Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase

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Abstract
1 Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 μm) 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 μm). 2 The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 μg ml−1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 μg 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 μm). Preincubation of the SMC with NG-monomethyl-l-arginine (l-NMMA, 30–300 μm) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with l-arginine (l-Arg, 100 μm) but not with d-arginine (d-Arg, 100 μm). 4 Exposure of the non-treated SMC (5 × 105 cells) to stirring (1000 r.p.m., 37°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 μm) 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 μm) but not d-Arg (100 μm). 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 μm) 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 μg ml−1, 18 h)-treated cells (5 × 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.