Study of the Role of Cyclic Nucleotides in Vascular Cell Proliferation

1. Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cell (PAEC) homogenates, a cyclic GMP- stimulated PDE (Type II) which hydrolyses both cyclic AMP and cyclic GMP and a cyclic AMP-specific PDE (Type IV). The role of these PDE isozymes present in PAEC was evaluated by examining the effects of selective PDE inhibitors on cyclic AMP and cyclic GMP content. 2. Inhibitors of the calcium/ calmodulin- dependent PDE (Type I) and of the cyclic GMP-inhibited PDE (Type III), M & B 22948 and SK & F 94120, respectively, only weakly inhibited the two PDE isozymes. In contrast, dipyridamole and trequinsin, two non-selective PDE inhibitors, potently inhibited both isozymes, whereas rolipram, selectively inhibited the cyclic AMP-specific PDE . 3. Incubation of intact cells with the non- selective PDE inhibitors , dipyridamole (25muM) and trequinsin (25muM), induced large increases in intracellular cyclic GMP content, which were completely blocked by the addition of haemoglobin (10muM). The selective cyclic AMP PDE inhibitor, rolipram (25muM), was without effect on the cyclic GMP content. 4. Dipyridamole (25muM) enhanced the increase in cyclic GMP content induced by the nitrovasodilator and stimulant of soluble guanylate cyclase, sodium nitroprusside (1muM). 5. Atriopeptin II (0.1-100nM), which activates particulate guanylate cyclase, increased the cyclic GMP content in a concentration-dependent manner. Dipyridamole (25muM) and trequinsin (25muM), but not rolipram (25muM), enhanced the increase in cyclic GMP content induced by atriopeptin II (10nM). 6. The non- selective PDE inhibitor, dipyridamole increased cyclic AMP content at 100muM but not at 25muM. The selective cyclic AMP PDE inhibitor, rolipram (25muM) induced, a large increase in cyclic AMP content. 7. Dipyridamole (25muM and 100muM) enhanced the increase in cyclic AMP content stimulated by the beta-adrenoceptor agonist, isoprenaline (25muM), or the activator of adenylate cyclase, forskolin (10muM). Furthermore, rolipram (25muM) enhanced the increase in cyclic AMP content induced by forskolin (30muM). 8. These results suggest that the cyclic GMP-stimulated PDE present in PAEC regulates the cyclic GMP content and the cyclic AMP PDE regulates the cyclic AMP content. Whether or not the cyclic GMP- stimulated PDE also contributes to the regulation of the cyclic AMP content could not be determined. 9. The effects of stimulation of protein kinase C and of cyclic nucleotides on proliferation of PAEC in culture was investigated. 10. Phorbol 12-myristate 13-acetate (PMA. 0.1nM-1muM), which activates protein kinase C, inhibited proliferation in a concentration-dependent manner. No early stimulation of proliferation was seen with PMA (0.3muM). The inactive phorbol ester, 4alpha-phorbol-12,13-didecanoate (0.3muM), lacked the ability of PMA to inhibit proliferation of PAEC. 11. Staurosporine (10nM and 100nM) inhibited serum- induced proliferation of PAEC but had no effect on the antiproliferative effects of PMA (0.3muM). 12. 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 scavangers, superoxide dismutase (30 units/ml) and catalase (30 units/ml), vitamin E (30muM), or butylated hydroxytoluene (30muM). The antiproliferative actions of paraquat (10muM), an agent which generates free radicals intracellularly, was, in contrast, inhibited by vitamin E (30muM) or butylated hydroxytoluene (30muM) but not by the extracellular radical scavangers, superoxide dismutase (30 units/ml) and catalase (30 units/ml). 13. Neither dibutyryl cyclic AMP (30muM), nor 8 bromo cyclic GMP (30muM) had any effect on the ability of PMA (0.3muM) to inhibit proliferation of PAEC. 14. Dibutyryl cyclic AMP (30muM) inhibited proliferation, but 8 bromo cyclic GMP (30muM) had no effect. Four other stimuli known to increase PAEC cyclic GMP content by stimulating particulate or soluble guanylate cyclase, atriopeptin II (10nM), bradykinin (0. 1muM), sodium nitroprusside (1muM) and glyceryl trinitrate (1muM), were also without effect on proliferation. (Abstract shortened by ProQuest.)