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 f or the study of the effect of variations in cytoplasmic free Ca2+ on t he activity of phospholipase C, a key enzyme in agonist- stimulated si gnal transduction through the phosphoinositide pathway. Cells prelabel led with [H-3]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [H-3]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endoth elin-1 (10(-8) M) induced [H-3]inositolphosphate production (15 min) w as stimulated 1.54 and 1.43-fold by A23187 (10 mu M 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 1.3 S-f old) by A23187, however if was 43% inhibited by high K+. Ouabain (10 m u M) treatment of cells did not affect either basal or agonist stimula ted phosphoinositide turnover. On the other hand, total removal of ext ernal free Ca2+ by addition of 50 mu M ethylene glycol bis(beta-aminoe thyl ether) (N,N,N',N'-tetraacetic acid strongly inhibited (75%) the e ndothelin-l induced but not the basal phospholipase C activity. Endoth elin-l binding to its receptor was shown not to be inhibited by the ab sence of external Ca2+ while resynthesis of [H-3]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 i nduced Ptdlns 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 n ifedipine (1 mu M) or Ca2+-channel agonist Bay K8644 (1 mu M) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-a) measurements showed that total removal of external free Ca2 for a short period decreased, while short exposure to high K+ increas ed cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine no r Bay K8644 had any effect. Furthermore, in saponin-permeabilized card iomyocytes we could demonstrate that basal as well as GTPyS (30 mu M) stimulated phospholipase C activity was strongly activated by free Ca2 + in the concentration range of 0.1-10 mu M. We conclude that in the i ntact cardiomyocyte the signalling pathway through phospholipase C/pho sphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor int eraction that activates GTP-binding proteins as does GTPyS, is likely be a Ca2+ dependent process.