Fluid Flow Rapidly Activates G Proteins in Human Endothelial Cells

Abstract

Fluid shear stimulates endothelial cells, with the external hemodynamic forces transduced across the plasma membrane to modulate intracellular events. We report the first direct evidence that identifies specific GTP binding proteins (G proteins) activated within 1 second of flow onset, representing one of the earliest mechanochemical signal transduction events reported to date in shear-stimulated endothelium. A nonhydrolyzable GTP photoreactive analogue, azidoanilido [alpha-32P]GTP (AAGTP), allowed irreversible labeling of flow-stimulated G proteins, with two protein bands (42 kD and 31 kD) identified in human umbilical vein endothelial cells (HUVECs) subjected to laminar flow (10 dyne/cm2) in a parallel-plate flow chamber. Immunoprecipitation of labeled whole-cell lysates identified the specific G-protein subunits G q zero/alpha 11 and G alpha i3/alpha 0) as being activated by flow. Endothelial 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 components. Our results describe one of the earliest flow-induced signaling events reported in HUVECs, providing insight into the primary mechanosensing and signal transduction mechanisms.

Hypoxia-induced Proliferative Response of Vascular Adventitial Fibroblasts Is Dependent on G Protein-mediated Activation of Mitogen-activated Protein Kinases

“…Stimulation of the sphingosine 1-phosphate receptor has also recently been shown to be coupled to activation of ERK and p38 by pertussis toxin-sensitive and -insensitive mechanisms, respectively (50). Pertussis toxin-sensitive G proteins have also been implicated in activation of ERK in cardiac and endothelial cells subjected to changes in mechanical forces (51,52). Thus, our results indicate that G␣ i/o proteins are a necessary upstream component of hypoxia-induced proliferation in neonatal fibroblasts.…”

Biomechanical activation of vascular endothelium as a determinant of its functional phenotype

“…This list contains 28 genes of diverse functions, including genes involved in lipid metabolism (apolipoprotein E, long chain fatty acid-CoA ligase 3, megalin); the purinoreceptor, P2X4, which mediates fluid shear stress-dependent activation of calcium influx in endothelial cells (19); as well as isoforms of ␣, ␤ and ␥ subunits of G proteins. The coordinated up-regulation of trimeric G proteins is interesting because these molecules have been implicated in mechanosignaling in response to fluid shear stress (20,21). Certain genes involved in vascular responses to injury also showed significant regulation, including fibrogenic lymphokine (fibrosin), thioredoxin reductase, and matrix-gla protein.…”

MAPKs (ERK½, p38) and AKT Can Be Phosphorylated by Shear Stress Independently of Platelet Endothelial Cell Adhesion Molecule-1 (CD31) in Vascular Endothelial Cells

“…These mechanical forces induce morphological, physiological, and biochemical changes of ECs (1,2). Although mechanisms for mechanosensing and subsequent signaling events in ECs have not yet been well characterized, activation of trimeric G proteins (3)(4)(5)(6), Ca 2ϩ mobilization (7)(8)(9)(10), and increased K ϩ efflux (11,12) are known as early signaling events. Other investigators have recently demonstrated that a glycoprotein, platelet endothelial cell adhesion molecule-1 (PECAM-1) is rapidly tyrosine-phosphorylated in bovine ECs (BAEC) exposed to fluid flow; this is thought to play important roles in transducing chemical stimuli received on the cell surface to the cytoplasmic aspects of the cell (13,14).…”