Elevated blood pressures in mice lacking endothelial nitric oxide synthase - PubMed (original) (raw)
Elevated blood pressures in mice lacking endothelial nitric oxide synthase
E G Shesely et al. Proc Natl Acad Sci U S A. 1996.
Abstract
Nitric oxide produced in endothelial cells affects vascular tone. To investigate the role of endothelial nitric oxide synthase (eNOS) in blood pressure regulation, we have generated mice heterozygous (+/-) or homozygous (-/-) for disruption of the eNOS gene. Immunohistochemical staining with anti-eNOS antibodies showed reduced amounts of eNOS protein in +/- mice and absence of eNOS protein in -/- mutant mice. Male or female mice of all three eNOS genotypes were indistinguishable in general appearance and histology, except that -/- mice had lower body weights than +/+ or +/- mice. Blood pressures tended to be increased (by approximately 4 mmHg) in +/- mice compared with +/+, while -/- mice had a significant increase in pressure compared with +/+ mice (approximately 18 mmHg) or +/- mice (approximately 14 mmHg). Plasma renin concentration in the -/- mice was nearly twice that of +/+ mice, although kidney renin mRNA was modestly decreased in the -/- mice. Heart rates in the -/- mice were significantly lower than in +/- or +/+ mice. Appropriate genetic controls show that these phenotypes in F2 mice are due to the eNOS mutation and are not due to sequences that might differ between the two parental strains (129 and C57BL/6J) and are linked either to the eNOS locus or to an unlinked chromosomal region containing the renin locus. Thus eNOS is essential for maintenance of normal blood pressures and heart rates. Comparisons between the current eNOS mutant mice and previously generated inducible nitric oxide synthase mutants showed that homozygous mutants for the latter differ in having unaltered blood pressures and heart rates; both are susceptible to lipopolysaccharide-induced death.
Figures
Figure 1
eNOS targeting strategy. (A) Targeting construct pENOSX. The heavy line represents eNOS genomic sequences with the disrupted exon 12 indicated by solid bars. Shaded and open boxes represent the neo (neomycin resistance) gene and herpes simplex virus thymidine kinase genes, respectively. The thin line is plasmid vector (not to scale). (B) Portion of the endogenous murine eNOS gene. (C) Correctly targeted eNOS gene. (D) Southern blot, hybridized to probe A (indicated by a horizontal bar), of _Bam_HI digested genomic DNA from offspring of an eNOS +/− × eNOS +/− mating. The 5.3-kb band indicates a wild-type eNOS gene while the 6.4-kb band indicates a disrupted eNOS gene. B, _Bam_HI; N,_Not_I; X, _Xba_I.
Figure 2
Immunohistochemical localization of eNOS. Heart and kidney sections from eNOS +/+ and −/− animals incubated with an anti-eNOS polyclonal antiserum, washed, and developed with the Vectastain ABC system using diaminobenzidine. The +/+ mice show intense staining in capillaries surrounding individual myocytes (arrowheads) (Upper Left) and in glomerular tufts (Lower Left). Staining is virtually absent in the capillaries (arrowheads) (Upper Right) or glomerular tufts (Lower Right) of the −/− mice. No eNOS staining is visible in the cardiac myocytes of either genotype.
Figure 3
Effect of eNOS genotypes (+/+, +/−, and −/−) on blood pressure (A) and heart rate (B). Error bars indicate SEM. Numbers of mice are as follows: 34, +/+; 38, +/−; 21, −/−.
Figure 4
Effect of eNOS genotypes (+/+ and −/−) on kidney renin mRNA (A) and plasma renin concentration (B). Kidney renin mRNA units are pg of mRNA per μg of total RNA. Plasma renin concentration units are angiotensin I production (ng/hr) per ml of plasma. Error bars indicate SEM. Numbers of mice are as follows: 5 male and 5 female, +/+; 4 male and 4 female, −/−.
Figure 5
Survival of LPS-treated mice. Ten F2 mice of each eNOS genotype (+/+, +/−, and −/−) were treated i.p. with LPS at 12.5 mg/kg and survival was monitored over 4 days.
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