Intracellular pH and Tyrosine Phosphorylation but Not Calcium Determine Shear Stress–Induced Nitric Oxide Production in Native Endothelial Cells

Abstract

Signalling pathways determining the shear stress-induced production of NO from endothelial cells in situ were investigated using a bioassay system in which shear stress was increased by inducing vasoconstriction in an endothelium-intact donor segment (rabbit iliac artery) while maintaining a constant luminal perfusion rate. Shear stress-induced NO production, as assessed by changes in the tone of a preconstricted endothelium-denuded detector ring, was biphasic and consisted of an initial transient (20- to 25-minute) Ca(2+)-dependent phase followed by a Ca(2+)-independent plateau phase, which was maintained as long as the donor segment remained constricted. Stretching the donor segments to their in vivo length abolished the initial phase without affecting the plateau phase of NO release. Inhibition of the Na(+)-H+ exchanger using HOE 694 elicited an intracellular acidification which attenuated shear stress-induced NO production. The specific protein kinase C inhibitor, Ro 31-8220, was without effect, whereas the unspecific inhibitors, staurosporine and calphostin C, abolished the shear stress-induced production of NO. Erbstatin A, a tyrosine kinase inhibitor, attenuated the shear stress-induced tyrosine phosphorylation of specific cellular proteins and abrogated the associated NO production. In summary, these data indicate that shear stress activates the NO synthase at basal levels of [Ca2+]i via a mechanotransduction cascade that involves tyrosine phosphorylation and can be modulated by changes in pHi. The apparent fundamental alteration of the endothelial NO synthase under shear stress that renders its maintained activation independent of an increase in [Ca2+]i is probably the consequence of a change in the enzyme microenvironment.

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

“…Indeed, most humoral ligands including bradykinin, acetylcholine, and ATP stimulate NO production from eNOS by raising the level of intracellular Ca 2ϩ , which forms Ca 2ϩ /CaM complex (34). On the other hand, mechanical forces such as fluid shear stress and stretching stimulate NO production from eNOS by Ca 2ϩ -independent mechanisms (1,10). Moreover, eNOS has been shown to be regulated by interactions with other positive and negative protein modulators such as caveolin and heat shock protein 90 (20,41).…”

Effects of Shear Stress on [Ca2+]I and Membrane Potential of Vascular Endothelium of Intact Rat Blood Vessels

“…The mechanism of the shear stress-evoked relaxation in intact rat aorta is therefore at present unclear. It may involve changes in cytosolic pH as demonstrated in response to changes in shear stress in cultured cells from this vessel (Ziegelstein et al 1992(Ziegelstein et al , 1998 or activation of tyrosine kinases as suggested in cultured endothelium from other species (Ayajiki et al 1996). Al-Mehdi, A.…”

“…However, a number of investigators were unable to detect any changes in endothelial [Ca¥]é and have proposed other mechanisms of mechanotransduction. These include shear stress-evoked protein tyrosine phosphorylation (Ayajiki et al 1996) and changes in endothelial cytosolic pH. In cultured rat aortic endothelium, increased shear stress has been reported to evoke cytosolic acidification (Ziegelstein et al 1992), whilst an abrupt reduction in shear stress results in alkalinisation due to activation of Na¤-dependent Cl¦-HCOצ exchange and Na¤-H¤ exchange (Ziegelstein et al 1998).…”

“…In cultured rat aortic endothelium, increased shear stress has been reported to evoke cytosolic acidification (Ziegelstein et al 1992), whilst an abrupt reduction in shear stress results in alkalinisation due to activation of Na¤-dependent Cl¦-HCOצ exchange and Na¤-H¤ exchange (Ziegelstein et al 1998). In cultured porcine aortic endothelium, modest increases in shear stress are reported to evoke initial acidification followed by alkalinisation (Ayajiki et al 1996). Cytosolic alkalinisation has been shown to activate NO synthesis (Fleming et al 1994).…”

A Neurovascular Blood-Flow Modulation Model via Acupuncture Induced Nitric Oxide

“…Furthermore, both transient and sustained release of NO has been reported in response to this shear stress. It is seems that shear stress regulates NO release in a Ca 2+ -independent mode through phosporylation of eNOS, which results in a sustained basal NO production irrespective of the presence or absence of the Ca 2+ transient effect [87][88][89]. Kutchan and Frangos [90] measured the NO end-oxidation products ( -2 NO and -3 NO ) released from endothelial cells exposed to laminar flow and reported the presence of transient Ca 2+ -dependent NO release at the initial moment but sustained release in the presence of a constant shear stress level.…”