Flow-Mediated Regulation of G-Protein Expression in Cocultured Vascular Smooth Muscle and Endothelial Cells

From the Georgetown University Medical Center, Department of Surgery, Washington, DC.

Abstract—G-proteins have been implicated in the transduction of a number of flow-induced responses. We determined whether flow 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 flow up to (1) 2 mL/min (low flow: 0.5 dyne/cm²), or (2) 44 mL/min (high flow: 15 dyne/cm²) and maintained for 72 hours before SMCs and ECs were harvested separately. Using Western blot analysis, EC Gi₃ expression was significantly increased (41±2.9%) by high-flow conditions compared with low-flow. 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 substrates, and enhanced agonist-stimulated GTPase activity in cocultured ECs. In contrast, high flow induced a significant decrease in Gi_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 substrates, reduced agonist-stimulated GTPase activity, and enhanced basal and G-protein–stimulated adenylyl cyclase activity. These data demonstrate that flow mediates selective changes in EC and SMC G-protein expression concomitant with changes in G-protein functionality and cellular signaling capacity. Moreover, flow-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.

Key Words: shear stress • flow • vascular endothelial cells • vascular smooth muscle • G-proteins

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