KAWASHIMA Seinosuke Associate Professor, First Department of Internal Medicine, Kobe University Scho, 医学部・付属病院, 講師 (10177678)
KAWAHARA Yasuhiro Associate Professor, First Department of Internal Medicine, Kobe University Scho, 医学部, 講師 (80169755)
AKITA Hozuka Associate Professor, First Department of Internal Medicine, Kobe University Scho, 医学部, 助教授 (60175792)
|Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1996: ¥6,400,000 (Direct Cost: ¥6,400,000)
Nitric oxide (NO), constitutively produced by endothelial cell nitric oxide synthase (eNOS), plays a major role in regulation of blood pressure and vascular tone and also acts as an anti-atherogenic molecule.
To study chronic effects of lifelong overexpression of eNOS in endothelial cells, we generated transgenic mice overexpressing eNOS by pronuclear microinjection and analyzed their phenotypic changes. We constracted a fusion gene comprising 9.2kb murine preproendothelin-1 gene promoter and 4.1kb bovine eNOS cDNA to direct the expression specifically tp vascular wall. Five founder mice with 9-13 transgene copies out of 104 pups were identified by PCR and subsequent Southem blot analysis. The specific espression of transgene transcript was confirmed in all organs examined by RNase protection assay using bovine eNOS specific riboprobe. In immunohistochemical studies using polyclonal eNOS antibody, eNOS protein was identified predominantly in the vascular endothelial cells in most organs, and also in epithelial cells in the lung and the uterus. Immunoblotting showed significantly increased eNOS protein in particulate fractions of the aorta and the lung, and consistent increase in -calcium-dependent NOS activity, assayd by the conversion of ^3H-arginine to ^3H-citrulline, was identified especially in aorta (53.9 nmol/mg/min, in transgenic to 7.76 nmol/mg/min, in wild-type). General appearance and orgen (heart, lung, and kidney) weights were not significantly different between the transgenic and wild-type mice but blood pressure, measured by direct femoral arterial catheterization, was lower in the transgenic mice. In addition, isotonic tension meserement in aorta revealed the attenuated vasorelaxation to acetylcholine or substance P in transgenic mice compared with wild-type mice.
Thus, transgenic mice overexpressing eNOS will be a new experimental model to study the role of NO in cardiovascular system.