Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase.

Abstract

Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), plays a major role in the regulation of blood pressure and vascular tone.We generated transgenic mice overexpressing bovine eNOS in the vascular wall using murine preproendothelin-1 promoter.In transgenic lineages with three to eight transgene copies, bovine eNOS-specific mRNA, protein expression in the particulate fractions, and calcium-dependent NOS activity were confirmed by RNase protection assay, immunoblotting, and L -arginine/citrulline conversion.Immunohistochemical studies revealed that eNOS protein was predominantly localized in the endothelial cells of aorta, heart, and lung.Blood pressure was significantly lower in eNOS-overexpressing mice than in control littermates.In the transgenic aorta, basal NO release (estimated by N -nitro-L -arginine-induced facilitation of the contraction by prostaglandin F 2 ␣ ) and basal cGMP levels (measured by enzyme immunoassay) were significantly increased.In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas.Thus, our novel mouse model of chronic eNOS overexpression demonstrates that, in addition to the essential role of eNOS in blood pressure regulation, tonic NO release by eNOS in the endothelium induces the reduced vascular reactivity to NO-mediated vasodilators, providing several insights into the pathogenesis of nitrate tolerance.( J. Clin.Invest.1998.102:2061-2071.)Key words: transgenic animals • nitric oxide • endothelium • blood pressure • vascular reactivity Methods Materials.N -nitro-L -arginine methyl ester (L-NAME), N -nitro-L -arginine (L-NA), NADPH, flavin adenine dinucleotide, calmodulin, nitrate reductase ( Aspergillus species), PGF 2 ␣ , acetylcholine chloride (ACh), ATP ␥ S, sodium nitroprusside (SNP), forskolin, and N 6 ,2 Ј -O -dibutyryladenosine 3 Ј ,5 Ј -cyclic monophosphate (dibutyryl cAMP) were purchased from Sigma Chemical Co.(St.Louis, MO).Nitroglycerin (NTG) was obtained from Nippon Kayaku (Tokyo, Japan).EGTA was purchased from Dojindo Laboratories (Kumamoto, Japan).A rabbit polyclonal anti-human eNOS Ab was obtained from