FLUID-FLOW RAPIDLY ACTIVATES G-PROTEINS IN HUMAN ENDOTHELIAL-CELLS - INVOLVEMENT OF G-PROTEINS IN MECHANOCHEMICAL SIGNAL-TRANSDUCTION

Fluid shear stimulates endothelial cells, with the external hemodynami c forces transduced across the plasma membrane to modulate intracellul ar events. We report fhe first direct evidence that identifies specifi c GTP binding proteins (G proteins) activated within 1 second of flow onset, representing one of the earliest mechanochemical signal transdu ction events reported to date in shear-stimulated endothelium. A nonhy drolyzable GTP photoreactive analogue, azidoanilido [alpha-P-32]GTP (A AGTP), allowed irreversible labeling of flow-stimulated G proteins, wi th two protein bands (42 kD and 31 kD) identified in human umbilical v ein endothelial cells (HUVECs) subjected to laminar flow (10 dyne/cm(2 )) in a parallel-plate flow chamber. Immunoprecipitation of labeled wh ole-cell lysates identified the specific G-protein subunits G(alpha q/ alpha ll) and G(alpha i3/alpha o) as being activated by flow. Endothel ial cell membrane vesicles were sheared in a cone-and-plate viscometer . with the 42-kD protein band labeled by AAGTP, but the 31-kD protein absent, indicating that the 42-kD G protein is membrane associated and activated independently of intact cytoskeletal or cytosolic component s. Our results describe one of the earliest flow induced signaling eve nts reported in HUVECs, providing insight into the primary mechanosens ing and signal transduction mechanisms.