ALPHA-ADRENERGIC AGONIST AND ENDOTHELIN-1 INDUCED INTRACELLULAR CA2+ RESPONSE IN THE PRESENCE OF A CA2+ ENTRY BLOCKER IN CULTURED RAT VENTRICULAR MYOCYTES

Previously we demonstrated that stimulation of cultured neonatal rat ventricular myocytes by either alpha(1)-adrenergic agonist or endothelin-l resulted in a rapid formation of total inositolphosphates, although the levels of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate did not rise significantly. The aim of this study was to examine whether stimulation by alpha(1)-adrenergic agonist and endothelin-1 could still elicit phosphatidylinositol cycle mediated intracellular Ca2+ mobilization in these cells. The intracellular free Ca2+ concentration ([Ca2+](i)) was measured by single cell imaging dual wavelength fluorescence microscopy in Fura-2-loaded cardiomyocytes. The interference of agonist induced [Ca2+](i) responses by the beat to beat variation of [Ca-i(2+) was prevented by arresting the cells with the Ca2+ entry blocker diltiazem (10 mu M). The [Ca2+](i) response (expressed as % of baseline ratio of fluorescence intensities of Fura-P at 340 nm and 380 nm excitation wavelength), induced by phenylephrine (10(-4) M) and endotherin-1 (10(-8) M) was small, up to 20% of baseline after 9-20 min. In contrast, Ca2+-influx induced by incubation in Na+-free buffer caused a steep increase of [Ca2+](i) up to 150% of baseline after 30 s. Analysis of single cells following stimulation with phenylephrine or endothelin-l showed heterogeneity with respect to a rise in Ca2+](i). However, if rapid Ca2+-influx was induced by incubation in Na+-free buffer, [Ca+](i) responses in individual myocytes occurred homogeneously. It is concluded that the alpha(1)-adrenergic agonist and endothelin-l induced [Ca2+](i) responses are delayed in time, small and quite heterogeneous among cells. The findings are in agreement with earlier observations which revealed no detectable overall increase of the Ca2+ releasing inositolphosphates under these conditions and suggest that other second messengers, such as 1,2-diacylglycerol, are involved in the agonist mediated Ca2+ signals.