APOPTOSIS OF VASCULAR SMOOTH-MUSCLE CELLS - PROTEIN-KINASE-C AND ONCOPROTEIN BCL-2 ARE INVOLVED IN REGULATION OF APOPTOSIS IN NONTRANSFORMED RAT VASCULAR SMOOTH-MUSCLE CELLS

We examined the effect of several inhibitors/activators of various protein kinases on the proliferation and apoptosis of nontransformed rat coronary vascular smooth muscle cells (SMC). As expected all the compounds (calphostin C, KT5720, KT5823, verapamil, W7, and dibutyryl-cAMP) inhibited SMC proliferation, as judged by [H-3]thymidine incorporation Three (calphostin C, verapamil and dibutyryl-cAMP) of the six compounds caused occurrence of the classical apoptotic morphology in SMC. The effect of calphostin C, an inhibitor of protein kinase C, was examined in more detail due to the known involvement of this kinase in regulation of apoptosis in a variety of cell types. In SMC cultures exposed for 1, 2, and 3 days to 0.1 mu mol/l; calphostin C, 7 +/- 1%, 32 +/- 3%, and 29 +/- 3% of cells underwent apoptosis, respectively, as assessed by cell morphology (control cultures had I to 3% of apoptotic cells). The effect of calphostin C was transient in that on day 6 following exposure to this compound the number of apoptotic cells declined to control values. Simultaneous with the induction of apoptotic morphology in SMC, a decline was seen (within 24 hours) in expression of the oncoprotein Bcl-2 in morphologically nonapoptotic SMC. An altered distribution of Bcl-2 was seen in the apoptotic cells. The calphostin C-induced generation of apoptotic cells in SMC cultures and the decline/alteration of Bcl-2 expression were not accompanied by degradation of DNA into nucleosomal fragments. In conclusion, normal, nontransformed rat coronary artery vascular SMC undergo apoptosis when exposed to an inhibitor of protein kianse C (calphostin C), to a calcium channel blocker (verapamil), and to a stimulator of cAMP-dependent protein kinase (dibutyryL-cAMP). The induction of apoptosis by the inhibitor of protein kinase C is accompanied by alterations in the Bcl-2 expression but not by DNA fragmentation.