Interactions between NO, CO and an endothelium-derived hyperpolarizing factor (EDHF) in maintaining patency of the ductus arteriosus in the mouse.

BACKGROUND AND PURPOSE: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E(2), possibly along with nitric oxide (NO) and carbon monoxide (CO), and cyclooxygenase (COX) deletion upregulates NO. Here, we have examined enzyme source and action of NO for ductus patency and whether NO and CO are upregulated by deletion of, respectively, heme oxygenase 2 (HO-2) and COX1 or COX2. EXPERIMENTAL APPROACH: Experiments were performed in vitro and in vivo with wild-type and gene-deleted, near-term mouse fetuses. KEY RESULTS: N(G)-nitro-L-arginine methyl ester (L-NAME) contracted the isolated ductus and its effect was reduced by eNOS, but not iNOS, deletion. L-NAME contraction was not modified by HO-2 deletion. Zinc protoporphyrin (ZnPP) also contracted the ductus, an action unaffected by deletion of either COX isoform. Bradykinin (BK) relaxed indomethacin-contracted ductus similarly in wild-type and eNOS-/- or iNOS-/-. BK relaxation was suppressed by either L-NAME or ZnPP. However, it reappeared with combined L-NAME and ZnPP to subside again with K(+) increase or K(+) channel inhibition. In vivo, the ductus was patent in wild-type and NOS-deleted fetuses. Likewise, no genotype-related difference was noted in postnatal closure. CONCLUSIONS AND IMPLICATIONS: NO, formed mainly via eNOS, regulates ductal tone. NO and CO cooperatively mediate BK-induced relaxation in the absence of PGE(2). However, in the absence of PGE(2), NO and CO, BK induces a relaxant substance behaving as an endothelium-derived hyperpolarizing factor. Ductus patency is, therefore, sustained by a cohort of agents with PGE(2) and NO being preferentially coupled for reciprocal compensation.

Nitric oxide synthase gene knockout mice do not become hypertensive during pregnancy.

OBJECTIVE: The purpose of this study was to test whether omitting the vasodilator nitric oxide that is derived from any 1 of the 3 isoforms of nitric oxide synthase results in hypertension during pregnancy. STUDY DESIGN: We measured systolic blood pressure before, during, and after pregnancy using an automated tail cuff method in 3 mutant (gene knockout) mouse strains in which the gene for neuronal nitric oxide, inducible nitric oxide, or endothelial nitric oxide was disrupted by gene targeting. RESULTS: In neuronal nitric oxide gene knockout mice (n = 10), blood pressure was 100 +/- 3 mm Hg, not significantly different from 101 +/- 3 mm Hg in matched wild-type control mice (n = 10). Pregnancy did not change blood pressure or heart rate in either group. In inducible nitric oxide gene knockout mice (n = 9), blood pressure was 110 +/- 3 mm Hg, the same as in the wild-type control mice (110 +/- 2 mm Hg; n = 14). Blood pressure was unaffected by pregnancy in either group of mice. However, heart rate was significantly less in knockout mice (647 +/- 11 beats/min vs 666 +/- 9 beats/min; P <.005); this difference persisted through pregnancy. In endothelial nitric oxide gene knockout mice (n = 8), blood pressure was higher before pregnancy (114 +/- 4 mm Hg vs 103 +/- 4 mm Hg; P <.05) than in wild-type control mice (n = 9), but this difference disappeared during pregnancy, returning only after delivery. Heart rates were not different before pregnancy and were unaffected by pregnancy. CONCLUSION: There was no apparent increase in systolic blood pressure in any of the 3 nitric oxide synthase gene knockout strains during pregnancy compared to the wild-type control mice. This suggests that, at least in the mouse, genetic deficiency of any 1 isoform of nitric oxide synthase does not result in pregnancy-induced hypertension.

EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice.

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.

Potential role of eNOS in the therapeutic control of myocardial oxygen consumption by ACE inhibitors and amlodipine.

OBJECTIVES: Our aim was to investigate the potential therapeutic role of endothelial nitric oxide synthase (eNOS) in the modulation of cardiac O(2) consumption induced by the angiotensin converting enzyme (ACE) inhibitor ramiprilat and amlodipine. METHODS: Three different groups of mice were used; wild type, wild type in the presence of N-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/l) or genetically altered mice lacking the eNOS gene (eNOS -/-). Myocardial O(2) consumption was measured using a Clark-type O(2) electrode in an air-tight stirred bath. Concentration-response curves to ramiprilat (RAM), amlodipine (AMLO), diltiazem (DIL), carbachol (CCL), substance P (SP) and S-nitroso-N-acetyl-penicillamine (SNAP) were performed. The rate of decrease in O(2) concentration was expressed as a percentage of the baseline. RESULTS: Baseline O(2) consumption was not different between the three groups of mice. In tissues from wild type mice, RAM (10(-5) mol/l), AMLO (10(-5) mol/l), DIL (10(-4) mol/l), CCL (10(-4) mol/l), SP (10(-7) mol/l) and SNAP (10(-4) mol/l) reduced myocardial O(2) consumption by -32+/-4, -27+/-10, -20+/-6, -25+/-2, -22+/-4 and -42+/-4%, respectively. The responses to RAM, AMLO, CCL and SP were absent in tissues taken from eNOS -/- mice (-7.1+/-4.3, -5.0+/-6.0, -5.2+/-5.1 and -0.4+/-0.2%, respectively). In addition, L-NAME significantly (P<0.05) inhibited the reduction in O(2) consumption induced by RAM (-9.8+/-4.4%), AMLO (-1.0+/-6.0%), CCL (-8.8+/-3.7%) and SP (-6.6+/-4.9%) in cardiac tissues from wild type mice. In contrast, NO-independent responses to the calcium channel antagonist, DIL, and responses to the NO donor, SNAP, were not affected in cardiac tissues taken from eNOS -/- (DIL: -20+/-4%; SNAP: -46+/-6%) or L-NAME-treated (DIL: -17+/-2%; SNAP: -33+/-5%) mice. CONCLUSIONS: These results suggest that endogenous endothelial NO synthase derived NO serves an important role in the regulation of myocardial O(2) consumption. This action may contribute to the therapeutic action of ACE inhibitors and amlodipine.

eNOS-deficient mice show reduced pulmonary vascular proliferation and remodeling to chronic hypoxia.

Pulmonary hypertension is characterized by structural and morphological changes to the lung vasculature. To determine the potential role of nitric oxide in the vascular remodeling induced by hypoxia, we exposed wild-type [WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient [(-/-)] mice to normoxia or hypoxia (10% O(2)) for 2, 4, and 6 days or for 3 wk. Smooth muscle alpha-actin and von Willebrand factor immunohistochemistry revealed significantly less muscularization of small vessels in hypoxic eNOS(-/-) mouse lungs than in WT(+/+) mouse lungs at early time points, a finding that correlated with decreases in proliferating vascular cells (5-bromo-2'-deoxyuridine positive) at 4 and 6 days of hypoxia in the eNOS(-/-) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of muscularized small vessels; however, only the WT(+/+) mice exhibited an increase in the percentage of fully muscularized vessels and increased vessel wall thickness. eNOS protein expression was increased in hypoxic WT(+/+) mouse lung homogenates at all time points examined, with significantly increased percentages of small vessels expressing eNOS protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely attributable to the decrease in vascular cell proliferation observed in these mice.

Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice.

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF.

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.

Myocardial glucose uptake is regulated by nitric oxide via endothelial nitric oxide synthase in Langendorff mouse heart.

Although the role of nitric oxide (NO) in the modulation of vascular tone has been studied and well understood, its potential role in the control of myocardial metabolism is only recently evident. Several lines of evidence indicate that NO regulates myocardial glucose metabolism; however, the details and mechanisms responsible are still unknown. The aim of this study was to further define the role of NO in the control of myocardial glucose metabolism and the nitric oxide synthase (NOS) isoform responsible using transgenic animals lacking endothelial NOS (ecNOS). In the present study, we examined the regulation of myocardial glucose uptake using isometrically contracting Langendorff-perfused hearts from normal mice (C57BL/6J), mice with defects in the expression of ecNOS [ecNOS (-/-)], and its heterozygote [ecNOS (+/-)], and wild-type mice [ecNOS (+/+)] (n=6, respectively). In hearts from normal mice, little myocardial glucose uptake was observed. This myocardial glucose uptake increased significantly in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Similarly, in the hearts from ecNOS (-/-), glucose uptake was much greater than in normal mice, whereas myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice was not different from normal mice. In addition, myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice increased significantly in the presence of L-NAME. At a workload of 800 g. beats/min, L-NAME increased glucose uptake from 0.1+/-0.1 to 3+/-0.4 microg/min x mg in ecNOS (+/-) mice and from 0.2+/-0.1 to 2.7+/-0.7 microg/min x mg in ecNOS (+/+) mice. Furthermore, in the hearts from ecNOS (-/-) mice, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog or S-nitroso-N-acetylpenicillamine (SNAP), a NO donor essentially shut off glucose uptake, and in hearts from ecNOS (+/-) mice, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), an inhibitor of cGMP, increased the glucose uptake significantly. These results indicate clearly that cardiac NO production regulates myocardial glucose uptake via a cGMP-dependent mechanism and strongly suggest that ecNOS plays a pivotal role in this regulation. These findings may be important in the understanding of the pathogenesis of the diseases such as ischemic heart disease, heart failure, diabetes mellitus, hypertension, and hypercholesterolemia, in which NO synthesis is altered and substrate utilization by the heart changes.

Enhanced atherosclerosis and kidney dysfunction in eNOS(-/-)Apoe(-/-) mice are ameliorated by enalapril treatment.

Hypertension and atherosclerosis are each important causes of morbidity and mortality in the developed world. We have investigated the interaction between these conditions by breeding mice that are atherosclerotic due to lack of apolipoprotein (apo) E with mice that are hypertensive due to lack of endothelial nitric oxide synthase (eNOS). The doubly deficient mice (nnee) have higher blood pressure (BP) and increased atherosclerotic lesion size but no change in plasma lipoprotein profiles compared with normotensive but atherosclerotic (NNee) mice. The nnee mice also develop kidney damage, evidenced by increased plasma creatinine, decreased kidney weight/body weight ratio, and glomerular lipid deposition and calcification. Enalapril treatment abolishes the deleterious effects of eNOS deficiency on BP, atherosclerosis, and kidney dysfunction in nnee mice. In striking contrast, a genetic lack of inducible NOS, which does not affect BP, has no effect on the development of atherosclerotic lesions in Apoe(-/-) mice. We also observed a positive relationship between BP and size of atherosclerotic lesions These results suggest that the atherogenic effects of eNOS deficiency can be partially explained by an increase in BP and reemphasize the importance of controlling hypertension in preventing atherosclerosis.

eNOS mediates L-arginine-induced inhibition of thick ascending limb chloride flux.

We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J(Cl)) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0. 5 mmol/L L-Arg to the bath decreased J(Cl) from 105.8+/-17.5 to 79. 2+/-15.8 pmol/mm per minute (P<0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J(Cl.) In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J(Cl) of THALs from eNOS knockout mice. When 10 micromol/L SPM was added to the bath of eNOS knockout THALs, J(Cl) decreased from 89.1+/-8.6 to 74.8+/-7.5 pmol/mm/min (P<0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J(Cl) in THALs from iNOS and nNOS knockout mice by 37.7+/-6.4% (P<0.05) and 31.8+/-8.3% (P<0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J(Cl).

Role of nitric oxide in the control of cardiac oxygen consumption in B(2)-kinin receptor knockout mice.

The aim of this study was to determine whether bradykinin, the angiotensin-converting enzyme inhibitor ramiprilat, and the calcium-channel antagonist amlodipine reduce myocardial oxygen consumption (MV(O2)) via a B(2)-kinin receptor/nitric oxide-dependent mechanism. Left ventricular free wall and septum were isolated from normal and B(2)-kinin receptor knockout (B(2) -/-) mice. Myocardial tissue oxygen consumption was measured in an airtight chamber with a Clark-type oxygen electrode. Baseline MV(O2) was not significantly different between normal (239+/-13 nmol of O(2). min(-1). g(-1)) and B(2) -/- (263+/-24 nmol of O(2). min(-1). g(-1)) mice. S-nitroso-N-acetyl-penicillamine (10(-7) to 10(-4) mol/L) reduced oxygen consumption in a concentration-dependent manner in both normal (maximum, 36+/-3%) and B(2) -/- mice (28+/-3%). This was also true for the endothelium-dependent vasodilator substance P (10(-10) to 10(-7) mol/L; 22+/-7% in normal mice and 20+/-4% in B(2) -/- mice). Bradykinin (10(-7) to 10(-4) mol/L), ramiprilat (10(-7) to 10(-4) mol/L), and amlodipine (10(-7) to 10(-5) mol/L) all caused concentration-dependent decreases in MV(O2)in normal mice. At the highest concentration, tissue O(2) consumption was decreased by 18+/-3%, 20+/-5%, and 28+/-3%, respectively. The reduction in MV(O2) to all 3 drugs was attenuated in the presence of N(G)-nitro-L-arginine-methyl ester. However, in the B(2) -/- mice, bradykinin, ramiprilat, and amlodipine had virtually no effect on MV(O2). Therefore, nitric oxide, through a bradykinin-receptor-dependent mechanism, regulates cardiac oxygen consumption. This physiological mechanism is absent in B(2) -/- mice and may be evidence of an important therapeutic mechanism of action of angiotensin-converting enzyme inhibitors and amlodipine.

Enhanced release of prostaglandins contributes to flow-induced arteriolar dilation in eNOS knockout mice.

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 ( approximately 80 micrometer) 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 microq/min were similar in arterioles from eNOS-KO (n=28) and WT (n=22) mice ( approximately 20 micrometer at 10 microL/min flow). Removal of the endothelium eliminated flow-induced dilations in vessels of both strains of mice. N(omega)-nitro-L-arginine (L-NNA, 10(-4) mol/L) significantly inhibited flow-induced dilation in arterioles of WT mice by approximately 51% but had no effect on responses of arterioles from eNOS-KO mice. Indomethacin (INDO, 10(-5) mol/L) inhibited flow-induced dilation of WT mice by approximately 49%, 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(-8) and 3x10(-8) mol/L but decreased significantly at 10(-7) 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.

Protein expression, vascular reactivity and soluble guanylate cyclase activity in mice lacking the endothelial cell nitric oxide synthase: contributions of NOS isoforms to blood pressure and heart rate control.

OBJECTIVE: Both disruption of the endothelial nitric oxide synthase (eNOS) gene and pharmacological inhibition of the NOS produce modest hypertension. It is unclear if and to what extent NOS isoforms other than eNOS contribute to this effect and how loss of one copy of the eNOS gene might impact on vascular reactivity or eNOS protein expression. METHODS: We examined protein expression, vascular reactivity, activity of soluble guanylate cyclase, blood pressure and heart rate in mice completely lacking the eNOS gene (eNOS-/-), wild-type mice (eNOS+/+) and mice heterozygotic for the eNOS gene (eNOS+/-). RESULTS: While eNOS-/- mice had mild hypertension and bradycardia, eNOS+/- mice were normotensive. In control mice, oral administration of L-NAME (approximately 100 mg/kg/day x 21 days) increased blood pressure to levels observed in eNOS-/- mice. In eNOS-/- mice, chronic oral administration of L-NAME had no effect on blood pressure, suggesting that inhibition of other NOS isoforms unlikely contribute to hypertension. L-NAME treatment induced bradycardia in both control and eNOS-/- mice, suggesting that both eNOS and other isoforms of NOS might be involved in heart rate control. Studies of aortic rings from eNOS-/- mice revealed a complete lack of endothelium-dependent vascular relaxation in response to acetylcholine and the calcium ionophore A23187 and an increase in sensitivity to phenylephrine, serotonin and nitroglycerin. Aortic rings from eNOS+/- mice demonstrated only minor alterations of responses to nitroglycerin and a normal relaxation to either acetylcholine or A23187 compared to vessels from eNOS-/+. Western analysis demonstrated that eNOS expression was virtually identical between eNOS+/+ and eNOS+/- mice and was absent in eNOS-/- mice. The activity of lung-isolated soluble guanylate cyclase was identical in the three strains of mice. CONCLUSIONS: We conclude that loss of one copy of the eNOS gene, as observed in heterozygotic animals, has no effect on vascular reactivity, blood pressure or eNOS protein expression. Isoforms of NOS, other than eNOS are unlikely involved in blood pressure regulation but may participate in heart rate control.

Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice.

Relaxation to acetylcholine (ACh) and calcium ionophore (A-23187) is absent in aortas from endothelial nitric oxide synthase (eNOS)-deficient (eNOS -/-) mice. We hypothesized that gene transfer of eNOS would restore relaxation to ACh and A-23187 in eNOS -/- mice. Aortic rings from eNOS -/- and eNOS +/+ mice were exposed in vitro to vehicle or adenoviral vectors encoding beta-galactosidase (lacZ) or eNOS. Histochemical staining for beta-galactosidase and eNOS demonstrated transduction of endothelial cells and adventitia. Vehicle-treated vessels from eNOS -/- mice did not relax to ACh or A-23187 compared with eNOS +/+ mice. In contrast, relaxation to nitroprusside (NP) was significantly greater in eNOS -/- mice than in eNOS +/+ mice. Gene transfer of eNOS, but not lacZ, to vascular rings of eNOS -/- mice restored relaxation to ACh and A-23187. In vessels from eNOS -/- mice that were transduced with eNOS, N(omega)-nitro-L-arginine (10(-4) M) inhibited relaxation to ACh and A-23187 but not NP. Thus vascular function can be significantly improved by gene transfer in vessels where a major relaxation mechanism is genetically absent.

Endothelial nitric oxide gene knockout mice: cardiac phenotypes and the effect of angiotensin-converting enzyme inhibitor on myocardial ischemia/reperfusion injury.

We tested the hypothesis that nitric oxide (NO) released by endothelial NO synthase (eNOS) is not only important in blood pressure regulation but also involved in cardiac function and remodeling and in the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEi). With the use of a 2D Doppler echocardiography system equipped with a 15-MHz linear transducer, we evaluated left ventricular (LV) morphology and function in conscious eNOS knockout mice (eNOS(-/-); n=15) and their wild-type littermates (eNOS(+/+); n=16). We also studied whether in eNOS(-/-) mice (1) myocardial ischemia/reperfusion injury is more severe and (2) the cardioprotective effect of ACEi is diminished or absent. In comparison with the wild type, eNOS(-/-) mice had significantly increased systolic blood pressure (128+/-3 versus 108+/-5 mm Hg; P<0.001) and decreased heart rate (531+/-22 versus 629+/-18 bpm; P<0.001) associated with increased LV posterior wall thickness (0.80+/-0.04 versus 0.64+/-0.02 mm; P<0.001) and LV mass (18.3+/-0.9 versus 13.1+/-0.5 mg/10 g body weight; P<0.01). Despite hypertension and LV hypertrophy, LV chamber dimension, shortening fraction and ejection fraction (indicators of LV contractility), and cardiac output did not differ between the 2 strains, which indicates that LV function in eNOS(-/-) mice is well compensated. We also found that in eNOS(+/+) mice, ACEi decreased the ratio of myocardial infarct size to area at risk from 62.7+/-3.9% to 36.3+/-1.6% (P<0. 001), whereas in eNOS(-/-) mice this effect of ACEi was almost abolished: the ratio of myocardial infarct size to area at risk was 67.2+/-2.9% in the vehicle-treated group and 62.7+/-3.9% in mice treated with ACEi. Moreover, infarct size in vehicle-treated eNOS(-/-) mice was not significantly different from eNOS(+/+) mice given the same treatment. We concluded that (1) endothelium-derived NO plays an important role in the regulation of blood pressure homeostasis; (2) NO released under basal conditions has no significant impact on cardiac function; and (3) ACEi protect the heart against ischemia/reperfusion injury in mice and that this effect is mediated in part by endothelium-derived NO.

An enhanced effect of arginine vasopressin in bradykinin B2 receptor null mutant mice.

Under water restriction, arginine vasopressin (AVP) is released and promotes water reabsorption in the distal nephron, mainly through AVP V2-receptors. It has been proposed that renal kinins counteract the hydro-osmotic effect of AVP. We hypothesized that kinins acting through B2 receptors antagonize the urinary concentrating effect of AVP. To test this, bradykinin B2 receptor knockout mice (B2-KO) and 129/SvEv mice (controls) were placed in metabolic cages and urine collected for 24 hours (water ad libitum). After that, urine was again collected from the same mice during 24 hours of water restriction. Urinary volume (UV), urinary osmolarity (UOsm), and urinary Na+ (UNaV) and K+ (UKV) excretion were determined. On water restriction, UV in controls decreased by approximately 25%, whereas in B2-KO mice there was almost a 60% drop in urinary output (P=0.001 versus controls). In the controls, water restriction increased UOsm by 347 mOsm/kg H2O, approximately 14% above baseline (NS), whereas in knockout mice the increase was 3 times that seen in the controls: >1000 mOsm/kg H2O (P=0.001 versus controls). Compared with normohydration, UNaV and UKV in the water-restricted state increased more in controls than in B2-KO mice. This difference in electrolyte excretion could be explained by greater dehydration in the controls (dehydration natriuresis). In a second protocol, we tried to mimic the effect of endogenous AVP by exogenous administration of an AVP V2-receptor agonist, desmopressin (DDAVP). To suppress endogenous AVP levels before DDAVP administration, mice were volume-overloaded with dextrose and alcohol. UOsm was 685+/-125 and 561+/-58 mOsm/kg H2O in water-loaded controls and B2-KO mice, respectively. After DDAVP was injected subcutaneously at a dose of 1 microgram/kg, UOsm increased to 1175+/-86 mOsm/kg H2O (Delta+490 mOsm) in the controls and 2347+/-518 mOsm/kg H2O (Delta+1786 mOsm) in B2-KO mice (P<0.05 versus controls). We concluded that water restriction or exogenous administration of an AVP V2-receptor agonist has a greater urinary concentrating effect in B2-KO mice than in controls, suggesting that endogenous kinins acting through B2 receptors oppose the antidiuretic effect of AVP in vivo.

Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption.

Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O2 consumption and to further examine the role of nitric oxide (NO) in the control of mitochondrial respiration. Left ventricle O2 consumption in mice with defects in the expression of eNOS [eNOS (-/-)] and inducible NOS [iNOS (-/-)] was measured with a Clark-type O2 electrode. The rate of decreases in O2 concentration was expressed as a percentage of the baseline. Baseline O2 consumption was not significantly different between groups of mice. Bradykinin (10(-4) mol/L) induced significant decreases in O2 consumption in tissues taken from iNOS (-/-) (-28+/-4%), wild-type eNOS (+/+) (-22+/-4%), and heterozygous eNOS(+/-) (-22+/-5%) but not homozygous eNOS (-/-) (-3+/-4%) mice. Responses to bradykinin in iNOS (-/-) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with N-nitro-L-arginine methyl ester (L-NAME) (-2+/-5%, -3+/-2%, and -6+/-5%, respectively, P<0.05). In contrast, S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4) mol/L), which releases NO spontaneously, induced decreases in myocardial O2 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 O2 consumption in tissues taken from normal but not iNOS (-/-)-deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O2 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.

Effect of ACE inhibitor on DOCA-salt- and aortic coarctation-induced hypertension in mice: do kinin B2 receptors play a role?

Kinins have been shown to play an important role in the cardioprotective effect of ACE inhibitors (ACEi) during heart failure and ischemia-reperfusion. However, it is controversial as to whether kinins oppose the hypertensinogenic effect of deoxycorticosterone acetate plus salt (DOCA-salt) or aortic coarctation and whether they mediate both chronic antihypertensive and cardiac antihypertrophic effects of ACEi in hypertension. Using normal 129/SvEvTac mice and mice lacking the bradykinin B2 receptor gene (B2-KO), we investigated whether (1) the hypertensinogenic effect of DOCA-salt or aortic coarctation is enhanced in B2-KO mice and (2) the chronic antihypertensive and antihypertrophic effects of an ACEi (ramipril, 4 mg. kg-1. d-1) are mediated by B2 receptors in aortic coarctation (6 weeks)- and DOCA-salt (4 weeks)-induced hypertension. Before surgery, there was no difference between 129/SvEvTac and B2-KO mice in terms of blood pressure and heart weight, suggesting that kinins are not essential to maintaining normal blood pressure. DOCA-salt (volume expansion) or aortic coarctation (renin-dependent) induced similar hypertension and left ventricular hypertrophy (LVH) in 129/SvEvTac and B2-KO mice, suggesting that kinins do not play an essential role in the development of DOCA-salt- or aortic coarctation-induced hypertension. We found that B2 receptors mediate only the early (1 week) but not the late phase (4 weeks) of the chronic hypotensive effect of ACEi in DOCA-salt hypertension. On the other hand, chronic ACE inhibition prevented the development of hypertension and LVH in both 129/SvEvTac and B2-KO mice given DOCA-salt or subjected to aortic coarctation, suggesting that kinins do not participate in the chronic antihypertensive and antihypertrophic effects of ACEi in these 2 models of hypertension. Thus, in mice, kinins acting via B2 receptors do not participate in (1) maintenance of normal basal blood pressure, (2) establishment and maintenance of hypertension induced by DOCA-salt or aortic coarctation, and (3) chronic antihypertensive and cardiac antihypertrophic effects of ACEi in DOCA-salt and aortic coarctation hypertension.

Role of nNOS in blood pressure regulation in eNOS null mutant mice.

The role of neural nitric oxide synthase (nNOS) in regulating blood pressure (BP) remains uncertain. Recently it was reported that in mice lacking functional endothelial NOS (eNOS) genes (-/-), acute administration of a nonselective NOS inhibitor, Nw-nitro-L-arginine, decreased mean BP, suggesting that NO released by non-eNOS isoforms increases BP. Because the inducible NOS isoform is not constitutively expressed and when induced causes hypotension, we hypothesize that it is NO produced by nNOS that increases BP in the absence of eNOS activity. To test this hypothesis, we studied the acute effect of selective and nonselective nNOS inhibitors on BP and cerebellar NOS activity in eNOS (-/-), wild-type (+/+), and heterozygous (+/-) mice as well as in +/+ mice with renovascular hypertension. Because it is not known whether the decrease in BP caused by acute NOS inhibition in -/- mice can occur chronically, we also studied the effect of chronic NOS inhibition on both BP and cerebellar NOS activity. eNOS (-/-) mice had higher BP than +/+ or +/-mice, and acute administration of the selective nNOS inhibitor 7-nitroindazole (7-NI) decreased their mean BP from 137+/-13 to 124+/-12 mm Hg (P<0.01). In +/+, +/-, or renovascular hypertensive +/+ mice, 7-NI caused a small but insignificant rise from 105+/-5 to 110+/-6 mm Hg, from 115+/-9 to 119+/-13 mm Hg, and from 146+/-6 to 150+/-6 mm Hg, respectively. Fifteen minutes after administration of 7-NI, cerebellar NOS activity decreased by 70%; however, this inhibitory effect was brief, since 2 hours after 7-NI administration NOS returned toward control values. Chronic oral or intraperitoneal administration of 7-NI did not inhibit cerebellar NOS activity, whereas the nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) decreased this activity by 50%. Therefore, we studied the effect of chronic L-NAME administration (4 weeks) on BP. In -/- mice, chronic L-NAME administration decreased BP from 135+/-4 to 120+/-3 mm Hg (P<0.05), whereas in +/+ and +/-mice, as expected, it increased BP from 109+/-2 to 125+/-3 mm Hg (P<0.001) and from 107+/-6 to 119+/-5 mm Hg (P<0.02), respectively. After L-NAME administration was stopped, BP returned to baseline. These results suggest that in eNOS -/- mice, NO derived from nNOS increases BP both acutely and chronically.

Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling.

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.