Changes in NO bioavailability regulate cardiac O2 consumption: control by intramitochondrial SOD2 and intracellular myoglobin

The aim of this study was to investigate the significance of two intracellular scavengers of nitric oxide (NO): 1) superoxide dismutase (SOD) (SOD2) to scavenge intramitochondrial superoxide anion, and 2) cytosolic myoglobin (Mb) in the regulation of tissue O2 consumption. O2 consumption was measured in vitro using a Clark-type O2 electrode. SOD heterozygous mice (SODHZ) (n = 13) and SOD wild-type (SODWT) (n = 5) mice were used. Bradykinin (BK, 10-4 mol/l) reduced O2 consumption by 15% +/- 1 in hearts of SODHZ mice, which was significantly different from SODWT (reduced by 24 +/- 0.4%). Tiron significantly increased the inhibition of O2 consumption by BK in male mice from 15 +/- 1% (n = 13) to 29 +/- 1.2% (n = 4) at 10-4 mol/l concentration (P < 0.05). The effect of carbachol was similar to BK. S-nitroso-N-acetyl penicillamine (SNAP, 10-4 mol/l) reduced O2 consumption by 39 +/- 1.3% in hearts of SODHZ mice, which was not significantly different from SODWT. But at 10-7 mol/l, SNAP caused significantly less inhibition of O2 consumption in SODHZ mice. Mb knockout (MbKO; Mb wild-type n = 6) and (MbWT) mice (n = 6) were also used. Kidney cortex was studied as the negative control because it does not contain Mb. BK (10-4 mol/l) reduced O2 consumption by 32 +/- 2, 29 +/- 1, and 26 +/- 1% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. SNAP (10-4 mol/l) reduced O2 consumption by 39 +/- 3, 42 +/- 4, and 46 +/- 2% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. NG-nitro-l-arginine methyl ester (P < 0.05) inhibited the reduction in O2 consumption induced by BK in the MbKO mouse heart (15 +/- 1%), skeletal muscle (17 +/- 1%), and kidney (17 +/- 1%) as in the MbWT mice. These results suggest that the role of Mb as an intracellular NO scavenger is small, and the increase in mitochondrial superoxide in SODHZ mice may cause a decrease NO bioavailability and alter the control of myocardial O2 consumption by NO.     

Displacement-encoded and manganese-enhanced cardiac MRI reveal that nNOS, not eNOS, plays a dominant role in modulating contraction and calcium influx in the mammalian heart

Within cardiomyocytes, endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are thought to modulate L-type calcium channel (LTCC) function and sarcoplasmic reticulum calcium cycling, respectively. However, divergent results from mostly invasive prior studies suggest more complex roles. To elucidate the roles of nNOS and eNOS in vivo, we applied noninvasive cardiac MRI to study wild-type (WT), eNOS(-/-), and nNOS(-/-) mice. An in vivo index of LTCC flux (LTCCI) was measured at baseline (Bsl), dobutamine (Dob), and dobutamine + carbacholamine (Dob + CCh) using manganese-enhanced MRI. Displacement-encoded MRI assessed contractile function by measuring circumferential strain (E(cc)) and systolic (dE(cc)/dt) and diastolic (dE(cc)/dt(diastolic)) strain rates at Bsl, Dob, and Dob + CCh. Bsl LTCCI was highest in nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)) and increased only in WT and eNOS(-/-) mice with Dob (P < 0.05 vs. Bsl). LTCCI decreased significantly from Dob levels with Dob + CCh in all mice. Contractile function, as assessed by E(cc), was similar in all mice at Bsl. With Dob, E(cc) increased significantly in WT and eNOS(-/-) but not nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)). With Dob + CCh, E(cc) returned to baseline levels in all mice. Systolic blood pressure, measured via tail plethysmography, was highest in eNOS(-/-) mice (P < 0.05 vs. WT and nNOS(-/-)). Mice deficient in nNOS demonstrate increased Bsl LTCC function and an attenuated contractile reserve to Dob, whereas eNOS(-/-) mice demonstrate normal LTCC and contractile function under all conditions. These results suggest that nNOS, not eNOS, plays the dominant role in modulating Ca(2+) cycling in the heart.     

Voluntary physical activity alterations in endothelial nitric oxide synthase knockout mice

One of the main factors that control vasoreactivity and angiogenesis is nitric oxide produced by endothelial nitric oxide synthase (eNOS). We recently showed that knocking out eNOS induces an important reduction of mitochondrial oxidative capacity in slow-twitch skeletal muscle. Here we investigated eNOS's role in physical activity and contribution to adaptation of muscle energy metabolism to exercise conditions. Physical capacity of mice null for the eNOS isoform (eNOS-/-) was estimated for 8 wk with a voluntary wheel-running protocol. In parallel, we studied energy metabolism enzyme profiles and their response to voluntary exercise in cardiac and slow-twitch soleus (Sol) and fast-twitch gastrocnemius (Gast) skeletal muscles. Weekly averaged running distance was two times lower for eNOS-/- (4.09 +/- 0.42 km/day) than for wild-type (WT; 7.74 +/- 0.42 km/day; P < 0.01) mice. Average maximal speed of running was also lower in eNOS-/- (17.2 +/- 1.4 m/min) than WT (21.2 +/- 0.9 m/min; P < 0.01) mice. Voluntary exercise influenced adaptation to exercise specifically in Sol muscle. Physical activity significantly increased Sol weight by 22% (P < 0.05) in WT but not eNOS-/- mice. WT Sol muscle did not change its metabolic profile in response to exercise, in contrast to eNOS-/- muscle, in which physical activity decreased cytochrome-c oxidase (COX; -36%; P < 0.05), citrate synthase (-37%; P < 0.06), and creatine kinase (-24%, P < 0.01) activities. Voluntary exercise did not change energy enzyme profile in heart (except for 39% increase in COX activity in WT) or Gast muscle. These results suggest that eNOS is necessary for maintaining a suitable physical capacity and that when eNOS is downregulated, even moderate exercise could worsen energy metabolism specifically in oxidative skeletal muscle.     

Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in beta-cells

Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in beta-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 +/- 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 +/- 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 +/- 311; WT, 425 +/- 231 pg x islet(-1) x 35 min(-1); P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 +/- 0.8 mM glucose, compared with 11.8 +/- 0.7 mM in WT (P < 0.05). Older Tg mice secreted less insulin in response to 20 mM glucose (Tg, 2,256 +/- 342; WT, 3,493 +/- 367 pg x islet(-1) x 35 min(-1); P < 0.05). Secretion in response to carbachol was similar in WT and Tg at both ages. Glucose oxidation was blunted in older Tg islets. At 5 mM glucose, islet CO2 production was comparable between Tg and WT. However, WT mice increased islet CO2 production 2.7 +/- 0.4-fold in 20 mM glucose, compared with only 1.4 +/- 0.1-fold in Tg (P < 0.02). Results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, acting at least in part by modulating glucose oxidation pathways. Prolonged excess hexosamine flux results in glucose desensitization and mimics glucose toxicity.     

Growth hormone receptor deficiency in mice results in reduced systolic blood pressure and plasma renin, increased aortic eNOS expression, and altered cardiovascular structure and function

To study the role of the growth hormone receptor (GHR) in the development of cardiovascular structure and function, female GHR gene-disrupted or knockout (KO) and wild-type (WT) mice at age 18 wk were used. GHR KO mice had lower plasma renin levels (12 +/- 2 vs. 20 +/- 4 mGU/ml, P < 0.05) and increased aortic endothelial NO synthase (eNOS) expression (146%, P < 0.05) accompanied by a 25% reduction in systolic blood pressure (BP, 110 +/- 4 vs. 147 +/- 3 mmHg, P < 0.001) compared with WT mice. Aldosterone levels were unchanged, whereas the plasma potassium concentration was elevated by 14% (P < 0.05) in GHR KO. Relative left ventricular weight was 14% lower in GHR KO mice (P < 0.05), and cardiac dimensions as analyzed by echocardiography were similarly reduced. Myograph studies revealed a reduced maximum contractile response in the aorta to norepinephrine (NE) and K(+) (P < 0.05), and aorta media thickness was decreased in GHR KO (P < 0.05). However, contractile force was normal in mesenteric arteries, whereas sensitivity to NE was increased (P < 0.05). Maximal acetylcholine-mediated dilatation was similar in WT and GHR KO mice, whereas the aorta of GHR KO mice showed an increased sensitivity to acetylcholine (P < 0.05). In conclusion, loss of GHR leads to low BP and decreased levels of renin in plasma as well as increase in aortic eNOS expression. Furthermore, GHR deficiency causes functional and morphological changes in both heart and vasculature that are beyond the observed alterations in body size. These data suggest an important role for an intact GH/IGF-I axis in the maintenance of a normal cardiovascular system.     

Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs

Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg. kg(-1). day(-1)) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control (P < 0.05) and veratrine- (activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced (P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NO(x)) levels increased from 5.22 +/- 1.2 to 7. 79 +/- 1.3 microM (P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented (P < 0.05); resting in vivo myocardial oxygen consumption (MVO(2)) decreased from 6.8 +/- 0.6 to 5.9 +/- 0.4 ml/min (P < 0.05); NO-dependent regulation of MVO(2) in response to NO agonists was augmented in isolated myocardial segments (P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MVO(2).     

Mice lacking the gene encoding for MMP-9 and resistance artery reactivity

OBJECTIVES: To define the link between the deletion of gene encoding for metalloproteinase 9 and resistance artery reactivity, we studied in vitro smooth muscle and endothelial cell function in response to pressure, shear stress, and pharmacological agents. BACKGROUND: Matrix metalloproteinases play a crucial role in the regulation of extracellular matrix turnover and structural artery wall remodeling. METHODS: Resistance arteries were isolated from mice lacking gene encoding for MMP-9 (KO) and their control (WT). Hemodynamic, pharmacology approaches, and Western blot analysis were used in this study. RESULTS: The measurement of blood pressure in vivo was similar in KO and WT mice. Pressure-induced myogenic tone, contractions to angiotensin-II and phenylephrine were similar in both groups. The inhibition of MMP2/9 ((2R)-2-[(4-biphenylylsulfonyl) amino]-3-phenylpropionic acid) significantly decreased myogenic tone in WT and had no effect in KO mice. Relaxation endothelium-dependent (flow-induced- dilation 41.3+/-0.6 vs. 21+/-1.6 at 10 microl/min in KO and WT mice, respectively, P<0.05) and eNOS expression were increased in KO compared to WT mice. The inhibition of eNOS with L-NAME significantly decreased endothelium response to shear stress, which was more pronounced in KO mice resistance arteries (-26.83+/-2.5 vs. -15.84+/-2.3 at 10 microl/min in KO and WT, respectively, P<0.05). However, the relaxation to exogenous nitric oxide-donor was similar in both groups. CONCLUSION: Our study provides evidence of a selective effect of MMP-9 on endothelium function. Thus, MMP-9 gene deletion specifically increased resistance artery dilation endothelium-dependent and eNOS expression. Based on our results, MMP-9 could be a potential therapeutic target in cardiovascular disease associated with resistance arteries dysfunction.     

Nitric oxide increases fluid extravasation from the splenic circulation of the rat

We hypothesized that nitric oxide (NO) contributes to intrasplenic fluid extravasation by inducing greater relaxation in splenic resistance arteries than veins such that intrasplenic microvascular pressure (P(C)) rises. Fluid efflux was estimated by measuring the difference between splenic blood inflow and outflow. Intrasplenic infusion of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (0.3 microg. 10 microl(-1). min(-1)) caused a significant increase in intrasplenic fluid efflux (baseline: 0.8 +/- 0.4 ml/min, n = 10 vs. peak rise during SNAP infusion: 1.3 +/- 0.4 ml/min, n = 10; P < 0.05). Intrasplenic P(C) was measured in the isolated, blood-perfused rat spleen. Intrasplenic infusion of SNAP (0.1 microg. 10 microl(-1). min(-1)) caused a significant increase in P(C) (saline: 10.9 +/- 0.2 mmHg, n = 3 vs. SNAP: 12.2 +/- 0.2 mmHg, n = 3; P < 0.05). Vasoreactivity of preconstricted splenic resistance vessels to sodium nitroprusside (SNP) (1 x 10(-12)-1 x 10(-4) M) and SNAP (1 x 10(-10)-3 x 10(-4) M) was investigated with the use of a wire myograph system. Significantly greater relaxation of arterioles than of venules occurred with both SNP (%maximal vasorelaxation: artery 96 +/- 2.3, n = 9 vs. vein 26 +/- 1.9, n = 10) and SNAP (%maximal vasorelaxation: artery 50 +/- 3.5, n = 11 vs. vein 32 +/- 1.7, n = 8). These results are consistent with our proposal that differential vasoreactivity of splenic resistance arteries and veins to NO elevates intrasplenic P(C) and increases fluid extravasation into the systemic lymphatic system.     

A novel approach to assess insulin sensitivity reveals no increased insulin sensitivity in mice with a dominant-negative mutant hepatocyte nuclear factor-1alpha

In phenotype experiments in mice, determination of dynamic insulin sensitivity often uses the insulin tolerance test. However, the interpretation of this test is complicated by the counterregulation occurring at low glucose. To overcome this problem, we determined the dynamic insulin sensitivity after inhibition of endogenous insulin secretion by diazoxide (25 mg/kg) in association with intravenous administration of glucose plus insulin (the DSGIT technique). Estimation of insulin sensitivity index (SI) by this technique showed good correlation to SI from a regular intravenous glucose tolerance test (r = 0.87; P < 0.001; n = 15). With DSGIT, we evaluated dynamic insulin sensitivity in mice with a rat insulin promoter (beta-cell-targeted) dominant-negative mutation of hepatic nuclear factor (HNF)-1alpha [RIP-DN HNF-1alpha (Tg) mice]. When insulin was administered exogenously at the same dose in Tg and wild-type (WT) mice, plasma insulin levels were higher in WT, indicating an increased insulin clearance in Tg mice. When the diazoxide test was used, different doses of insulin were therefore administered (0.1 and 0.15 U/kg in WT and 0.2 and 0.25 U/kg in Tg) to achieve similar insulin levels in the groups. Minimal model analysis showed that SI was the same in the two groups (0.78 +/- 0.21 x 10(-4) min.pmol(-1).l(-1) in WT vs. 0.60 +/- 0.11 in Tg; P = 0.45) as was the glucose elimination rate (P = 0.27). We conclude that 1) the DSGIT technique determines the in vivo dynamic insulin action in mice, 2) insulin clearance is increased in Tg mice, and 3) chronic islet dysfunction in RIP-DN HNF-1alpha mice is not compensated with increased insulin sensitivity.     

Role of nitric oxide in the coupling of myocardial oxygen consumption and coronary vascular dynamics during pregnancy in the dog

We examined the ability of cardiac endothelial nitric oxide synthase (eNOS) to couple myocardial oxygen consumption (MVo(2)) and oxygen delivery during pregnancy. Awake dogs were studied using echocardiography before and at 40 days, 50 days, and 60 days (60D) of pregnancy and at approximately 14 days postpartum. Left ventricular eNOS, phosphorylated eNOS, and copper, zinc-superoxide dismutase (CuZnSOD or SOD-1) were determined by immunoblotting. MVO(2) of left ventricular tissue samples was measured in vitro in response to increasing doses of bradykinin, enalapril maleate, and amlodipine. We examined the changes in passive diameter and flow-dependant arteriolar dilation of coronary arterioles. Echocardiography indicated increases in cardiac output ( approximately 60%) during pregnancy. Myocardial eNOS (21 +/- 4%), phosphorylated eNOS (19 +/- 3%), and SOD-1 (61 +/- 2.7%) protein levels were significantly increased at 60D. Bradykinin, enalapril maleate, and amlodipine (10(-4) mol/l) decreased MVO(2) in a nitric oxide-dependant manner (by 24 +/- 1.3% in controls and 34 +/- 2.2% at 60D; by 21 +/- 1.1% in controls and 29 +/- 1.1 at 60D; and by 22 +/- 2.5% in controls and 26 +/- 1.0% at 60D, respectively). Arterioles from pregnant dogs showed increased flow-dependant dilation in response to increased shear stress and larger passive diameter. Nitrite production was stimulated by bradykinin and carbachol in microvessels in vitro; pregnancy enhanced nitrite release. Myocardial eNOS, phosphorylated eNOS, and SOD-1 protein expression are increased during pregnancy, and this increase is associated with enhanced nitric oxide-dependant control of MVO(2). Thus increases in eNOS and SOD-1 promote the coupling of oxygen delivery and efficiency in the heart during pregnancy.     

NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice

Increased nitric oxide (NO) production is the cause of hypotension and shock during sepsis. In the present experiments, we have measured the contribution of endothelial (e) and inducible (i) nitric oxide synthase (NOS) to systemic NO production in mice under baseline conditions and upon LPS treatment (100 microg/10 g ip LPS). NO synthesis was measured by the rate of conversion of l-[guanidino-15N2]arginine to l-[ureido-15N]citrulline, and the contribution of the specific NOS isoforms was evaluated by comparing NO production in eNOS-deficient [(-/-)] and iNOS(-/-) mice with that in wild-type (WT) mice. Under baseline conditions, NO production was similar in WT and iNOS(-/-) mice but lower in eNOS(-/-) mice [WT: 1.2 +/- 0.2; iNOS(-/-): 1.2 +/- 0.2; eNOS(-/-): 0.6 +/- 0.3 nmol. 10 g body wt-1. min-1]. In response to the challenge with LPS (5 h), systemic NO production increased in WT and eNOS(-/-) mice but fell in iNOS(-/-) mice [WT: 2.7 +/- 0.3; eNOS(-/-): 2.2 +/- 0.6; iNOS(-/-): 0.7 +/- 0.1 nmol. 10 g body wt-1. min-1]. After 5 h of LPS treatment, blood pressure had dropped 14 mmHg in WT but not in iNOS(-/-) mice. The present findings provide firm evidence that, upon treatment with bacterial LPS, the increase of NO production is solely dependent on iNOS, whereas that mediated by cNOS is reduced. Furthermore, the data show that the LPS-induced blood pressure response is dependent on iNOS.     

Inhibition of NO production increases myocardial blood flow and oxygen consumption in congestive heart failure

Coronary blood flow (CBF) and myocardial oxygen consumption (MVO(2)) are reduced in dogs with pacing-induced congestive heart failure (CHF), which suggests that energy metabolism is downregulated. Because nitric oxide (NO) can inhibit mitochondrial respiration, we examined the effects of NO inhibition on CBF and MVO(2) in dogs with CHF. CBF and MVO(2) were measured at rest and during treadmill exercise in 10 dogs with CHF produced by rapid ventricular pacing before and after inhibition of NO production with N(G)-nitro-L-arginine (L-NNA, 10 mg/kg iv). The development of CHF was accompanied by decreases in aortic and left ventricular (LV) systolic pressure and an increase in LV end-diastolic pressure (25 +/- 2 mmHg). L-NNA increased MVO(2) at rest (from 3.07 +/- 0.61 to 4.15 +/- 0.80 ml/min) and during exercise; this was accompanied by an increase in CBF at rest (from 31 +/- 2 to 40 +/- 4 ml/min) and during exercise (both P < 0.05). Although L-NNA significantly increased LV systolic pressure, similar increases in pressure produced by phenylephrine did not increase MVO(2). The findings suggest that NO exerts tonic inhibition on respiration in the failing heart.     

Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na(+)/Ca(2+) exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 +/- 2; TG, 58 +/- 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/dt(max)): WT, 3,750 +/- 346; TG, 5,075 +/- 334 mmHg/s; P < 0.05]. The isometric contractile response to beta-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 +/- 0.9; TG, 16.3 +/- 1.0 mN/mm(2) at 10(-4) M isoproterenol). The sarcoplasmic reticulum (SR) Ca(2+) content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca(2+) content via overexpression of the Na(+)/Ca(2+) exchanger.     

Preserved ventricular contractility in infarcted mouse heart overexpressing beta(2)-adrenergic receptors

Effects of cardiac specific overexpression of beta(2)-adrenergic receptors (beta(2)-AR) on the development of heart failure (HF) were studied in wild-type (WT) and transgenic (TG) mice following myocardial infarction (MI) by coronary artery occlusion. Animals were studied by echocardiography at weeks 7 to 8 and by catheterization at week 9 after surgery. Post-infarct mortality, due to HF or cardiac rupture, was not different among WT mice, and there was no difference in infarct size (IS). Compared with the sham-operated group (all P < 0.01), WT mice with moderate (<36%) and large (>36%) IS developed lung congestion, cardiac hypertrophy, left ventricular (LV) dilatation, elevated LV end-diastolic pressure (LVEDP), and suppressed maximal rate of increase of LV pressure (LV dP/dt(max)) and fractional shortening (FS). Whereas changes in organ weights and echo parameters were similar to those in infarcted WT groups, TG mice had significantly higher levels of LV contractility in both moderate (dP/dt(max) 4,862 +/- 133 vs. 3,694 +/- 191 mmHg/s) and large IS groups (dP/dt(max) 4,556 +/- 252 vs. 3,145 +/- 312 mmHg/s, both P < 0.01). Incidence of pleural effusion (36% vs. 85%, P < 0.05) and LVEDP levels (6 +/- 0.3 vs. 9 +/- 0.8 mmHg, P < 0.05) were also lower in TG than in WT mice with large IS. Thus beta(2)-AR overexpression preserved LV contractility following MI without adverse consequence.     

Loss of alpha2B-adrenoceptors increases magnitude of hypertension following nitric oxide synthase inhibition

Vascular alpha(2B)-adrenoceptors (alpha(2B)-AR) may mediate vasoconstriction and contribute to the development of hypertension. Therefore, we hypothesized that blood pressure would not increase as much in mice with mutated alpha(2B)-AR as in wild-type (WT) mice following nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NNA, 250 mg/l in drinking water). Mean arterial pressure (MAP) was recorded in heterozygous (HET) alpha(2B)-AR knockout mice and WT littermates using telemetry devices for 7 control and 14 l-NNA treatment days. MAP in HET mice was increased significantly on treatment days 1 and 4 to 14, whereas MAP did not change in WT mice (days 0 and 14 = 113 +/- 3 and 114 +/- 4 mmHg in WT, 108 +/- 0.3 and 135 +/- 13 mmHg in HET, P < 0.05). MAP was significantly higher in HET than in WT mice days 10 through 14 (P < 0.05). Thus blood pressure increased more rather than less in mice with decreased alpha(2B)-AR expression. We therefore examined constrictor responses to phenylephrine (PE, 10(-9) to 10(-4) M) with and without NOS inhibition to determine basal NO contributions to arterial tone. In small pressurized mesenteric arteries (inner diameter = 177 +/- 5 microm), PE constriction was decreased in untreated HET arteries compared with WT (P < 0.05). l-NNA (100 microM) augmented PE constriction more in HET arteries than in WT arteries, and responses were not different between groups in the presence of l-NNA. Acetylcholine dilated preconstricted arteries from HET mice more than arteries from WT mice. Endothelial NOS expression was increased in HET compared with WT mesenteric arteries by Western analysis. Griess assay showed increased NO(x) concentrations in HET plasma compared with those in WT plasma. These data demonstrate that diminished alpha(2B)-AR expression increases the dependence of arterial pressure and vascular tone on NO production and that vascular alpha(2B)-AR either directly or indirectly regulates vascular endothelial NOS function.     

Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.     

Pioglitazone protects the myocardium against ischemia-reperfusion injury in eNOS and iNOS knockout mice

Endothelial nitric oxide synthase (eNOS) activation with subsequent inducible NOS (iNOS), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX2) activation is essential to statin inhibition of myocardial infarct size (IS). In the rat, the peroxisome proliferator-activated receptor-gamma agonist pioglitazone (Pio) limits IS, upregulates and activates cPLA2 and COX2, and increases myocardial 6-keto-PGF1alpha levels without activating eNOS and iNOS. We asked whether Pio also limits IS in eNOS-/- and iNOS-/- mice. Male C57BL/6 wild-type (WT), eNOS-/-, and iNOS-/- mice received 10 mg.kg(-1).day(-1) Pio (Pio+) or water alone (Pio-) for 3 days. Mice underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts were harvested and subjected to ELISA and immunoblotting. As a result, Pio reduced IS in the WT (15.4+/-1.4% vs. 39.0+/-1.1%; P<0.001), as well as in the eNOS-/- (32.0+/-1.6% vs. 44.2+/-1.9%; P<0.001) and iNOS-/- (18.0+/-1.2% vs. 45.5+/-2.3%; P<0.001) mice. The protective effect of Pio in eNOS-/- mice was smaller than in the WT (P<0.001) and iNOS-/- (P<0.001) mice. Pio increased myocardial Ser633 and Ser1177 phosphorylated eNOS levels in the WT and iNOS-/- mice. iNOS was undetectable in all six groups. Pio increased cPLA2, COX2, and PGI2 synthase levels in the WT, as well as in the eNOS-/- and iNOS-/-, mice. Pio increased the myocardial 6-keto-PGF1alpha levels and cPLA2 and COX2 activity in the WT, eNOS-/-, and iNOS-/- mice. In conclusion, the myocardial protective effect of Pio is iNOS independent and may be only partially dependent on eNOS. Because eNOS activity decreases with age, diabetes, and advanced atherosclerosis, this effect may be relevant in a clinical setting and should be further characterized.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)alpha-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)alpha and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARalpha ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARalpha activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARalpha ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARalpha knockout (KO) mice compared with its wild type (WT) litter mates (130+/-10 mmHg versus 107+/-4 mmHg). L-NAME (100mg/L p.o.), the inhibitor of NO production abolished the difference between PPARalpha KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8+/-1.4 pM/mg versus 8.3+/-0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46+/-6%, p<0.05) and a approximately 3 fold greater Ca2+-dependent NOS activity in kidney homogenates of untreated PPARalpha WT compared with the KO mice. Clofibrate, a PPARalpha ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19+/-4%, p<0.05), increased urinary NO excretion (4.06+/-0.53-7.07+/-1.59 microM/24 h; p<0.05) and reduced plasma 8-isoprostane level (45.8+/-15 microM versus 31.4+/-8 microM), and NADP(H) oxidase activity (16+/-5%). Implantation of DOCA pellet (20mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193+/-13 mmHg versus 130+/-12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARalpha activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.     

Altered sinus nodal and atrioventricular nodal function in freely moving mice overexpressing the A1 adenosine receptor

To investigate whether altered function of adenosine receptors could contribute to sinus node or atrioventricular (AV) nodal dysfunction in conscious mammals, we studied transgenic (TG) mice with cardiac-specific overexpression of the A1 adenosine receptor (A1AR). A Holter ECG was recorded in seven freely moving littermate pairs of mice during normal activity, exercise (5 min of swimming), and 1 h after exercise. TG mice had lower maximal heart rates (HR) than wild-type (WT) mice (normal activity: 437 +/- 18 vs. 522 +/- 24 beats/min, P < 0.05; exercise: 650 +/- 13 vs. 765 +/- 28 beats/min, P < 0.05; 1 h after exercise: 588 +/- 18 vs. 720 +/- 12 beats/min, P < 0.05; all values are means +/- SE). Mean HR was lower during exercise (589 +/- 16 vs. 698 +/- 34 beats/min, P < 0.05) and after exercise (495 +/- 16 vs. 592 +/- 27 beats/min, P < 0.05). Minimal HR was not different between genotypes. HR variability (SD of RR intervals) was reduced by 30% (P < 0.05) in TG compared with WT mice. Pertussis toxin (n = 4 pairs, 150 microg/kg ip) reversed bradycardia after 48 h. TG mice showed first-degree AV nodal block (PQ interval: 42 +/- 2 vs. 37 +/- 2 ms, P < 0.05), which was diminished but not abolished by pertussis toxin. Isolated Langendorff-perfused TG hearts developed spontaneous atrial arrhythmias (3 of 6 TG mice vs. 0 of 9 WT mice, P < 0.05). In conclusion, A1AR regulate sinus nodal and AV nodal function in the mammalian heart in vivo. Enhanced expression of A1AR causes sinus nodal and AV nodal dysfunction and supraventricular arrhythmias.     

Attenuated renovascular constrictor responses to angiotensin II in adenosine 1 receptor knockout mice

In the present experiments we examined the renovascular constrictor effects of ANG II in the chronic and complete absence of A1 adenosine receptors (A1AR) using mice with targeted deletion of the A1AR gene. Glomerular filtration rate (GFR) was not different between A1AR +/+ and A1AR -/- mice under control conditions (450.5 +/- 60 vs. 475.2 +/- 62.5 microl/min) but fell significantly less in A1AR -/- mice during infusion of ANG II at 1.5 ng/min (A1AR +/+: 242 +/- 32.5 microl/min, A1AR -/-: 371 +/- 42 microl/min; P = 0.03). Bolus injection of 1, 10, and 100 ng of ANG II reduced renal blood flow and increased renal vascular resistance significantly more in A1AR +/+ than in A1AR -/- mice. Perfused afferent arterioles isolated from A1AR +/+ mice constricted in response to bath ANG II with an EC50 of 1.5 +/- 0.4 x 10(-10) mol/l, whereas a right shift in the dose-response relationship with an EC50 of 7.3 +/- 1.2 x 10(-10) mol/l (P < 0.05) was obtained in arterioles from A1AR -/- mice (P < 0.05). The expression of AT1A receptor mRNA was not different in kidney RNA from A1AR +/+ or A1AR -/- mice. We conclude that chronic A1AR deficiency diminishes the effectiveness of ANG II to constrict renal resistance vessels and to reduce GFR.