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Whole‐cell calcium current in guinea‐pig ventricular myocytes dialysed with guanine nucleotides.
Author(s) -
Shuba Y M,
Hesslinger B,
Trautwein W,
McDonald T F,
Pelzer D
Publication year - 1990
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1990.sp018063
Subject(s) - gtp' , guanosine , chemistry , g protein , biophysics , forskolin , pertussis toxin , biochemistry , medicine , endocrinology , pharmacology , biology , signal transduction , enzyme , receptor
1. Whole‐cell calcium current (ICa) was recorded in guinea‐pig ventricular myocytes superfused with Na+,K(+)‐free solution and dialysed with a substrate‐free solution (minimum intracellular solution, MICS). A dual tight‐seal pipette method was often used to permit pressure‐enhanced dialysis of a test solution after a given pre‐dialysis. 2. In dual‐pipette experiments, test dialysates contained 100 mM‐GTP‐gamma‐S (guanosine 5'‐O‐(3‐thiotriphosphate] or 100 microM‐GMP‐PNP (guanyl‐5'‐imidodiphosphate). These non‐hydrolysable analogues of guanosine triphosphate (GTP) enhanced ICa amplitude (+ 10 mV) by 20‐40%. Dialysates containing 100 microM‐GTP or GDP‐beta‐S (guanosine 5'‐O‐(2‐thiodiphosphate] were ineffective, and pre‐dialysis with GDP‐beta‐S blocked stimulation by GTP‐gamma‐S. 3. Non‐hydrolysable GTP analogues slowed the inactivation of ICa and shifted the voltage eliciting maximum ICa by 5‐10 mV in the negative direction. 4. ICa enhancement by GTP analogues was attributed to the activation of three GTP‐binding regulatory (G) proteins (Gi, Gp and Gs). In single‐pipette experiments, the inactivation of Gi by pre‐treatment with pertussis toxin did not block enhancement, and a Gp‐activating regimen (external acetylcholine‐internal GTP) was without effect. Thus, it is probable that the effects of GTP analogues on ICa were primarily mediated by Gs activation. 5. PI‐MICS dialysates contained phosphorylation‐pathway inhibitors and were used to inhibit Ca2+ channel phosphorylation via the adenyl cyclase pathway. These were deemed effective since forskolin (1‐5 microM) doubled ICa during control dialysis but was without effect after 8 min PI‐MICS dialysis. However, 0.1 microM‐isoprenaline increased ICa by 35% in myocytes totally unresponsive to forskolin, suggesting that beta‐adrenergic receptor occupation can stimulate ICa even when the phosphorylation pathway is blocked. 6. After prolonged dialysis of myocytes with PI‐MICS, ICa was still enhanced by pressure‐assisted dialysis of 100 microM‐GTP‐gamma‐S or GMP‐PNP. We conclude that activated Gs has a direct effect on cardiac Ca2+ channels.