| Abstrakt: |
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 (10-8M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10-8 μM external Ca2+) and 50 mM K+(1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10-4M)induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10-8 μM) treatment of cells did not affect either or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+by addition of 50 μM ethylene glycol bis (β-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 μM) or Ca2+-channel agonist Bay K8644 (1 μM) 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 GTPγS (30 μM) stimulated phospholipase C activity was strongly activated by free Ca2+in the concentration range of 0.1-10 μM. 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 GTPγS, is likely be a Ca2+dependent process. |
