A Gs protein couples P2-purinergic stimulation to cardiac Ca channels without cyclic AMP production.
P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.
Scamps
F
F
Laboratoire de Physiologie Cardiaque, Institut National de la Sante et de la Recherche Medicale U-241, Université de Paris-Sud, Orsay, France.
Rybin
V
V
Puceat
M
M
Tkachuk
V
V
Vassort
G
G
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D015151:Immunoblotting; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000187:drug effects / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
D015220:Calcium Channels; D011983:Receptors, Purinergic; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins