Manuel Ochoa

Role of nitric oxide in the regulation of oxygen consumption in conscious dogs.

The role of nitric oxide (NO) in the regulation of O2 consumption was studied in chronically instrumented conscious dogs. A specific NO synthesis inhibitor, nitro-L-arginine (NLA, 30 mg/kg i.v.), significantly increased mean arterial pressure from 100 +/- 4 to 134 +/- 5 mm Hg (mean +/- SEM) and total peripheral resistance by 157 +/- 16% and reduced cardiac output by 47 +/- 3% and heart rate by 34 +/- 6% after 120 minutes. Changes in arterial blood gases were not observed. There were significant changes in PO2 (-14 +/- 2 mm Hg), O2 saturation (-21 +/- 2%), the percentage of hemoglobin as oxyhemoglobin (-21 +/- 2%), and O2 content (-3.0 +/- 0.9 vol%) and a significant increase in percent reduced hemoglobin (21 +/- 1%) in mixed venous blood, associated with an increase in O2 extraction (5.1 +/- 0.2 vol%) (all P < .01). O2 consumption was increased from 124 +/- 6 to 155 +/- 9 mL/min (P < .05). Methoxamine, titrated to have hemodynamic effects similar to those of NLA (eg, mean arterial pressure increased from 97 +/- 4 to 131 +/- 5 mm Hg), had much smaller effects on venous blood gases, hemoglobin, and O2 extraction (2.3 +/- 0.7 vol%) and no significant effect on O2 consumption. NLA also caused an increase in O2 consumption of 37 +/- 8% (P < .01) in quietly resting conscious dogs that had undergone pretreatment with hexamethonium and atropine, but no significant change in O2 consumption in dogs anesthetized with barbiturate.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Nitric Oxide in the Control of Renal Oxygen Consumption and the Regulation of Chemical Work in the Kidney

Inhibition of NO synthesis has recently been shown to increase oxygen extraction in vivo, and NO has been proposed to play a significant role in the regulation of oxygen consumption by both skeletal and cardiac muscle in vivo and in vitro. It was our aim to determine whether NO also has such a role in the kidney, a tissue with a relatively low basal oxygen extraction. In chronically instrumented conscious dogs, administration of an inhibitor of NO synthase, nitro-L-arginine (NLA, 30 mg/kg i.v.), caused a maintained increase in mean arterial pressure and renal vascular resistance and a decrease in heart rate (all P<0.05). At 60 minutes, urine flow rate and glomerular flow rate decreased by 44+/-12% and 45+/-7%, respectively; moreover, the amount of sodium reabsorbed fell from 16+/-1.7 to 8.5+/-1.1 mmol/min (all P<0.05). At this time, oxygen uptake and extraction increased markedly by 115+/-37% and 102+/-34%, respectively (P<0.05). Oxygen consumption also significantly increased from 4.5+/-0.6 to 7.1+/-0.9 mL O2/min. Most important, the ratio of oxygen consumption to sodium reabsorbed increased dramatically from 0.33+/-0.07 to 0.75+/-0.11 mL O2/mmol Na+ (P<0.05), suggesting a reduction in renal efficiency for transporting sodium. In vitro, both a NO-donating agent and the NO synthase-stimulating agonist bradykinin significantly decreased both cortical and medullary renal oxygen consumption. In conclusion, NO plays a role in maintaining a balance between oxygen consumption and sodium reabsorption, the major ATP-consuming process in the kidney, in conscious dogs, and NO can inhibit mitochondrial oxygen consumption in canine renal slices in vitro.

Nitric oxide controls cardiac substrate utilization in the conscious dog

OBJECTIVES The aim of this study was to determine whether the acute inhibition of nitric oxide (NO) synthase causes changes in cardiac substrate utilization which can be reversed by a NO donor. METHODS NO synthase was blocked by giving 30 mg/kg of nitro-L-arginine (NLA) i.v. to 15 chronically instrumented dogs. Hemodynamics and blood samples from aorta and coronary sinus were taken at control and at 1 and 2 h after NLA. In five dogs, 0.4 mg/kg of the NO donor 3754 was given i.v. 1 h after NLA. In six dogs, angiotensin II was infused over 2 h (20-40 ng/kg/min) to mimic the hemodynamic effects of NLA. RESULTS Two h after NLA: mean arterial pressure was 153 +/- 4 mmHg; MVO2 increased by 38%; cardiac uptake of lactate and glucose increased, respectively, from 20.0 +/- 5.0 to 41.0 +/- 9.3 mumol/min and from 1.1 +/- 0.7 to 6.8 +/- 1.5 mg/min (all P < 0.05 vs. control). Cardiac uptake of free fatty acids decreased by 43% after 1 h (P < 0.05) and returned to control values at 2 h. Cardiac respiratory quotient increased from 0.76 +/- 0.03 to 1.05 +/- 0.07, indicating a shift to carbohydrate oxidation. All these changes were reversed by the NO donor. In the dogs receiving angiotensin II infusion, MVO2 increased by 28% and lactate uptake doubled (both P < 0.05), but no other metabolic changes where observed. CONCLUSIONS The acute inhibition of NO synthase by NLA causes a switch from fatty acids to lactate and glucose utilization by the heart which can be reversed by a NO donor, suggesting an important regulatory action of NO on cardiac metabolism.

Selective Impairment of Vagally Mediated, Nitric Oxide–Dependent Coronary Vasodilation in Conscious Dogs After Pacing-Induced Heart Failure

BACKGROUND Activation in conscious dogs of the carotid chemoreflex or cardiac receptors results in coronary vasodilation that is mediated by a vagal cholinergic mechanism. Our previous study showed that the coronary vasodilation following activation of carotid chemoreflex is also mediated by nitric oxide (NO). In addition, NO production is depressed after the development of heart failure. Therefore, we hypothesized that the coronary vasodilation after activation of reflexes that elicit efferent vagal coronary vasodilation would be blunted in conscious dogs after pacing-induced heart failure due to the disappearance of NO. METHODS AND RESULTS Mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and left circumflex coronary blood flow (CBF). Without the heart rate controlled, intra-atrial injection of veratrine (4 micrograms/kg) caused bradycardia (-36 +/- 3 beats per minute). With the heart rate controlled, veratrine increased CBF in a dose-dependent manner: for example, 4 micrograms/kg of veratrine increased CBF by 54 +/- 5% from 38 +/- 4.9 mL/min (P < .05). The increases in CBF induced by veratrine were markedly blunted by nitro-L-arginine (NLA). Activation of carotid chemoreflex by nicotine increased CBF by 121 +/- 9% from 32 +/- 4 mL++/min (P < .05) with the heart rate controlled and caused bradycardia (-32 +/- 5 beats per minute) without the heart rate controlled. After the development of heart failure, in response to activation of carotid chemoreflex or cardiac receptors the coronary vasodilation was almost abolished (CBF increased by only 23 +/- 8% or 11 +/- 3%, P < .05 compared with control). There still was a marked bradycardia after injections of nicotine or veratrine (-50 +/- 11 or -48 +/- 7 beats per minute). CONCLUSIONS Our results indicate that vagally mediated coronary vasodilation is selectively attenuated in conscious dogs after pacing-induced heart failure, whereas the vagally mediated bradycardia is preserved. Since muscarinic receptor-induced coronary vasodilation is mediated by NO, the disappearance of NO from blood vessels leads to a defect in the integrated neural regulation of coronary blood flow and myocardial function during heart failure.

Reduced coronary NO production in conscious dogs after the development of alloxan-induced diabetes.

The role of nitric oxide (NO) in the control of coronary blood flow (CBF) during the development of diabetes is unknown. To study this, mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and CBF. With heart rate controlled (150 beats/min), veratrine (1-10 micrograms/kg) caused dose-dependent increases in CBF; e.g., 5 mirograms/kg of veratrine increased CBF by 57 +/- 7% from 41 +/- 1.3 ml/min (P < 0.05). The dogs developed diabetes 4-5 wk after injection of alloxan (40-60 mg/kg iv, blood glucose levels were 384 +/- 18 mg/dl). After diabetes the same doses of veratrine caused smaller increases in CBF; i.e., 5 micrograms/kg of veratrine increased CBF by 32 +/- 2% (P < 0.05 compared with control) from 28 +/- 4 ml/min. ACh- and adenosine-induced coronary vasodilation were reduced after diabetes as well. In anesthetized dogs after diabetes, vagal stimulation caused smaller increases in CBF. ACh and bradykinin caused smaller increases in NO(-)(2) production in coronary microvessels from diabetic dogs. Furthermore, despite the fact that mRNA for endothelial cell NO synthase from the aorta was increased twofold with the use of Northern blotting, the protein for aortic endothelial constitutive NO synthase was reduced by 66% after diabetes, as determined by Western blotting. Our results indicate that the NO-dependent coronary vasodilation by the Bezold-Jarisch reflex is impaired in conscious dogs after diabetes. The mechanism responsible for the impaired endothelium-dependent coronary vasodilation is most likely the decreased release of NO from the endothelium.The role of nitric oxide (NO) in the control of coronary blood flow (CBF) during the development of diabetes is unknown. To study this, mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and CBF. With heart rate controlled (150 beats/min), veratrine (1-10 μg/kg) caused dose-dependent increases in CBF; e.g., 5 μg/kg of veratrine increased CBF by 57 ± 7% from 41 ± 1.3 ml/min ( P < 0.05). The dogs developed diabetes 4-5 wk after injection of alloxan (40-60 mg/kg iv, blood glucose levels were 384 ± 18 mg/dl). After diabetes the same doses of veratrine caused smaller increases in CBF; i.e., 5 μg/kg of veratrine increased CBF by 32 ± 2% ( P < 0.05 compared with control) from 28 ± 4 ml/min. ACh- and adenosine-induced coronary vasodilation were reduced after diabetes as well. In anesthetized dogs after diabetes, vagal stimulation caused smaller increases in CBF. ACh and bradykinin caused smaller increases in[Formula: see text] production in coronary microvessels from diabetic dogs. Furthermore, despite the fact that mRNA for endothelial cell NO synthase from the aorta was increased twofold with the use of Northern blotting, the protein for aortic endothelial constitutive NO synthase was reduced by 66% after diabetes, as determined by Western blotting. Our results indicate that the NO-dependent coronary vasodilation by the Bezold-Jarisch reflex is impaired in conscious dogs after diabetes. The mechanism responsible for the impaired endothelium-dependent coronary vasodilation is most likely the decreased release of NO from the endothelium.

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.

Caffeine attenuates the duration of coronary vasodilation and changes in hemodynamics induced by regadenoson (CVT-3146), a novel adenosine A2A receptor agonist.

Effects of caffeine on regadenoson-induced coronary vasodilation and changes in hemodynamics were examined in conscious dogs. Sixteen dogs were chronically instrumented for measurements of coronary blood flow (CBF), mean arterial pressure (MAP), and heart rate (HR). Regadenoson (5 μg/kg, IV) increased CBF from 34 ± 2 to 191 ± 7 mL/min. The duration of the 2-fold increase in CBF was 515 ± 71 seconds. Regadenoson decreased MAP by 15 ± 2% and increased HR by 114 ± 14%. Regadenoson-induced maximum increases in CBF were not significantly lower in the presence of caffeine at 1, 2, 4, and 10 mg/kg (2 ± 3, 0.7 ± 3, 16 ± 5, and 13 ± 8%, respectively; all P > 0.05). Caffeine at 1, 2, 4, and 10 mg/kg significantly decreased the duration of the 2-fold increase in CBF induced by regadenoson by 17% ± 4%, 48% ± 8%, 62% ± 5%, and 82% ± 5%, respectively (all P < 0.05). Caffeine at 4 and 10 mg/kg significantly attenuated the effects of regadenoson on MAP and HR. The results indicate that 1 to 10 mg/kg caffeine dose-dependently reduced the duration, but not the peak increase of CBF caused by 5 μg/kg regadenoson.

Antiadrenergic and Hemodynamic Effects of Ranolazine in Conscious Dogs

Effects of ranolazine alone and in the presence of phenylephrine (PE) or isoproterenol (ISO) on hemodynamics, coronary blood flow and heart rate (HR) in the absence and presence of hexamethonium (a ganglionic blocker) were studied in conscious dogs. Ranolazine (0.4, 1.2, 3.6, and 6 mg/kg, intravenous) alone caused transient (<1 minute) and reversible hemodynamic changes. PE (0.3-10 μg/kg) caused a dose-dependent increase in blood pressure and decrease in HR. ISO (0.01-0.3 μg/kg) caused a dose-dependent decrease in blood pressure and an increase in HR. Ranolazine at high (11-13 mM), but not at moderate (4-5 mM) concentrations partially attenuated changes in mean arterial blood pressure and HR caused by either PE or ISO in normal conscious dogs. However, in dogs treated with hexamethonium (20 mg/kg) to cause autonomic blockade, ranolazine (both 4-5 and 11-13 μM) significantly attenuated both the PE- and ISO-induced changes in mean arterial blood pressure. The results suggest that a potential antiadrenergic effect of ranolazine was masked by autonomic control mechanisms in conscious dogs but could be observed when these mechanisms were inhibited (eg, in the hexamethonium-treated dog). Ranolazine, at plasma concentrations <10 μM and in conscious dogs with intact autonomic regulation, had minimal antiadrenergic (α and β) effects.

Relationship between plasma NOx and cardiac and vascular dysfunction after LPS injection in anesthetized dogs

The relationship between plasma nitrite, nitrate, and nitric oxide (NOx), cytokines, and cardiac and vascular dysfunction after lipopolysaccharide (LPS) was studied in chronically instrumented anesthetized dogs. LPS was administered (1 mg/kg iv), and hemodynamics were recorded at baseline, every 15 min for 1 h, and every hour for an additional 14 h. Dramatic reductions in mean arterial pressure (-48 ± 6%), cardiac output (-40 ± 8%), stroke volume (-42 ± 9%), and first derivative of left ventricular pressure (LV dP/d t, -38 ± 7%) were seen within 1 h after injection of endotoxin. Cardiac output was not different from control by 9 h, whereas mean arterial pressure (-19 ± 7%), stroke volume (-32 ± 8%), and LV dP/d t (-21 ± 10%) remained significantly depressed from control. Total peripheral resistance was not significantly different from control. Therefore, the hypotension appears to be due to a reduction in cardiac function and not to vasodilation. Levels of plasma NOx were not different from control until 4 h after LPS reached levels 597 ± 126% higher than control at 15 h. In vitro production of nitrite by coronary microvessels was also elevated, supporting our in vivo findings. In contrast, production of tumor necrosis factor-α and interleukin-6 occurred shortly after endotoxin injection, reaching peak levels at 45 and 150 min, respectively. Our data suggest that inducible nitric oxide synthase induction occurred after LPS injection. It is unlikely that nitric oxide contributed significantly to the hypotension and cardiac dysfunction early in our study, whereas cardiodepressive cytokines, particularly tumor necrosis factor-α, may be important. In contrast, the hemodynamic effects seen late after injection of endotoxin may be the result of an overproduction of nitric oxide, since there was a sixfold increase in plasma NOx levels at this time and a marked production of nitric oxide in isolated coronary microvessels in vitro.The relationship between plasma nitrite, nitrate, and nitric oxide (NOx), cytokines, and cardiac and vascular dysfunction after lipopolysaccharide (LPS) was studied in chronically instrumented anesthetized dogs. LPS was administered (1 mg/kg i.v.), and hemodynamics were recorded at baseline, every 15 min for 1 h, and every hour for an additional 14 h. Dramatic reductions in mean arterial pressure (-48 +/- 6%), cardiac output (-40 +/- 8%), stroke volume (-42 +/- 9%), and first derivative of left ventricular pressure (LV dP/dt, -38 +/- 7%) were seen within 1 h after injection of endotoxin. Cardiac output was not different from control by 9 h, whereas mean arterial pressure (-19 +/- 7%), stroke volume (-32 +/- 8%), and LV dP/dt (-21 +/- 10%) remained significantly depressed from control. Total peripheral resistance was not significantly different from control. Therefore, the hypotension appears to be due to a reduction in cardiac function and not to vasodilation. Levels of plasma NOx were not different from control until 4 h after LPS reached levels 597 +/- 126% higher than control at 15 h. In vitro production of nitrite by coronary microvessels was also elevated, supporting our in vivo findings. In contrast, production of tumor necrosis factor-alpha and interleukin-6 occurred shortly after endotoxin injection, reaching peak levels at 45 and 150 min, respectively. Our data suggest that inducible nitric oxide synthase induction occurred after LPS injection. It is unlikely that nitric oxide contributed significantly to the hypotension and cardiac dysfunction early in our study, whereas cardiodepressive cytokines, particularly tumor necrosis factor-alpha, may be important. In contrast, the hemodynamic effects seen late after injection of endotoxin may be the result of an overproduction of nitric oxide, since there was a sixfold increase in plasma NOx levels at this time and a marked production of nitric oxide in isolated coronary microvessels in vitro.

Short-term Exercise Training Enhances Reflex Cholinergic Nitric Oxide–Dependent Coronary Vasodilation in Conscious Dogs

The effects of exercise training on the coronary vasodilation following activation of the Bezold-Jarisch reflex were examined in conscious dogs. Mongrel dogs were chronically instrumented using sterile techniques for measurements of systemic hemodynamics and left circumflex coronary blood flow (CBF). With the heart rate controlled (150 bpm), veratrine (0.5 to 20 micrograms/kg) caused dose-dependent increases in CBF; eg, 5 micrograms/kg of veratrine increased CBF by 61 +/- 6% from 31 +/- 1.3 mL/min (P < .05). After exercise training, the dose-response curve of CBF in response to veratrine was shifted to the left; eg, 5 micrograms/kg of veratrine increased CBF by 101 +/- 12% (P < .05 compared with control) from 34 +/- 2.3 mL/min. The enhanced coronary vasodilation was blunted by nitro-L-arginine (NLA, 35 mg/kg). In anesthetized dogs after exercise training, electrical stimulation of the left vagus nerve caused greater increases in CBF, and NLA inhibited increases in CBF. Acetylcholine, norepinephrine, angiotensin II, and bradykinin caused greater increases in NO2- production in coronary microvessels from exercise-trained dogs compared with those from normal dogs. Our results indicate that the coronary vasodilation following activation of the Bezold-Jarisch reflex is enhanced in conscious dogs after exercise training. Since electrical stimulation of the vagus nerve caused greater coronary vasodilation and since the agonists resulted in greater increases in NO production in coronary microvessels from exercise-trained dogs, the mechanism responsible for the enhanced coronary vasodilation following activation of the Bezold-Jarisch reflex is most likely due to the increased release of NO from the endothelial cells.