Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells.
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.
Ayajiki
K
K
Zentrum der Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt/Main, Germany.
Kindermann
M
M
Hecker
M
M
Fleming
I
I
Busse
R
R
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013314:Stress, Mechanical; D013552:Swine; D014443:Tyrosine / Q000378:metabolism*
D006873:Hydroquinones; D017923:Sodium-Hydrogen Exchangers; D009569:Nitric Oxide; D014443:Tyrosine; D006859:Hydrogen; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D002118:Calcium; C048212:erbstatin