Whole-cell calcium current in guinea-pig ventricular myocytes dialysed with guanine nucleotides
| Autor: | Yaroslav M. Shuba, B Hesslinger, T. F. McDonald, D. Pelzer, W. Trautwein |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
medicine.medical_specialty
GTP' Physiology G protein Heart Ventricles Guinea Pigs Action Potentials Guanosine chemistry.chemical_element Calcium Guanosine triphosphate Pertussis toxin chemistry.chemical_compound GTP-Binding Proteins Internal medicine medicine Animals Myocyte Phosphorylation Forskolin Myocardium Colforsin Isoproterenol Guanine Nucleotides Enzyme Activation Endocrinology chemistry Calcium Channels Guanosine Triphosphate Ion Channel Gating Research Article Adenylyl Cyclases Signal Transduction |
| Zdroj: | The Journal of Physiology. 424:205-228 |
| ISSN: | 0022-3751 |
| DOI: | 10.1113/jphysiol.1990.sp018063 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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