Harer Huang

Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.

To determine the effects of acute myocardial infarction on the regulation of angiotensin II (Ang II) receptors and contractile performance of left and right ventricular myocytes, coronary artery ligation was surgically induced in rats, and Ang II receptor density and affinity and the mechanical properties of surviving muscle cells were examined 1 week later. Physiological determinations of cardiac pump function revealed the presence of ventricular failure, which was associated at the cellular level with a depression in the velocity of myocyte shortening and relengthening, a prolongation of time to peak shortening, and a reduction in the extent of cell shortening. These abnormalities in single-cell function were more prominent in left than in right ventricular myocytes. Cellular hypertrophy was documented by increases in cell length and width, which were also greater in the spared myocytes of the infarcted left ventricle. Reactive hypertrophy was accompanied by a 1.84- and 1.85-fold increase in the density of Ang II receptors on left and right myocytes, respectively. On the other hand, the affinity of Ang II receptors for the radiolabeled antagonist was not altered. However, Ang II-stimulated phosphoinositol turnover was enhanced by 3.7- and 2.5-fold in left and right myocytes, respectively, after infarction. Ventricular myocytes were found to possess the AT1 receptor subtype exclusively. In conclusion, myocardial infarction leads to impairment in the contractile behavior of the remaining cells and to the activation of Ang II receptors and effector pathway associated with these receptors, which may be involved in the reactive growth adaptation of the viable myocytes.

A Defect of Neuronal Nitric Oxide Synthase Increases Xanthine Oxidase-Derived Superoxide Anion and Attenuates the Control of Myocardial Oxygen Consumption by Nitric Oxide Derived From Endothelial Nitric Oxide Synthase

Endothelial nitric oxide synthase (eNOS) plays an important role in the control of myocardial oxygen consumption (MVO2) by nitric oxide (NO). A NOS isoform is present in cardiac mitochondria and it is derived from neuronal NOS (nNOS). However, the role of nNOS in the control of MVO2 remains unknown. MVO2 in left ventricular tissues from nNOS−/− mice was measured in vitro. Stimulation of NO production by bradykinin or carbachol induced a significant reduction in MVO2 in wild-type (WT) mice. In contrast to WT, bradykinin- or carbachol-induced reduction in MVO2 was attenuated in nNOS−/−. S-methyl-l-thiocitrulline, a potent isoform selective inhibitor of nNOS, had no effect on bradykinin-induced reduction in MVO2 in WT. Bradykinin-induced reduction in MVO2 in eNOS−/− mice, in which nNOS still exists, was also attenuated. The attenuated bradykinin-induced reduction in MVO2 in nNOS−/− was restored by preincubation with Tiron, ascorbic acid, Tempol, oxypurinol, or SB203850, an inhibitor of p38 kinase, but not apocynin. There was an increase in lucigenin-detectable superoxide anion (O2−) in cardiac tissues from nNOS−/− compared with WT. Tempol, oxypurinol, or SB203850 decreased O2− in all groups to levels that were not different from each other. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOS−/− compared with WT mice. These results indicate that a defect of nNOS increases O2− through the activation of xanthine oxidase, which is mediated by the activation of p38 kinase, and attenuates the control of MVO2 by NO derived from eNOS.

Coronary Microvascular Endothelial Stunning After Acute Pressure Overload in the Conscious Dog Is Caused by Oxidant Processes The Role of Angiotensin II Type 1 Receptor and NAD(P)H Oxidase

Background—Few studies have examined the effect of acute pressure overload on endothelial function in the coronary microcirculation. Methods and Results—In instrumented conscious dogs with heart rate held constant, veratrine caused a cholinergic nitric oxide (NO)–dependent increase in coronary blood flow by 23±3 mL/min (Bezold-Jarisch reflex). Ten minutes after release of constriction of the ascending aorta to increase left ventricular (LV) systolic pressure to 214±5 mm Hg for 30 minutes, the veratrine-induced increase in coronary blood flow (7±1 mL/min) was reduced by 66% and remained depressed for 2 hours (ie, endothelial stunning [ES]). Nitrite production from isolated coronary microvessels during ES was not different from normal. Ascorbic acid (AA), losartan, or apocynin prevented ES. Myocardial oxygen consumption (M&OV0312;o2) of LV tissue was measured in vitro in response to bradykinin with preincubation of angiotensin II for 30 minutes. Bradykinin (10−4 mol/L)–induced reduction in M&OV0312;o2 was reversed in a concentration-dependent manner by angiotensin II (38±1% versus 19±2% at 10−8 mol/L) and restored by coincubation of AA (37±2%), tempol (33±2%), losartan (34±2%), or apocynin (36±1%). Exogenous NO-induced reduction in M&OV0312;o2 was not altered by angiotensin II. Angiotensin II increased lucigenin-detectable superoxide anion in LV tissue in a manner that was inhibited by bradykinin, AA, tempol, losartan, or apocynin. Conclusions—Endothelial stunning is caused by oxidant processes inhibited by ascorbate, and the activation of NAD(P)H oxidase by increased angiotensin II plays an important role in this process.

Hyperhomocysteinemia, a Cardiac Metabolic Disease: Role of Nitric Oxide and the p22phox Subunit of NADPH Oxidase

Background—Hyperhomocysteinemia (HHcy) is a reliable indicator of cardiovascular disease, in part because of the production of superoxide and scavenging of nitric oxide (NO). The present study assessed the impact of HHcy on the NO-dependent control of cardiac O2 consumption and examined enzymatic sources of superoxide. Methods and Results—Rats and mice were fed methionine in drinking water for 5 to 9 weeks to increase plasma homocysteine, a process that did not cause significant changes in hemodynamic function. The ability of the NO agonists bradykinin and carbachol to reduce myocardial O2 consumption in vitro was impaired by ≈40% in methionine-fed rats, and this impairment was proportional to their individual plasma homocysteine concentration. However, responses were restored in the presence of ascorbic acid, tempol, and apocynin, which inhibits NADPH oxidase assembly. Western blots showed no difference in Cu/Zn or Mn superoxide dismutase, endothelial NO synthase, or inducible NO synthase protein, but HHcy caused a 100% increase in the p22phox subunit of NADPH oxidase. Western blots with plasma membrane–enriched fractions of cell lysate detected elevated levels of p22phox, p67phox, and rac-1, which indicates increased oxidase assembly. Finally, mice lacking a functional gp91phox subunit of NADPH oxidase demonstrated normal NO-dependent regulation of myocardial O2 consumption after methionine feeding. Conclusions—In HHcy, superoxide produced by NADPH oxidase reduces the ability of NO to regulate mitochondrial function in the myocardium. The severity of this effect is proportional to the increase in homocysteine.

Impaired Regulation of Renal Oxygen Consumption in Spontaneously Hypertensive Rats

Abnormalities of nitric oxide (NO) and oxygen radical synthesis and of oxygen consumption have been described in the spontaneously hypertensive rat (SHR) and may contribute to the pathogenesis of hypertension. NO plays a role in the regulation of renal oxygen consumption in normal kidney, so the response of renal cortical oxygen consumption to stimulators of NO production before and after the addition of the superoxide scavenging agent tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) was studied. Baseline cortical oxygen consumption was similar in SHR and Wistar-Kyoto (WKY) rats (SHR: 600 +/- 55 nmol O(2)/min per g, WKY: 611 +/- 51 nmol O(2)/min per g, P > 0.05). Addition of bradykinin, enalaprilat, and amlodipine decreased oxygen consumption significantly less in SHR than WKY (SHR: bradykinin -13.9 +/- 1.9%, enalaprilat -15.3 +/- 1.6%, amlodipine -11.9 +/- 0.7%; WKY: bradykinin -22.8 +/- 1.0%, enalaprilat -24.1 +/- 2.0%, amlodipine -20.7 +/- 2.3%; P < 0.05), consistent with less NO effect in SHR. Addition of tempol reversed the defects in responsiveness to enalaprilat and amlodipine, suggesting that inactivation of NO by superoxide contributes to decreased NO availability. The response to an NO donor was similar in both groups and was unaffected by the addition of tempol. These results demonstrate that NO availability in the kidney is decreased in SHR, resulting in increased oxygen consumption. This effect is due to enhanced production of superoxide in SHR. By lowering intrarenal oxygen levels, reduced NO may contribute to susceptibility to injury and renal fibrosis. Increasing NO production, decreasing oxidant stress, or both might prevent these changes by improving renal oxygenation.

Oxidant Stress Leads to Impaired Regulation of Renal Cortical Oxygen Consumption by Nitric Oxide in the Aging Kidney

Structural and functional changes occur in the kidney with aging. Previous studies have suggested that loss of nitric oxide production contributes to these changes. The authors therefore explored regulation of renal cortical oxygen consumption, a nitric oxide mediated effect, in tissue from Fischer 344 rats at different ages (4, 13, and 23 mo) to characterize changes in renal nitric oxide production with age. Bradykinin, enalaprilat, and amlodipine significantly suppressed cortical oxygen consumption in 4-mo-old rats (bradykinin: -2.5 +/- 0.9% to -21 +/- 1.5%; enalaprilat: -0.7 +/- 0.5% to -26 +/- 1.2%; amlodipine: -1.3 +/- 0.9% to -18 +/- 1.2%; P < 0.05). Similar results were obtained in 13-mo-old animals. However, in 23-mo-old animals, the responses to bradykinin and enalaprilat were attenuated (bradykinin: 0 +/- 0% to -13 +/- 0.9%; enalaprilat: -0.3 +/- 0.3% to -17 +/- 2.1%; P < 0.05), whereas the response to an NO donor was unaffected, suggesting decreased bioavailability of NO. Addition of the superoxide radical scavenger tempol restored the ability of bradykinin, enalaprilat, and amlodipine to suppress oxygen consumption in tissue from 23-mo-old animals to levels seen in younger animals, suggesting NO destruction by superoxide as the reason for decreased NO availability. Apocynin, an inhibitor of NAD(P)H oxidase, similarly restored the ability of all three drugs to suppress oxygen consumption, suggesting NAD(P)H oxidase as the enzyme responsible for enhanced superoxide production in aging. Levels of eNOS protein, assessed by immunoblotting, did not change significantly with age. These results suggest that NO availability is decreased in the aging kidney and that this is due to scavenging of NO by superoxide produced by NAD(P)H oxidase. Oxidant stress, by depleting NO, may contribute to the structural and hemodynamic changes characteristic of the aging kidney.

Modulation of renal oxygen consumption by nitric oxide is impaired after development of congestive heart failure in dogs.

We investigated the role of nitric oxide (NO) in the modulation of renal O2 consumption in dogs with pacing-induced congestive heart failure (CHF). O2 consumption in the renal cortex (C) and medulla (M) of normal dogs and dogs with CHF was measured under control conditions and in the presence of increasing concentrations of three stimulators of NO production, bradykinin, ramiprilat, and amlodipine, or the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). Baseline O2 consumption (nmol O2/min per gram) was similar in the CHF group (C: 637 ± 65; M: 618 ± 83) and the control group (C: 601 ± 58, M: 534 ± 55). In normal dogs, bradykinin (10−4 M), ramiprilat (10−4 M), amlodipine (10−5 M) and SNAP (10−4 M) all significantly reduced O2 consumption in the cortex (−31.5 ± 3.5%, −33 ± 2.5%, −28.4 ± 4.9%, −49.3 ± 3.1%) and medulla (−26.9 ± 2.2%, −31.4 ± 2.2%, −23.1 ± 1.3%, −48.3 ± 4%), respectively. The responses to bradykinin, ramiprilat and amlodipine were significantly attenuated in dogs with CHF (C: −22.2 ± 1.8%, −20.1 ± 2.6%, −14.2 ± 2.5%; M: −20.8 ± 1.7%, −17.8 ± 1.9%, −15.6 ± 2.6%, respectively; p < 0.05). The responses in dogs with CHF were not altered by NO synthase blockade with L-NAME (10−4 M). In contrast, in normal kidneys treatment with L-NAME significantly attenuated the response to all three stimuli of NO production. Responses to SNAP were not affected either by CHF or L-NAME. These data indicate that the role of NO production in the modulation of tissue O2 consumption in the kidney is impaired after the development of pacing-induced heart failure in dogs.