Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells

Abstract

Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.

Shear stress stimulates phosphorylation of eNOS at Ser635by a protein kinase A-dependent mechanism

“…Unlike in BAEC, however, VEGF stimulated dephosphorylation of eNOS-Thr 497 in endothelial cells obtained from a different vascular bed, human umbilical vein endothelial cells (35). These differences in the behavior of endothelial cells depending on vascular origin are not unusual (28,40). These results also provide an example that the response of cells to a physiological stimulus such as shear stress and VEGF are not necessarily identical compared with that induced by a bolus addition of 8-BRcAMP.…”

Ventilation and oxygenation induce endothelial nitric oxide synthase gene expression in the lungs of fetal lambs.

“…Shear stress is increased in the pulmonary circulation of the fetus or newborn if: (a) increased pulmonary blood flow increases pulmonary blood flow velocity; (b) blood hematocrit is increased; and (c) there is underdevelopment of the pulmonary circulation. Increases in shear stress stimulate endothelial cells to produce several modulators of vascular tone, including NO (20)(21)(22)(23). When endothelial cells are subjected to shear stress, a diverse set of responses is initiated, some of which occur within minutes, others which occur within hours or days (39).…”

“…In vivo, the acute compression of the ductus arteriosus increases fetal pulmonary blood flow; this increase is blocked by inhibition of NO synthesis, suggesting that the increase in pulmonary blood flow has increased shear stress, increasing NOS activity and NO production (9). This increase in NO activity induced by shear stress is associated with rapid changes in endothelial cell calcium concentration, ionic conductance, adenylate cyclase activity, and inositol triphosphate generation (20)(21)(22)(23)39). If the shear stress is maintained, endothelial cells change their shape secondary to a structural reorganization of the cytoskeleton.…”

“…Initially, NOS activity and NO production are increased when endothelial cells are exposed to increased shear stress (12,14,15,(20)(21)(22)(23). If the shear is maintained there are then subsequent increases in eNOS mRNA and protein expression (24)(25)(26).…”

The hepatic circulation in health and disease: Report of a single-topic symposium

“…G proteins are activated within 1 second of flow onset and represent one of the earliest mechanochemical signal transduction events in flow-stimulated endothelium. 30,31 In recent studies using a nonhydrolyzable guanylate 5Ј-triphosphate photoreactive analogue, azidoanilido-guanylate 5Ј-triphos-phate, two distinct G proteins were stimulated by flow. 30 One of these proteins, a 42-kd G protein, was membraneassociated and activated independently of cytoskeletal or cytosolic components.…”