VIP-INDUCED RELAXATION OF GUINEA-PIG INTESTINAL SMOOTH-MUSCLE CELLS -SEQUENTIAL INVOLVEMENT OF CYCLIC-AMP AND NITRIC-OXIDE

1 A possible interaction between cyclic AMP and nitric oxide (NO) in m ediating the relaxant effect of vasoactive intestinal polypeptide (VIP ) on intestinal smooth muscle cells has been investigated. The effects of the inhibitor of NO synthesis, N-G-nitro-L-arginine methyl ester ( L-NAME), have been studied on VIP-, forskolin-, and 8 bromo-cyclic AMP - induced relaxation of cells, dispersed by enzymatic digestion of mus cle strips from the circular layer of guinea-pig ileum. 2 VIP alone di d not modify the length of isolated muscle cells. By contrast, when th e cells were contracted by cholecystokinin octapeptide, CCK8 (10 nM), VIP inhibited this contraction, inducing a concentration-dependent rel axation of the cells. Maximal relaxation was induced by 1 mu M VIP (EC (50)=408.2+/-16.7 pM). 3 N-ethylmaleimide, inhibitors of adenylate cyc lase or somatostatin, abolished the relaxing effect of VIP. (R)-p-cAMP s, an antagonist of cyclic AMP on protein kinase A also inhibited the VIP-induced relaxation by 92.1+/-6.3%. Inhibitors of nitric oxide synt hase (NOS), L-NAME and L-NMMA, partially inhibited VIP-induced relaxat ion. The effect of L-NAME was reversed by L-arginine but not by D-argi nine. 4 (R)-p-cAMPS and L-NAME also inhibited the cell relaxation indu ced either by forskolin which directly stimulates adenylate cyclase ac tivity or 8-bromo-cyclic AMP, an analogue of cyclic AMP. 5 When cells were incubated for 30 min with dexamethasone 10 mu M, a glucocorticoid known to decrease the synthesis of iNOS, the relaxing effect of a max imal concentration of VIP was decreased by 52+/-4% and L-NMMA had no f urther effect on this residual VIP-induced relaxation. Milrinone, a ph osphodiesterase type III inhibitor, potentiated the relaxant effect of VIP. 6 These data demonstrate that the intracellular pathway mediatin g the relaxant effect of VIP in intestinal smooth muscle cells include s the sequential activation of adenylate cyclase, protein kinase A, ac tivation of NOS and finally production of NO and cyclic GMP. NO could in turn regulate the cyclic AMP-dependent pathway of cell relaxation.