NITRIC-OXIDE FROM VASCULAR SMOOTH-MUSCLE CELLS - REGULATION OF PLATELET REACTIVITY AND SMOOTH-MUSCLE CELL GUANYLATE-CYCLASE

1 Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10-mu-M) promoted a cell number-related inhibition of platelet aggregation induced by thrombin (40 mu ml-1). This inhibition was not attributable to products of the cyclooxygenase pathway for the SMC were also treated with indomethacin (10-mu-M). 2 The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5-mu-g ml-1) for a period of 9 to 24h. This effect was attenuated when cycloheximide (10-mu-g ml-1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non-treated cells. 3 The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10-mu-M). Preincubation of the SMC with N(G)-monomethyl-L-arginine (L-NMMA, 30-300-mu-M) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100-mu-M) but not with D-arginine (D-Arg, 100-mu-M). 4 Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37-degrees-C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP). L-NMMA (300-mu-M) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells. The inhibitory activity of L-NMMA was reversed by co-incubation with L-Arg (100-mu-M) but not D-Arg (100-mu-M). L-Arg alone had no effect on the levels of cyclic GMP. In the absence of stirring, a 10 min stimulation of the non-treated SMC with glyceryl trinitrate (GTN, 200-mu-M) or atrial natriuretic factor (ANF, 10(-7) M) led to an increase in the levels of cyclic GMP but not cyclic AMP. The increase in cyclic GMP promoted by GTN or ANF was not affected by L-NMMA. The levels of cyclic GMP were higher in the LPS (0.5-mu-g ml-1, 18 h)-treated cells (5 x 10(-5)) and stirring was more effective in increasing the levels of cyclic GMP in these cells. 5 These findings support the idea that non-treated or LPS-treated cultured SMC can produce an NO-like factor. Production by the latter requires protein synthesis as evidenced by blockade with cycloheximide. This NO-like factor may play a role in the auto-regulation of smooth muscle cell reactivity through a cyclic GMP-dependent mechanism.