Flow-mediated regulation of G-protein expression in cocultured vascular smooth muscle and endothelial cells

G-proteins have been implicated in the transduction of a number of now-induced responses. We determined whether now can modulate vascular endothelial or smooth muscle cell G-protein signaling. By use of a perfused transcapillary coculture system that permits the chronic exposure of cultured endothelial cells (ECs) and smooth muscle cells (SMCs) to physiological shear stresses, cocultures were exposed to stepwise increases in now up to (1) 2 mL/min (low now: 0.5 dyne/cm(2)), or (2) 44 mL/min (high flow: 15 dyne/cm(2)) and maintained for 72 hours before SMCs and ECs were harvested separately. Using Western blot analysis, EC Gi alpha(3), expression was significantly increased (41+/-2.9%) by high-now conditions compared with low-ao cv. The changes in G-protein expression were associated with a significant increase in endothelial nitric oxide synthase (eNOS) activity, elevated prostacyclin levels in the perfusing media, increased pertussis toxin-catalyzed ADP ribosylation of Gi alpha substrates, and enhanced agonist-stimulated GTPase activity in cocultured ECs. In contrast, high now induced a significant decrease in Gi alpha(1-2) expression (57+/-5%) in SMCs cocultured with ECs, an effect that was endothelium dependent, inhibited by indomethacin, and correlated with a decrease in pertussis toxin-catalyzed ADP ribosylation of Gi alpha substrates, reduced agonist-stimulated GTPase activity, and enhanced basal and G-protein-stimulated adenylyl cyclase activity. These data demonstrate that now mediates selective changes in EC and SMC G-protein expression concomitant with changes in G-protein functionality and cellular signaling capacity. Moreover, now-induced changes in SMC G-protein signaling capacity are endothelium dependent and require a cyclooxygenase product. G-protein modulation may thus represent an important mechanism whereby hemodynamic forces regulate vessel wall function.