite, J,
Southan E,
Boulton CL,
Nielsen EB,
Schmidt K,
and
Mayer B.
Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one.
Mol Pharmacol
48:
184-188,
1995[Abstract].
24.
Goin, JC,
Borda E,
Perez-Leiros C,
Storino R,
and
Sterin-borda L.
Identification of antibodies with muscarinic cholinergic activity in human Chagas' disease: pathological implications.
J Auton Nerv Syst
47:
45-52,
1994[ISI][Medline].
25.
Goin, JC,
Leiros CP,
Borda E,
and
Sterin-Borda L.
Interaction of human chagasic IgG with the second extracellular loop of the human heart muscarinic acetylcholine receptor: functional and pathological implications.
FASEB J
10:
77-83,
1997.
26.
Haddad, GE,
Sperelakis N,
and
Bkaily G.
regulation of the slow channel by cyclic GMP dependent protein kinase in chick heart cells.
Mol Cell Biochem
148:
89-94,
1995[ISI][Medline].
27.
Han, X,
Kobzik L,
Balligand J-L,
Kelly RA,
and
Smith TW.
Nitric oxide synthase (NOS3)-mediated cholinergic modulation of Ca2+ current in adult rabbit atrioventricular nodal cells.
Circ Res
78:
998-1008,
1996
28.
Han, X,
Kubota I,
Feron O,
Opel DJ,
Arstall MA,
Zhao Y-Y,
Huang P,
Fishman MC,
Michel T,
and
Kelly RA.
Muscarinic cholinergic regulation of cardiac myocyte ICa,L is absent in mice with targeted disruption of endothelial nitric oxide synthase.
Proc Natl Acad Sci USA
95:
6510-6515,
1998
29.
Hartzell, HC.
Regulation of cardiac ion channels by catecholamines, acetylcholine and 2nd messenger systems.
Prog Biophys Mol Biol
52:
165-247,
1988[ISI][Medline].
30.
Hartzell, HC,
and
Fischmeister R.
Opposite effects of cyclic GMP and cyclic AMP on Ca current in single heart cells.
Nature
323:
273-275,
1986[ISI][Medline].
31.
Hebert, TE,
Moffett S,
Morello JP,
Loisel T,
Bichet DG,
Barret C,
and
Bouvier M.
A peptide derived from a 2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation.
J Biol Chem
271:
16384-16392,
1996
32.
Hescheler, J,
Kameyama M,
and
Trautwein W.
On the mechanism of muscarinic inhibition of the cardiac Ca current.
Pflügers Arch
407:
182-189,
1986[ISI][Medline].
33.
Kenakin, TP.
Receptor conformation induction versus selection: all part of the same energy landscape.
Trends Pharmacol Sci
17:
190-191,
1996[ISI].
34.
Le Guennec, JY,
Peineau N,
Esnard F,
Lacampagne A,
Gannier F,
Argibay J,
Gauthier F,
and
Garnier D.
A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation.
Biol Cell
79:
161-165,
1993[ISI][Medline].
35.
Levi, RC,
Alloatti G,
and
Fischmeister R.
Cyclic GMP regulates the Ca-channel current in guinea pig ventricular myocytes.
Pflügers Arch
413:
685-687,
1989[ISI][Medline].
36.
Limas, CJ,
Goldenberg IF,
and
Limas C.
Autoantibodies against cardiac -adrenergic receptors in human dilated cardiomyopathy.
Circ Res
64:
97-103,
1989[Abstract].
37.
Lindemann, JP,
and
Watanabe AM.
Mechanisms of adrenergic and cholinergic regulation of the myocardial contractility.
In: Physiology and Pathophysiology of the Heart (2nd ed.), edited by Sperelakis N.. Boston, MA: Kluwer Academic, 1991, p. 423-452.
38.
Maggio, R,
Vogel Z,
and
Wess J.
Co-expression studies with mutant muscarinic/adrenergic receptors provide evidence for intramolecular "cross-talk" between G protein linked receptors.
Proc Natl Acad Sci USA
90:
3103-3107,
1993[Abstract].
39.
Masuda, MO,
Levin M,
Oliveira SF,
Costa PCS,
Bergami PL,
Almeida NAS,
Pedrosa RC,
Ferrari I,
Hoebeke J,
and
Campos de Carvalho AC.
Functionally active cardiac antibodies in chronic Chagas' disease are specifically blocked by Trypanosoma cruzi antigens.
FASEB J
12:
1551-1558,
1998
40.
McDonald, TF,
Pelzer S,
Trautwein W,
and
Pelzer DJ.
Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells.
Physiol Rev
74:
365-507,
1994
41.
Méry, P-F,
Abi-Gerges N,
Vandecasteele G,
Jurevicius J,
Eschenhagen T,
and
Fischmeister R.
Muscarinic regulation of the L-type calcium current in isolated cardiac myocytes.
Life Sci
60:
1113-1120,
1997[ISI][Medline].
42.
Méry, P-F,
Lohmann SM,
Walter U,
and
Fischmeister R.
Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes.
Proc Natl Acad Sci USA
88:
1197-1201,
1991[Abstract].
43.
Mijares, A,
Verdot L,
Peineau N,
Vray B,
Hoebeke J,
and
Argibay J.
Antibodies from Trypanosoma cruzi infected mice recognize the second extracellular loop of the 1-adrenergic and M2-muscarinic receptors and regulate calcium channels in isolated cardiomyocytes.
Mol Cell Biochem
163:
107-112,
1996.
44.
Mubagwa, K,
Shiryama T,
Moreau M,
and
Pappano AJ.
Effect of PDE inhibitors and carbachol on the L-type Ca current in guinea pig ventricular myocytes.
Am J Physiol Heart Circ Physiol
265:
H1353-H1363,
1993
45.
Nawrath, H,
Bäumner D,
Rupp J,
and
Oelert H.
The ineffectiveness of the NO-cyclic GMP signaling pathway in the atrial myocardium.
Br J Pharmacol
116:
3061-3067,
1995[Abstract].
46.
Oliveira, SF,
Pedrosa RC,
Nascimento JHM,
Campos de Carvalho AC,
and
Masuda MO.
Sera from chronic chagasic patients with complex cardiac arrhytmias depress electrogenesis and conduction in isolated rabbit hearts.
Circulation
96:
2031-2037,
1997
47.
Ono, K,
and
Trautwein W.
Potentiation by cyclic GMP of -adrenergic effect on Ca2+ current in guinea pig ventricular cells.
J Physiol (Lond)
443:
387-404,
1991[Abstract].
48.
Peralta, EG,
Winslow JW,
Peterson GL,
Smith DH,
Ashkenazi A,
Ramachandran J,
Schimerlik MI,
and
Capon DJ.
Primary structure and biochemical properties of an M2 muscarinic receptor.
Science
236:
600-605,
1987[ISI][Medline].
49.
Petit-Jacques, J,
Bois P,
Bescond J,
and
Lenfant J.
Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes.
Pflügers Arch
423:
21-27,
1993[ISI][Medline].
50.
Salahpour, A,
Angers S,
and
Bouvier M.
Functional significance of oligomerization of G-protein-coupled receptors.
Trends Endocrinol Metab
11:
163-168,
2000[ISI][Medline].
51.
Schmidt, HHHW,
Lohmann SM,
and
Walter U.
The nitric oxide and cGMP signal transduction system: regulation and mechanism of action.
Biochim Biophys Acta
1178:
153-175,
1993[ISI][Medline].
52.
Sharma, VK,
Colecraft HM,
Wang DX,
Levey AI,
Grigorenko EV,
Yeh HH,
and
Sheu S-S.
Molecular and functional identification of m1 muscarinic acetylcholine receptors in rat ventricular myocytes.
Circ Res
79:
86-93,
1996
53.
Shirayama, T,
and
Pappano AJ.
Biphasic effects of intrapipette cyclic guanosine monophosphate on L-type calcium current and contraction of guinea pig ventricular myocytes.
J Pharmacol Exp Ther
279:
1274-1281,
1996[Abstract].
54.
Skomedal, T,
Fu ML,
Hjalmarson A,
Hoebeke J,
Schiander IG,
and
Osnes JB.
Anti-M2 muscarinic receptor antibodies inhibit -adrenoceptor-mediated inotropic response in rat myocardium.
Eur J Pharmacol
333:
169-175,
1997[ISI][Medline].
55.
Smith, TW,
Balligand JL,
Kaye DM,
Wiviott SD,
Simmons WW,
Han X,
Michel T,
Singh K,
and
Kelly RA.
The role of the NO pathway in the control of cardiac function.
J Card Fail
2:
S141-S147,
1996[Medline].
56.
Stein, B,
Drögemüller A,
Mülsch A,
Schmitz W,
and
Scholz H.
Ca2+-dependent constitutive nitric oxide synthase is not involved in the cyclic GMP-increasing effects of carbachol in ventricular cardiomyocytes.
J Pharmacol Exp Ther
266:
919-925,
1993[Abstract].
57.
Sterin-Borda, L,
Fink S,
Diez C,
Cossio P,
and
Debracco MME
Beta adrenergic effect of antibodies from chagasic patients and normal human lymphocytes on isolated atria.
Clin Exp Immunol
50:
534-540,
1982[ISI][Medline].
58.
Sterin-Borda, L,
Gorelik G,
and
Borda E.
Chagasic IgG binding with cardiac muscarinic cholinergic receptors modifies cholinergic-mediated cellular transmembrane signals.
Clin Immunol Immunopathol
61:
389-397,
1991.
59.
Sterin-Borda, L,
Leiros C,
Goin JC,
Gremashi G,
Genaro A,
Vila Echague A,
and
Borda E.
Participation of the nitric oxide signaling system in the cardiac muscarinic cholinergic effect of the human chagasic IgG.
J Mol Cell Cardiol
29:
1851-1865,
1997[ISI][Medline].
60.
Sterin-Borda, L,
Vila Echague A,
Leiros C,
Genaro A,
and
Borda E.
Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor-inotropic response.
Br J Pharmacol
115:
1525-1531,
1995[Abstract].
61.
Strada, SJ,
Martin MW,
and
Thompson WJ.
General properties of multiple molecular forms of cyclic nucleotide phosphodiesterase in the nervous system.
Adv Cyclic Nucleotide Protein Phosphorylation Res
16:
13-29,
1984[ISI][Medline].
62.
Sumii, K,
and
Sperelakis N.
cGMP-dependent protein kinase regulation of the L-type Ca2+ current in rat ventricular myocytes.
Circ Res
77:
803-812,
1995
63.
Tohse, N,
Nakaya H,
Takeda Y,
and
Kanno M.
Cyclic GMP-mediated inhibition of the L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells.
Br J Pharmacol
114:
1076-1082,
1995[Abstract].
64.
Tohse, N,
and
Sperelakis N.
cGMP inhibits the activity of single calcium channels in embryonic chick heart cells.
Circ Res
69:
325-331,
1991[Abstract].
65.
Trautwein, W,
and
Hescheler J.
Regulation of cardiac L-type calcium current by phosphorylation and G proteins.
Annu Rev Physiol
52:
257-274,
1990[ISI][Medline].
66.
Valenzuela, D,
Han X,
Mende U,
Fankhauser C,
Mashimo H,
Huang P,
Pfeffer J,
Neer EJ,
and
Fishman MC.
Go is necessary for muscarinic regulation of Ca2+ channels in mouse heart.
Proc Natl Acad Sci USA
94:
1727-1732,
1997
67.
Vandecasteele, G,
Eschenhagen T,
Scholz H,
Stein B,
Verde I,
and
Fischmeister R.
Muscarinic and -adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase.
Nat Med
5:
331-334,
1999[ISI][Medline].
68.
Wahler, GM,
and
Dollinger SJ.
Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase.
Am J Physiol Cell Physiol
268:
C45-C54,
1995
69.
Wallukat, G,
Fu HM,
Matsui S,
and
Hjalmarson
70.
Wallukat, G,
and
Wollenberger A.
Effect of gamma globulin fraction of patients with allergic asthma and dilated cardiomyopathy on chronotropic -adrenoceptor function in cultured neonatal rat heart myocytes.
Biomed Biochem Acta
46:
634-639,
1987.
71.
Wang, WZ,
Zhao RR,
Wu BW,
Jin XH,
Zhu L,
Hjalmarson A,
and
Fu ML.
Effects of anti-peptide antibodies against human M2 muscarinic receptors on cardiac function in rats in vivo.
Blood Press Suppl
3:
25-27,
1996[Medline].