SHEAR-STRESS INDUCTION OF THE ENDOTHELIAL NITRIC-OXIDE SYNTHASE GENE IS CALCIUM-DEPENDENT BUT NOT CALCIUM-ACTIVATED

Arterial levels of shear stress (25 dynes/cm(2)) can elevate constitut ive endothelial nitric oxide synthase (eNOS) gene expression in cultur ed endothelial cells (Ranjan et al., 1995). By Phosphorlmaging of Nort hern blots, we report that the eNOS/glyceraldehyde 3-phosphate dehydro genase (GAPDH) messenger RNA (mRNA) ratio in bovine aortic endothelial cells (BAEC) increased by 4.8- and 7.95-fold after 6-hr shear stress exposure of 4 and 25 dynes/cm(2), respectively. Incubation of BAEC wit h dexamethasone (1 mu M) had no effect on shear stress induction of eN OS mRNA. Buffering of intracellular calcium in BAEC with bis(o-aminoph enoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester ( BAPTA/AM) reduced shear stress induction of eNOS mRNA by 70%. Yet, sti mulation of BAEC with ionomycin (0.1-1.0 mu M) for 6-24 hr to elevate intracellular calcium had no effect on eNOS mRNA. These studies indica ted that the shear stress induction of eNOS mRNA was a calcium-depende nt, but not. calcium-activated, process. Shear stress was a very poten t ano rapid inducer of the eNOS mRNA, which could not be mimicked with phorbol myristrate acetate or endotoxin. Inhibition of tyrosine kinas es with genistein (10 mu M) or tyrphostin B46 (10 mu M) or inhibition of G-protein signaling with guanosine 5'-O-(2-thiodiphosphate) (GDP-be ta S) (600 mu M, 6-hr preincubation) did not block the shear stress el evation of eNOS mRNA. (C) 1997 Wiley-Liss, Inc.