Shear stress induction of the endothelial nitric oxide synthase gene is calcium-dependent but not calcium-activated.
Arterial levels of shear stress (25 dynes/cm2) can elevate constitutive endothelial nitric oxide synthase (eNOS) gene expression in cultured endothelial cells (Ranjan et al., 1995). By PhosphorImaging of Northern blots, we report that the eNOS/glyceraldehyde 3-phosphate dehydrogenase (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/cm2, respectively. Incubation of BAEC with dexamethasone (1 microM) had no effect on shear stress induction of eNOS mRNA. Buffering of intracellular calcium in BAEC with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA/AM) reduced shear stress induction of eNOS mRNA by 70%. Yet, stimulation of BAEC with ionomycin (0.1-1.0 microM) for 6-24 hr to elevate intracellular calcium had no effect on eNOS mRNA. These studies indicated that the shear stress induction of eNOS mRNA was a calcium-dependent, but not calcium-activated, process. Shear stress was a very potent and rapid inducer of the eNOS mRNA, which could not be mimicked with phorbol myristrate acetate or endotoxin. Inhibition of tyrosine kinases with genistein (10 microM) or tyrphostin B46 (10 microM) or inhibition of G-protein signaling with guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) (600 microM, 6-hr preincubation) did not block the shear stress elevation of eNOS mRNA.
Xiao
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Department of Chemical Engineering, State University of New York at Buffalo, USA.
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Ranjan
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D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics*; D011493:Protein Kinase C; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000502:physiology; D013314:Stress, Mechanical; D013873:Thionucleotides / Q000494:pharmacology
D002614:Chelating Agents; D007476:Ionophores; D012333:RNA, Messenger; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D004533:Egtazic Acid; D015759:Ionomycin; C023427:guanosine 5'-O-(2-thiodiphosphate); D003907:Dexamethasone; D019001:Nitric Oxide Synthase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium