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 (Ϸ18 mmHg) or ؉͞؊ mice (Ϸ14 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 F 2 mice are due to the eNOS mutation and are not due to sequences that might differ between the two parental strains (129 and C57BL͞6J) 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.

The vascular endothelium mediates the ability of blood vessels to alter their architecture in response to hemodynamic changes; however, the specific endothelial-derived factors that are responsible for vascular remodeling are poorly understood. Here we show that endothelial-derived nitric oxide (NO) is a major endothelial-derived mediator controlling vascular remodeling. In response to external carotid artery ligation, mice with targeted disruption of the endothelial nitric oxide synthase gene (eNOS) did not remodel their ipsilateral common carotid arteries whereas wild-type mice did. Rather, the eNOS mutant mice displayed a paradoxical increase in wall thickness accompanied by a hyperplastic response of the arterial wall. These findings demonstrate a critical role for endogenous NO as a negative regulator of vascular smooth muscle proliferation in response to a remodeling stimulus. Furthermore, our data suggests that a primary defect in the NOS/NO pathway can promote abnormal remodeling and may facilitate pathological changes in vessel wall morphology associated with complex diseases such as hypertension and atherosclerosis.

Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon y, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shockIn biological systems, nitric oxide (NO) is produced via the oxidation of L-arginine by enzymes known as nitric oxide synthases (NOSs). Three NOS isozymes have been described (for recent reviews see refs. 1 and 2). These include constitutively expressed neuronal (3, 4) and endothelial (5, 6) enzymes and a cytokine-induced enzyme found in macrophages (7), hepatocytes (8), and a variety of other cells (9, 10). Although the biological functions of these enzymes are not completely understood, neuronal NOS is thought to play a role in neurotransmission (11), endothelial NOS is involved in regulation ofvascular tone (12, 13), and inducible NOS (iNOS) is involved in immune defense (14,15). The constitutively synthesized neuronal and endothelial enzymes produce small amounts of NO in response to increases in intracellular calcium levels. In contrast, iNOS is synthesized de novo in response to a variety of inflammatory stimuli and produces large amounts of NO over prolonged periods of time (16). NO produced by iNOS has been shown to be beneficial through its antitumor (17, 18) and antimicrobial (15) activities, but it is also thought to cause tissue damage and contribute to pathology in a variety of inflammatory conditions including rheumatoid arthritis (19,20), inflammatory bowel disease (21), and septic shock (22,23).Septic shock is usually the result of a systemic Gram-negative bacterial infection and is characterized by hypotension and the failure of a number of organ systems, especially the liver, kidney, and heart (24). The bacterial membrane component, lipopolysaccharide (LPS), induces the production of host inflammatory mediators such as tumor necrosis factor a, interferon y (IFN-,y), and interleukin 1,B, which in turn cause an increase in the expression of iNOS. The large amount of NO produced by iNOS has been hypothesized to contribute to LPS-induced hypotension and mortality.To be...

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Abstract-Nitric oxide and prostaglandins were shown to contribute to the endothelial mediation of flow-induced dilation of skeletal muscle arterioles of rats. Thus, we hypothesized that flow-induced dilation and its mediation are altered in gracilis muscle arterioles of mice deficient in the gene for endothelial nitric oxide synthase (eNOS-KO) compared with control wild-type (WT) mice. Gracilis muscle arterioles (Ϸ80 m) of male mice were isolated, then cannulated and pressurized in a vessel chamber. The increases in diameter elicited by increases in perfusate flow from 0 to 10 L/min were similar in arterioles from eNOS-KO (nϭ28) and WT (nϭ22) mice (Ϸ20 m at 10 L/min flow). Removal of the endothelium eliminated flow-induced dilations in vessels of both strains of mice.Ϫ4 mol/L) significantly inhibited flow-induced dilation in arterioles of WT mice by Ϸ51% but had no effect on responses of arterioles from eNOS-KO mice. Indomethacin (INDO, 10 Ϫ5 mol/L) inhibited flow-induced dilation of WT mice by Ϸ49%, whereas it completely abolished this response in arterioles of eNOS-KO mice. Simultaneous administration of INDO and L-NNA eliminated flow-induced responses in arterioles of WT mice. Dilations to carbaprostacyclin were similar at concentrations of 10 Ϫ8 and 3ϫ10 Ϫ8 mol/L but decreased significantly at 10 Ϫ7 mol/L in arterioles of eNOS-KO compared with those of WT mice. These findings demonstrate that, despite the lack of nitric oxide mediation, flow-induced dilation is close to normal in arterioles of eNOS-KO mice because of an enhanced release of endothelial dilator prostaglandins and suggest that this vascular adaptation may contribute to the regulation of peripheral resistance in eNOS-KO mice. (Circ Res. 1999;85:288-293.) Key Words: transgenic mice Ⅲ intraluminal flow Ⅲ endothelium Ⅲ nitric oxide Ⅲ prostacyclin C onsiderable evidence demonstrates that endotheliumderived nitric oxide (NO) plays a critical role in the regulation of vascular tone. 1-3 One of the important mechanisms through which NO release is controlled is by alterations in wall shear stress during changes in blood flow. 3,4 Previous studies demonstrated that increases in perfusate flow elicit endotheliumdependent dilation of gracilis muscle arterioles, a response that is mediated by the combined release of NO and prostaglandins. 5 It has also been demonstrated that reduced release of NO could lead to an enhanced arteriolar resistance, which may be involved in the pathogenesis of cardiovascular disorders, such as hypertension and atherosclerosis. 6,7 The importance of the NO-related dilator mechanism is further underlined by studies showing a significant increase in blood pressure during systemic inhibition of nitric oxide synthase (NOS) with L-arginine analogs 8,9 or when the gene encoding endothelial nitric oxide synthase (eNOS) is disrupted. 10,11 Interestingly, however, there are quantitative differences in blood pressure in response to systemic administration of L-arginine analogs in wild-type (WT) mice compared with the level of blood pressur...

We examined the hypotheses that responses to acetylcholine are impaired and responses to NO are enhanced in carotid artery from mice made deficient in endothelial nitric oxide synthase (eNOS) by gene targeting (eNOS-deficient mice). We also tested the hypothesis that deletion of one copy of the eNOS gene is sufficient to alter vascular responses. Vessels were studied in vitro from heterozygous (+/−) and homozygous (−/−) eNOS-deficient mice as well as wild-type [eNOS(+/+)] littermates. After precontraction with prostaglandin F2α, acetylcholine produced marked relaxation of carotid arteries in eNOS(+/+) mice, with impaired vasorelaxation in eNOS(+/−) mice. For example, 1 μM acetylcholine relaxed carotid arteries by 55 ± 5% (mean ± SE) in eNOS(+/−) mice ( n = 13) compared with 83 ± 3% in eNOS(+/+) mice ( n = 14, P < 0.001 vs. +/−). In contrast, acetylcholine caused no relaxation in carotid arteries from eNOS(−/−) mice ( P < 0.001 vs. +/+ and +/−). Relaxation of the carotid artery in response to nitroprusside [a nitric oxide (NO) donor] was enhanced ( P < 0.001) in eNOS-deficient mice. For example, in response to 10 nM nitroprusside, the carotid artery relaxed by 18 ± 2% in eNOS(+/+) mice ( n = 14), 33 ± 2% in eNOS(+/−) mice ( n = 13), and 47 ± 4% in eNOS(−/−) mice ( n = 5). Thus relaxation of the carotid artery is impaired with acetylcholine and enhanced with the NO donor nitroprusside in eNOS-deficient mice. Enhanced responses to NO may represent a compensatory response expressed in the absence of eNOS. The findings that vascular responses to acetylcholine and NO are altered in eNOS(+/−) mice compared with those observed in eNOS(+/+) mice suggest a “gene-dosing” effect.

Vasodilation to increases in flow was studied in isolated gracilis muscle arterioles of female endothelial nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT) mice. Dilation to flow (0-10 microl/min) was similar in the two groups, yet calculated wall shear stress was significantly greater in arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin, which inhibited flow-induced dilation in vessels of WT mice by approximately 40%, did not affect the responses of eNOS-KO mice, whereas miconazole and 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses. Basal release of epoxyeicosatrienonic acids from arterioles was inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in arterioles of eNOS-KO mice but had no effect on arterioles of WT mice. In WT mice, neither N(omega)-nitro-L-arginine methyl ester nor miconazole alone affected flow-induced dilation. Combination of both inhibitors inhibited the responses by approximately 50%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone inhibited flow-induced dilation by approximately 49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT mice, nitric oxide and prostaglandins mediate flow-induced dilation. When eNOS is inhibited, endothelium-derived hyperpolarizing factor substitutes for nitric oxide. In female eNOS-KO mice, metabolites of cytochrome P-450, via activation of large-conductance Ca2+-activated K+ channels of smooth muscle, mediate entirely the arteriolar dilation to flow.

Abstract-Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O 2 consumption and to further examine the role of nitric oxide (NO) consumption in tissues taken from iNOS (Ϫ/Ϫ) (Ϫ28Ϯ4%), wild-type eNOS (ϩ/ϩ) (Ϫ22Ϯ4%), and heterozygous eNOS(ϩ/Ϫ) (Ϫ22Ϯ5%) but not homozygous eNOS (Ϫ/Ϫ) (Ϫ3Ϯ4%) mice. Responses to bradykinin in iNOS (Ϫ/Ϫ) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with N-nitro-L-arginine methyl ester (L-NAME) (Ϫ2Ϯ5%, Ϫ3Ϯ2%, and Ϫ6Ϯ5%, respectively, PϽ0.05). In contrast, S-nitroso-N-acetyl-penicillamine (SNAP, 10Ϫ4 mol/L), which releases NO spontaneously, induced decreases in myocardial O 2 consumption in all groups of mice, and such responses were not affected by L-NAME. In addition, pretreatment with bacterial endotoxin elicited a reduction in basal O 2 consumption in tissues taken from normal but not iNOS (Ϫ/Ϫ)-deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O 2 consumption and modulation of mitochondrial respiration by NO may have an important role in pathological conditions such as endotoxemia in which the production of NO is altered. . Their initial observations demonstrated that activated mouse peritoneal macrophages severely inhibited O 2 consumption in numerous tumor cell lines obtained from different tissues and animal species in cultures by an unknown mechanism. Evidence now suggests that the macrophage-induced cytotoxic effect on mitochondrial metabolism is NO related. 2,3 NO inhibits respiration by nitrosylating the iron-sulfur centers of aconitase, complexes I and II of the electron transport chain, and through a very potent reversible alteration in the activity of cytochrome c oxidase. 4 -6 Recently, we and others have provided direct evidence to suggest that under physiological conditions NO plays a modulatory role on mitochondrial respiration and tissue O 2 consumption. For instance, L-arginine analogues, which are nonspecific inhibitors of the 3 isoforms of nitric oxide synthase (NOS), 7 increase O 2 consumption in whole body, 8 heart, skeletal muscle, and kidney both in vivo 9 -12 and in vitro. [12][13][14] We have interpreted our previous studies to suggest that endothelial nitric oxide synthase (eNOS), the most highly expressed isoform of NOS in vascular tissue under physiological conditions, is responsible for the control of tissue O 2 consumption by NO. However, we have yet to determine which isoform of NOS regulates mitochondrial O 2 consumption, because almost all cells are capable of expressing all 3 different NOS isoforms. Studies of the effects of bacterial endotoxins have attributed a substantial role for inducible nitric oxide synthase (iNOS) in the development of shock and perhaps other pathological states. To address the role of NO in both physiological and pathophysiological states in the control of mitochondrial respiration, we used tissues from mice deficient in iNOS and eNOS and 3 additional groups, ie, control C57B...