Calcium and the endothelin-1 and alpha 1-adrenergic stimulated phosphatidylinositol cycle in cultured rat cardiomyocytes.
Cultured neonatal rat cardiac myocytes have been utilized as a model for the study of the effect of variations in cytoplasmic free Ca2+ on the activity of phospholipase C, a key enzyme in agonist-stimulated signal transduction through the phosphoinositide pathway. Cells prelabelled with [3H]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [3H]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endothelin-1 (10(-8) M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10 microM external Ca2+) and 50 mM K+ (1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10(-4) M) induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10 microM) treatment of cells did not affect either basal or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+ by addition of 50 microM ethylene glycol bis(beta-aminoethyl ether) (N,N,N',N'-tetraacetic acid strongly inhibited (75%) the endothelin-1 induced but not the basal phospholipase C activity. Endothelin-1 binding to its receptor was shown not to be inhibited by the absence of external Ca2+ while resynthesis of [3H]phosphatidylinositol 4,5-bisphosphate was not rate-limiting under this condition. The lack of external Ca2+ eventually resulted in total standstill of the ET-1 induced PtdIns turnover after 30 min. Although not always as predicted, effects on basal and agonist-activated phospholipase C were observed too when cells were treated with low Ca2+ medium, Ca2+ entry blocker nifedipine (1 microM) or Ca(2+)-channel agonist Bay K8644 (1 microM) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-2) measurements showed that total removal of external free Ca2+ for a short period decreased, while short exposure to high K+ increased cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine nor Bay K8644 had any effect. Furthermore, in saponin-permeabilized cardiomyocytes we could demonstrate that basal as well as GTP gamma S (30 microM) stimulated phospholipase C activity was strongly activated by free Ca2+ in the concentration range of 0.1-10 microM. We conclude that in the intact cardiomyocyte the signalling pathway through phospholipase C/phosphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor interaction that activates GTP-binding proteins as does GTP gamma S, is likely be a Ca2+ dependent process.
van Heugten
H A
HA
Department of Biochemistry, Cardiovascular Research Institute (COEUR), Faculty of Medicine, Erasmus University Rotterdam, The Netherlands.
de Jonge
H W
HW
Bezstarosti
K
K
Lamers
J M
JM
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D007295:Inositol Phosphates / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D008563:Membrane Lipids / Q000502:physiology*; D009124:Muscle Proteins / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009543:Nifedipine / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000502:physiology*; D010727:Phosphoric Diester Hydrolases / Q000502:physiology; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D013268:Stimulation, Chemical
D000316:Adrenergic alpha-Agonists; D015220:Calcium Channels; D016232:Endothelins; D007295:Inositol Phosphates; D008563:Membrane Lipids; D009124:Muscle Proteins; D010716:Phosphatidylinositols; D018340:Receptors, Adrenergic, alpha-1; D010656:Phenylephrine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000001:Calcimycin; D004533:Egtazic Acid; D010042:Ouabain; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D009543:Nifedipine; D011188:Potassium; D002118:Calcium