Control of myocardial oxygen consumption in transgenic mice overexpressing vascular eNOS.

Our objective was to investigate the potential role of selective endothelial nitric oxide (NO) synthase (eNOS) overexpression in coronary blood vessels in the control of myocardial oxygen consumption (MVO2). Transgenic (Tg) eNOS-overexpressing mice (eNOS Tg) (n=22) and wild-type (WT) mice (n=24) were studied. Western blot analysis indicated greater than sixfold increase of eNOS in cardiac tissue. Echocardiography in awake mice indicated no difference in cardiac function between WT and eNOS Tg; however, systolic pressure in eNOS Tg mice decreased significantly (126 +/- 2.3 to 109 +/- 2.3 mmHg; P <0.05), whereas heart rate (HR) was not different. Total peripheral resistance (TPR) was also decreased (9.8 +/- 0.8 to 7.6 +/- 0.4 4 mmHg.ml(-1).min; P <0.05) in eNOS Tg. Furthermore, female eNOS Tg mice showed even lower TPR (7.2 +/- 0.4 mmHg.ml(-1).min) compared with male eNOS mice (8.6 +/- 0.5, mmHg.ml.min(-1); P <0.05). Left ventricular slices were isolated from WT and eNOS Tg mice. With the use of a Clark-type oxygen electrode in an airtight bath, MVO2 was determined as the percent decrease during increasing doses (10(-10) to 10(-4) mol/l) of bradykinin (BK), carbachol (CCh), forskolin (10(-12) to 10(-6) mol/l), or S-nitroso-N-acetyl penicillamine (SNAP; 10(-7) to 10(-4) mol/l). Baseline MVO2 was not different between WT (181 +/- 13 nmol.g(-1).min(-1)) and eNOS Tg (188 +/- 14 nmol.g(-1).min(-1)). BK decreased MVO2 (10(-4) mol/l) in WT by 17% +/- 1.1 and 33% +/- 2.7 in eNOS Tg (P < 0.05). CCh also decreased MVO2, 10(-4) mol/l, in WT by 20% +/- 1.7 and 31% +/- 2.0 in eNOS Tg (P <0.05). Forskolin (10(-6) mol/l) or SNAP (10(-4) mol/l) also decreased MVO2 in WT by 24% +/- 2.8 and 36% +/- 1.8 versus eNOS 31% +/- 1.8 and 37% +/- 3.5, respectively. N-nitro-L-arginine methyl ester (10(-3) mol/l) inhibited the MVO2 reduction to BK, CCh, and forskolin by a similar degree (P <0.05), but not to SNAP. Thus selective overexpression of eNOS in cardiac blood vessels in mice enhances the control of MVO2 by eNOS-derived NO.

Novel vascular biology of third-generation L-type calcium channel antagonists: ancillary actions of amlodipine.

Calcium channel blockers (CCBs) were developed as vasodilators, and their use in cardiovascular disease treatment remains largely based on that mechanism of action. More recently, with the evolution of second- and third-generation CCBs, pleiotropic effects have been observed, and at least some of CCBs' benefit is attributable to these mechanisms. Understanding these effects has contributed greatly to elucidating disease mechanisms and the rationale for CCB use. Furthermore, this knowledge might clarify why drugs are useful in some disease states, such as atherosclerosis, but not in others, such as heart failure. Although numerous drugs used in the treatment of vascular disease, including statins and angiotensin-converting-enzyme inhibitors, have well-described pleiotropic effects universally accepted to contribute to their benefit, little attention has been paid to CCBs' potentially similar effects. Accumulating evidence that at least 1 CCB, amlodipine, has pharmacologic actions distinct from L-type calcium channel blockade prompted us to investigate the pleiotropic actions of amlodipine and CCBs in general. There are several areas of research; foci here are (1) the physicochemical properties of amlodipine and its interaction with cholesterol and oxidants; (2) the mechanism by which amlodipine regulates NO production and implications; and (3) amlodipine's role in controlling smooth muscle cell proliferation and matrix formation.

Simvastatin reverses impaired regulation of renal oxygen consumption in congestive heart failure.

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) regulates renal O(2) consumption. This mechanism is impaired in heart and kidney of dogs with heart failure (CHF). Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, increases eNOS expression in the endothelium. Therefore, we studied whether simvastatin treatment could restore the regulation of renal O(2) consumption by stimulators of NO production in dogs with CHF. Renal O(2) consumption was measured after stimulation of NO production with bradykinin, ramiprilat, or amlodipine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP). Simvastatin delayed the time to euthanasia in dogs with CHF (35 +/- 1.0 vs. 29 +/- 1.2 days; P < 0.01). In normal dogs, bradykinin (10(-4) M), ramiprilat (10(-4) M), amlodipine (10(-5) M), and SNAP (10(-4) M) significantly reduced O(2) consumption in the renal cortex (-31.8 +/- 0.9, -30.3 +/- 1.1, -30.1 +/- 2.0, -46.9 +/- 1.0%) and renal medulla (-29.7 +/- 2.1, -33.0 +/- 2.7, -30.8 +/- 2.2, -46.8 +/- 1.1%). Responses to bradykinin, ramiprilat, and amlodipine were significantly attenuated in CHF but were partially or completely restored by simvastatin. Responses to SNAP were unaffected. These data demonstrate that treatment with simvastatin improves renal production of NO in CHF, restoring the normal regulation of renal O(2) consumption by NO.

Oxidative stress-mediated cardiac cell death is a major determinant of ventricular dysfunction and failure in dog dilated cardiomyopathy.

Cell death has been questioned as a mechanism of ventricular failure. In this report, we tested the hypothesis that apoptotic death of myocytes, endothelial cells, and fibroblasts is implicated in the development of the dilated myopathy induced by ventricular pacing. Accumulation of reactive oxygen products such as nitrotyrosine, potentiation of the oxidative stress response by p66(shc) expression, formation of p53 fragments, release of cytochrome c, and caspase activation were examined to establish whether these events were coupled with apoptotic cell death in the paced dog heart. Myocyte, endothelial cell, and fibroblast apoptosis was detected before indices of severe impairment of cardiac function became apparent. Cell death increased with the duration of pacing, and myocyte death exceeded endothelial cell and fibroblast death throughout. Nitrotyrosine formation and p66(shc) levels progressively increased with pacing and were associated with cell apoptosis. Similarly, p50 (DeltaN) fragments augmented paralleling the degree of cell death in the failing heart. Moreover, cytochrome c release and activation of caspase-9 and -3 increased from 1 to 4 weeks of pacing. In conclusion, cardiac cell death precedes ventricular decompensation and correlates with the time-dependent deterioration of function in this model. Oxidative stress may be critical for activation of apoptosis in the overloaded heart.

Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart.

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity in vivo is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20--30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.

cAMP signal transduction cascade, a novel pathway for the regulation of endothelial nitric oxide production in coronary blood vessels.

The aim of this study was to determine whether cAMP signal transduction plays a role in the regulation of endothelial nitric oxide (NO) production. Canine coronary blood vessels were isolated, and nitrite, the hydration product of NO, from these vessels was quantified by using the Griess reaction. Forskolin (10(-4) mol/L), 8-bromo-cAMP (10(-2) mol/L), or isoproterenol (10(-4) mol/L) significantly increased nitrite release to 168+/-10, 162+/-13, or 149+/-13 pmol/mg, respectively, from isolated coronary microvessels (all P<0.05; control, 86+/-3 pmol/mg). Adrenomedullin and calcitonin gene-related peptide (CGRP), both potent vasodilator peptides, also increased coronary microvascular nitrite production. N(omega)-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, or Rp-cAMP, a protein kinase A inhibitor, markedly blocked the nitrite release induced by these agents. Forskolin and adrenomedullin also potentiated coronary NO production induced by bradykinin. In large coronary arteries, removal of the endothelium eliminated nitrite production to both forskolin and acetylcholine. Our data demonstrate that stimulation of cAMP signal transduction can substantially increase coronary NO production, indicating that there is a cAMP-mediated, endothelial NO-forming system in coronary blood vessels. Because the cAMP signal cascade can be activated by CGRP or adrenomedullin and enhance kinin-mediated nitrite production, the cAMP-NO pathway may play an important role in the regulation of cardiovascular function.

Endothelial nitric oxide synthase plays an essential role in regulation of renal oxygen consumption by NO.

Nitric oxide (NO) regulates renal O2 consumption, but the source of NO mediating this effect is unclear. We explored the effects of renal NO production on O2 consumption using renal cortex from mice deficient (-/-) in endothelial (e) nitric oxide synthase (NOS). O2 consumption was determined polarographically in slices of cortex from control and eNOS-/- mice. NO production was stimulated by bradykinin (BK) or ramiprilat (Ram) in the presence or absence of an NOS inhibitor. Basal O2 consumption was higher in eNOS-/- mice than in heterozygous controls (919 +/- 46 vs. 1,211 +/- 133 nmol O(2). min(-1). g(-1); P < 0.05). BK and Ram decreased O2 consumption significantly less in eNOS-/- mice [eNOS-/-: BK -19.0 +/- 2.8%, Ram -20.5 +/- 3.3% at 10(-4) M; control: BK -29.5 +/- 2.5%, Ram -34 +/- 1.6% at 10(-4) M]. The NO synthesis inhibitor nitro-L-arginine methyl ester (L-NAME) attenuated this decrease in control but not eNOS-/- mice. An NO donor inhibited O2 consumption similarly in both groups independent of the presence of L-NAME. These results demonstrate that NO production by eNOS is responsible for regulation of renal O2 consumption in mouse 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-acetylpenicillamine (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.

Plasma nitrate accumulation during the development of pacing-induced dilated cardiac myopathy in conscious dogs is due to renal impairment.

Heart failure is associated with an increase in plasma nitrate and nitrite (NOx). To date there is still some controversy regarding the causes of nitrate accumulation during the development of heart failure. The goal of this study was to analyze the underlying mechanisms that cause accumulation of plasma nitrates during the development of heart failure in dogs. Dogs were chronically instrumented for measurement of hemodynamics and renal function. Hearts were paced initially at 210 bpm for 3 weeks and then at 240 until the development of heart failure. Hemodynamics, renal function, renal blood flow, arterial blood gases, hemoglobin, plasma and urine NOx levels, and creatinine levels were measured weekly. Heart failure was assessed by hemodynamic alterations, physical signs such as lethargy, ascites, cachexia, and postmortem evidence of cardiac hypertrophy. LVSP (from 127 +/- 3 to 106 +/- 3 mmHg), LV dP/dt (from 2658 +/- 173 to 1439 +/- 217 mmHg/s), MAP (from 101 +/- 1.9 to 83 +/- 1.8 mmHg) fell, whereas LVEDP tripled (from 6.4 +/- 0.9 to 20 +/- 2.6 mmHg), and heart rate rose (from 101 +/- 4.2 to 117 +/- 6.3 bpm), all changes P < 0.05. RBF (from 146 +/- 10 to 96 +/- 9.9 ml/min), urine output (V) (from 0.26 +/- 0.02 to 0.16 +/- 0.02 ml/min), GFR (from 63 +/- 1.8 to 49 +/- 2 ml/min), and Na excretion (from 45 +/- 4.5 to 14 +/- 4.6 microEq/min) all decreased (P < 0.05), whereas RVR increased (from 0.68 +/- 0.05 to 0.94 +/- 0.1 mmHg/ml/min). These changes took place during a rise in plasma NOx (from 3.7 +/- 0.5 to 16+/-3.3 microM), a decrease in urine NOx (from 33 +/- 9.9 to 8.1 +/- 4.9 microM), and a concurrent increase in NOx reabsorption (from 221 +/- 31 to 818 +/- 166 nmol/min). There was a direct correlation between the increase in plasma NOx levels and an increase in filtered load (r(2) = 0.97, P = 0.02), a negative correlation between NOx levels and NOx excretion (r(2) = 0.65 P < 0.09), and a direct correlation between plasma NOx levels and NOx reabsorption (r(2) = 0.97, P = 0.02). These results indicate that elevated plasma NOx during heart failure are most likely the result of an impairment of the renal function and not increased NOx production. Furthermore, without knowing changes in renal function the measurement of plasma NOx in and of itself is a meaningless index of NO formation.

Canine ventricular myocytes possess a renin-angiotensin system that is upregulated with heart failure.

Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT(1) receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II-mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT(1) and AT(2) receptors was evaluated by reverse transcriptase-polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT(1) receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT(1). Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT(1) were markedly increased in paced myocytes. Conversely, chymase and AT(2) proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.

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.

Impaired nitric oxide modulation of myocardial oxygen consumption in genetically cardiomyopathic hamsters.

We investigated the role of kinin and nitric oxide (NO) in the modulation of cardiac O(2)consumption in Syrian hamsters with overt heart failure (HF) and age-matched normal hamsters. Using echocardiography, the hamsters with heart failure had reduced ejection fraction [31(+/-8) v 76(+/-5)%] and LV dilation [4.9(+/-0. 2) v 5.7(+/-0.3) mm, both P<0.05 from normal]. O(2)consumption in the left ventricular free wall was measured using a Clark-type O(2)electrode in an air-tight chamber, containing Krebs solution buffered with Hepes (37 degrees C, pH 7.4). Concentration response curves to bradykinin (BK), ramiprilat (RAM), amlodipine (AMLO) and the NO donor, S -nitroso- N -acetyl-penicillamine (SNAP) were performed. Basal myocardial O(2)consumption was lower in the HF group compared to normal [316(+/-21) v 404(+/-36) nmol O(2)/min/g, respectively, P<0.05]. In the hearts from normal hamsters BK (10(-4)mol/l), RAM (10(-4)mol/l), and AMLO (10(-5)mol/l) all significantly reduced myocardial O(2)consumption by 42(+/-6)%, 29(+/-7)% and 27(+/-5)% respectively. This reduction was attenuated in the presence of N -nitro- l -arginine methyl ester (l -NAME) [BK: 3.3(+/-1.5)%, RAM: 3.3(+/-1.2)%, AMLO: 2.3(+/-1.2)%, P<0.05]. Interestingly in the hearts from HF group, BK, RAM and AMLO caused a significantly smaller reduction in myocardial O(2)consumption [10(+/-2)%, 2.5(+/-1.3)%, 6.3(+/-2.3)%, P<0.05]. In contrast, the NO donor SNAP reduced myocardial O(2)consumption in both groups and all those responses were not affected by l -NAME. These data indicate that endogenous NO production through the kinin-dependent mechanism is impaired at end-stage heart failure. The loss of kinin and NO control of mitochondrial respiration may contribute to the pathogenesis of heart failure.

Left ventricular assist device implantation augments nitric oxide dependent control of mitochondrial respiration in failing human hearts.

OBJECTIVES: The objective of the study was to evaluate nitric oxide (NO) mediated regulation of mitochondrial respiration after implantation of a mechanical assist device in end-stage heart failure. BACKGROUND: Ventricular unloading using a left ventricular assist device (LVAD) can improve mitochondrial function in end-stage heart failure. Nitric oxide modulates the activity of the mitochondrial electron transport chain to regulate myocardial oxygen consumption (MVO2). METHODS: Myocardial oxygen consumption was measured polarographically using a Clark-type oxygen electrode in isolated left ventricular myocardium from 26 explanted failing human hearts obtained at the time of heart transplantation. RESULTS: The rate of decrease in oxygen concentration was expressed as a percentage of baseline. Results of the highest dose of drug are shown. Decrease in MVO2 was greater in LVAD hearts (n = 8) compared with heart failure controls (n = 18) in response to the following drugs: bradykinin (-34+/-3% vs. -24+/-5%), enalaprilat (-37+/-5% vs. -23+/-5%) and amlodipine (-43+/-13% vs. -16+/-5%; p<0.05 from controls). The decrease in MVO2 in LVAD hearts was not significantly different from controls in response to diltiazem (-22+/-5% in both groups) and exogenous NO donor, nitroglycerin (-33+/-7% vs. -30+/-3%). N(w)-nitro-L-arginine methyl ester, inhibitor of NO synthase, attenuated the response to bradykinin, enalaprilat and amlodipine. Reductions in MVO2 in response to diltiazem and nitroglycerin were not altered by inhibiting NO. CONCLUSIONS: Chronic LVAD support potentiates endogenous NO-mediated regulation of mitochondrial respiration. Use of medical or surgical interventions that augment NO bioavailability may promote myocardial recovery in end-stage heart failure.

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).

Enhanced cAMP-induced nitric oxide-dependent coronary dilation during myocardial stunning in conscious pigs.

The goal of the current study was to determine the effects of cAMP-mediated coronary reactivity in conscious pigs with stunned myocardium induced by 1.5 h coronary stenosis (CS) and 12 h coronary artery reperfusion (CAR). Domestic swine (n = 5) were chronically instrumented with a coronary artery blood flow (CBF) probe, hydraulic occluder, left ventricular pressure gauge, wall-thickening crystals in the ischemic and nonischemic zones, and a coronary sinus catheter. The hydraulic occluder was inflated to induce a CS with a stable 38 +/- 1% reduction in CBF for 1.5 h. Before flow reduction and during CAR, cAMP-induced coronary vasodilation was investigated by forskolin (20 nmol. kg(-1). min(-1)). Enhanced CBF responses [+62 +/- 9%, P < 0.05, compared with pre-CS (+37 +/- 3%)] were observed for forskolin at 12 h after CAR as well as for bradykinin and reactive hyperemia. With the use of a similar protocol during systemic nitric oxide (NO) synthase inhibition with N(omega)-nitro-L-arginine (30 mg. kg(-1). day(-1) for 3 days), the enhanced CBF responses to forskolin, bradykinin, and reactive hyperemia were not observed after CS. Isolated microvessel preparations from pigs (n = 8) also demonstrated enhanced NO production to direct stimulation of adenylyl cyclase with forskolin (+71 +/- 12%) or NKH-477 (+60 +/- 10%) and administration of 8-bromo-cAMP (+74 +/- 13%), which were abolished by protein kinase A or NO synthase inhibition. These data indicate that cAMP stimulation elicits direct coronary vasodilation and that this action is amplified in the presence of sustained myocardial stunning after recovery from CS. This enhanced cAMP coronary vasodilation is mediated by an NO mechanism that may be involved in myocardial protection from ischemic injury.

Simvastatin acts synergistically with ACE inhibitors or amlodipine to decrease oxygen consumption in rat hearts.

Statin drugs, which are cholesterol-lowering agents, can upregulate endothelial nitric oxide synthase (eNOS) in isolated endothelial cells independent of lipid lowering. We investigated the effect of short-term simvastatin administration on NO-mediated regulation of myocardial oxygen consumption (MV(O2)) in tissue from rat hearts. Male Wistar rats were divided into (a) control group (n = 14), and (b) simvastatin group (n = 10, 20 mg/kg/day by oral gavage). After 2 weeks, left ventricular myocardium was isolated to measure MV(O2) using a Clark-type oxygen electrode, and aortic plasma nitrates and nitrites (NOx) were measured. Baseline plasma NOx levels (19+/-2.6 in control vs. 20+/-2.5 microM/L in simvastatin) and baseline MV(O2) (288+/-23 in control vs. 252+/-11 nmol/g/min; p = 0.09) were not significantly different between the two groups. NO-dependent regulation of MV(O2) in response to bradykinin, ramipril, or amlodipine was augmented in simvastatin rats compared with controls (p < 0.05). Decrease of MV(O2) from baseline in response to highest doses in control versus simvastatin groups was as follows-bradykinin, -28+/-5% vs. -44+/-6%; ramipril, -35+/-5% vs. -50+/-8%; and amlodipine, -32+/-9% vs. -42+/-3%. Response to highest dose of NO donor S-nitroso N-acetyl penicillamine (SNAP) was not significantly different in the two groups (-55+/-5% vs. -52+/-7%). Treatment with Nw-nitro-L-arginine methyl ester, inhibitor of NO synthesis, attenuated the effect of bradykinin, ramipril, and amlodipine on MV(O2) (p < 0.05). In conclusion, short-term administration of simvastatin in rats potentiates the ability of angiotensin-converting enzyme (ACE) inhibitors and amlodipine to cause NO-mediated regulation of MV(O2).

Depressed modulation of oxygen consumption by endogenous nitric oxide in cardiac muscle from diabetic dogs.

Our previous study indicated that nitric oxide (NO)-dependent coronary vasodilation was impaired in conscious dogs with diabetes. Our goal was to determine whether modulation of O(2) consumption by NO is depressed in canine cardiac muscle after diabetes. Diabetes was induced by injection of alloxan (40-60 mg/kg iv), dogs were killed after diabetes was induced (4-5 wk), and the cardiac muscle from the left ventricle was cut into 15- to 30-mg slices. O(2) uptake by the muscle slices was measured polarographically with a Clark-type O(2) electrode. S-nitroso-N-acetylpenicillamine decreased O(2) consumption in normal and diabetic tissues (10(-4) M, 61 +/- 7 vs. 61 +/- 8%, P > 0.05). Bradykinin (10(-4) M)- or carbachol (CCh, 10(-4) M)-induced inhibition of O(2) consumption was impaired in diabetic tissues (51 +/- 6 vs. 17 +/- 4% or 48 +/- 4 vs. 19 +/- 3%, respectively, both P < 0.05 compared with normal). The inhibition of O(2) consumption by kininogen or kallikrein was depressed in diabetic tissues as well. In coronary microvessels from diabetic dogs, bradykinin or ACh (10(-5) M) caused smaller increases in NO production than those from normal dogs. Our results indicate that the modulation of O(2) consumption by endogenous, but not exogenous, NO is depressed in cardiac muscle from diabetic dogs, most likely because of decreased release of NO from the vascular endothelium.

NO metabolites accumulate in erythrocytes in proportion to carbon dioxide and bicarbonate concentration.

It is not known whether the ratio between the concentrations of NO metabolites (NOx) in plasma (pNOx) and in erythrocytes (eNOx) is constant or correlates with chemical parameters of the blood. We measured pH, PO(2), and PCO(2) and calculated bicarbonate concentration in 19 blood samples from the aorta, coronary sinus, and leg veins of 7 dogs. Erythrocytes were then separated by centrifugation and lysed with distilled water, and the lysate was ultrafiltered with a molecular cutoff of 50 kDa to remove the hemoglobin. NOx were measured in plasma and in the ultrafiltrate. NOx concentration was higher in erythrocytes, with eNOx/pNOx ranging from 4.38 to 14.60. Linear and significant correlations were found between the natural logarithm of eNOx/pNOx and PCO(2) (r = 0.70, P < 0.001) or bicarbonate concentration (r = 0.72, P < 0.001). These results demonstrate, for the first time, that plasma NOx cannot be considered as a constant fraction of the total NOx in blood but varies dramatically in proportion to the CO(2)/bicarbonate concentration. To prevent an underestimation of venous-arterial difference of NOx across organs, NOx should be measured in whole blood.