EFFECTS OF CYCLIC-NUCLEOTIDES AND PHORBOL-MYRISTATE ACETATE ON PROLIFERATION OF PIG AORTIC ENDOTHELIAL-CELLS

1 The role of cyclic nucleotides and protein kinase C in controlling proliferation of pig aortic endothelial cells (PAEC) in culture was investigated. 2 Dibutyryl cyclic AMP (30-mu-M), added twice daily, inhibited proliferation but 8 bromo cyclic GMP (30-mu-M) had no effect. Two other stimuli known to increase PAEC cyclic GMP content by stimulating particulate and soluble guanylate cyclase respectively, atriopeptin II (10 nM) and sodium nitroprusside (1-mu-M), were also without effect on proliferation. 3 Two agents known to inhibit soluble guanylate cyclase and lower intercellular cyclic GMP content, haemoglobin (10-mu-M) and methylene blue (10-mu-M), each inhibited proliferation of PAEC. 4 The inhibitory effect of haemoglobin (10-mu-M) was mediated by inhibition of soluble guanylate cyclase since it was reversed by agents known to increase cyclic GMP content, i.e. atriopeptin II (10-mu-M), 8 bromo cyclic GMP (30-mu-M) or sodium nitroprusside (1-mu-M). The inhibitory effect of methylene blue (10-mu-M) was not reversed by these agents. 5 Phorbol 12-myristate 13-acetate (PMA, 0.1 nM-1-mu-M), which activates protein kinase C, inhibited proliferation in a concentration-dependent manner. No early stimulation of proliferation was seen with PMA. The inactive isomer, 4-alpha-phorbol 12,13-didecanoate (0.3-mu-M), lacked the ability of PMA to inhibit proliferation of PAEC. 6 PMA-induced inhibition of proliferation appeared not to be due to stimulated production of destructive oxygen-derived free radicals since it was unaffected by the radical scavengers, vitamin E (30-mu-M) or butylated hydroxytoluene (30-mu-M). The antiproliferative actions of paraquat (10-mu-M), an agent which generates free radicals intracellularly, was, in contrast, inhibited by vitamin E or butylated hydroxytoluene. Furthermore, neither dibutyryl cyclic AMP (30-mu-M) nor 8 bromo cyclic GMP (30-mu-M) had any effect on the ability of PMA to inhibit proliferation. 7 This study suggests that cyclic AMP, cyclic GMP and protein kinase C play a role in controlling the proliferation of PAEC.