Role of nitric oxide and adenosine in control of coronary blood flow in exercising dogs.

BACKGROUND: Inhibition of nitric oxide (NO) synthesis results in very little change in coronary blood flow, but this is thought to be because cardiac adenosine concentration increases to compensate for the loss of NO vasodilation. Accordingly, in the present study, adenosine measurements were made before and during NO synthesis inhibition during exercise. METHODS AND RESULTS: Experiments were performed in chronically instrumented dogs at rest and during graded treadmill exercise before and during inhibition of NO synthesis with N(omega)-nitro-L-arginine (L-NNA, 35 mg/kg IV). Before inhibition of NO synthesis, myocardial oxygen consumption increased approximately 3.7-fold, and coronary blood flow increased approximately 3.2-fold from rest to the highest level of exercise, and this was not changed by NO synthesis inhibition. Coronary venous oxygen tension was modestly reduced by L-NNA at all levels of myocardial oxygen consumption. However, the slope of the relationship between myocardial oxygen consumption and coronary venous oxygen tension was not altered by L-NNA. Inhibition of NO synthesis did not increase coronary venous plasma or estimated interstitial adenosine concentration. During exercise, estimated interstitial adenosine remained well below the threshold concentration necessary for coronary vasodilation before or after L-NNA. CONCLUSIONS: NO causes a modest coronary vasodilation at rest and during exercise but does not act as a local metabolic vasodilator. Adenosine does not mediate a compensatory local metabolic coronary vasodilation when NO synthesis is inhibited.

Electrode and cuvette for rapid continuous CO2 tension and pH measurement in blood.

An electrode and cuvette system has been developed for the continuous and rapid measurement of either blood CO2 tension or pH. The CO2 electrode consists of a 1.5-mm-diameter flat-tip glass pH electrode covered by a film of carbonic anhydrase solution, over which a 25-micron-thick dimethyl silicone membrane is attached. Porous ceramic filled with 20% polyacrylamide, equilibrated with a salt solution, serves as a salt bridge between a Ag-AgCl reference electrode and the pH electrode surface. The electrode is housed in a four-port cuvette assembly. Blood from a vessel of interest is delivered to the cuvette by means of an occlusive roller pump. The cuvette maintains the electrode and blood at a constant temperature and directs a continuous jet of blood against the electrode surface. The cuvette also allows for easy and frequent calibration of the electrode with either gas or liquid standards. The 90% response time of the CO2 electrode is 3.0 s for liquids and 1.3 s for gases. Removal of the dimethyl silicone membrane and carbonic anhydrase film yields a pH electrode that can continuously measure blood pH with a 90% response time of 1.6 s.

Feedforward sympathetic coronary vasodilation in exercising dogs.

The hypothesis that exercise-induced coronary vasodilation is a result of sympathetic activation of coronary smooth muscle beta-adrenoceptors was tested. Ten dogs were chronically instrumented with a flow transducer on the circumflex coronary artery and catheters in the aorta and coronary sinus. During treadmill exercise, coronary venous oxygen tension decreased with increasing myocardial oxygen consumption, indicating an imperfect match between myocardial blood flow and oxygen consumption. This match was improved after alpha-adrenoceptor blockade with phentolamine but was significantly worse than control after alpha + beta-adrenoceptor blockade with phentolamine plus propranolol. The response after alpha-adrenoceptor blockade included local metabolic vasodilation plus a beta-adrenoceptor vasodilator component, whereas the response after alpha + beta-adrenoceptor blockade contained only the local metabolic vasodilator component. The large difference in coronary venous oxygen tensions during exercise between alpha-adrenoceptor blockade and alpha + beta-adrenoceptor blockade indicates that there is significant feedforward beta-adrenoceptor coronary vasodilation in exercising dogs. Coronary venous and estimated myocardial interstitial adenosine concentrations did not increase during exercise before or after alpha + beta-adrenoceptor blockade, indicating that adenosine levels did not increase to compensate for the loss of feedforward beta-adrenoceptor-mediated coronary vasodilation. These results indicate a meaningful role for feedforward beta-receptor-mediated sympathetic coronary vasodilation during exercise.

Quantitative analysis of feedforward sympathetic coronary vasodilation in exercising dogs.

Recent experiments demonstrate that feedforward sympathetic beta-adrenoceptor coronary vasodilation occurs during exercise. The present study quantitatively examined the contributions of epinephrine and norepinephrine to exercise coronary hyperemia and tested the hypothesis that circulating epinephrine causes feedforward beta-receptor-mediated coronary dilation. Dogs (n = 10) were chronically instrumented with a circumflex coronary artery flow transducer and catheters in the aorta and coronary sinus. During strenuous treadmill exercise, myocardial oxygen consumption increased by approximately 3.9-fold, coronary blood flow increased by approximately 3.6-fold, and arterial plasma epinephrine concentration increased by approximately 2.4-fold over resting levels. At arterial concentrations matching those during strenuous exercise, epinephrine infused at rest (n = 6) produced modest increases (18%) in flow and myocardial oxygen consumption but no evidence of direct beta-adrenoceptor-mediated coronary vasodilation. Arterial norepinephrine concentration increased by approximately 5. 4-fold during exercise, and coronary venous norepinephrine was always higher than arterial, indicating norepinephrine release from cardiac sympathetic nerves. With the use of a mathematical model of cardiac capillary norepinephrine transport, these norepinephrine concentrations predict an average interstitial norepinephrine concentration of approximately 12 nM during strenuous exercise. Published dose-response data indicate that this norepinephrine concentration increases isolated coronary arteriolar conductance by approximately 67%, which can account for approximately 25% of the increase in flow observed during exercise. It is concluded that a significant portion of coronary exercise hyperemia ( approximately 25%) can be accounted for by direct feedforward beta-adrenoceptor coronary vascular effects of norepinephrine, with little effect from circulating epinephrine.

Myoglobin oxygen dissociation by multiwavelength spectroscopy.

Multiwavelength optical spectroscopy was used to determine the oxygen-binding characteristics for equine myoglobin. Oxygen-binding relationships as a function of oxygen tension were determined for temperatures of 10, 25, 35, 37, and 40 degrees C, at pH 7.0. In addition, dissociation curves were determined at 37 degrees C for pH 6.5, 7.0, and 7.5. Equilibration was achieved with a myoglobin solution, at the desired temperature and pH, and 16 oxygen-nitrogen gas mixtures of known oxygen fraction. Correction for the inevitable presence of metmyoglobin was made by using a three-component least squares analysis and by correcting the end point oxymyoglobin spectra for the presence of metmyoglobin. The PO2 at which myoglobin is half-saturated with O2 (P50) was determined to be 2.39 Torr at pH 7.0 and 37 degrees C. The myoglobin dissociation curve was well fit by the Hill equation [saturation = PO2/(PO2 + P50)].

Role of K(ATP)(+) channels and adenosine in the control of coronary blood flow during exercise.

The present study was designed to examine the role of ATP-sensitive potassium (K(ATP)(+)) channels during exercise and to test the hypothesis that adenosine increases to compensate for the loss of K(ATP)(+) channel function and adenosine inhibition produced by glibenclamide. Graded treadmill exercise was used to increase myocardial O(2) consumption in dogs before and during K(ATP)(+) channel blockade with glibenclamide (1 mg/kg iv), which also blocks adenosine mediated coronary vasodilation. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous values by using a previously tested mathematical model (Kroll K and Stepp DW. Am J Physiol Heart Circ Physiol 270: H1469-H1483, 1996). Coronary venous O(2) tension was used as an index of the balance between O(2) delivery and myocardial O(2) consumption. During control exercise, myocardial O(2) consumption increased approximately 4-fold, and coronary venous O(2) tension fell from 19 to 14 Torr. After K(ATP)(+) channel blockade, coronary venous O(2) tension was decreased below control vehicle values at rest and during exercise. However, during exercise with glibenclamide, the slope of the line of coronary venous O(2) tension vs. myocardial O(2) consumption was the same as during control exercise. Estimated interstitial adenosine concentration with glibenclamide was not different from control vehicle and was well below the level necessary to overcome the 10-fold shift in the adenosine dose-response curve due to glibenclamide. In conclusion, K(ATP)(+) channel blockade decreases the balance between resting coronary O(2) delivery and myocardial O(2) consumption, but K(ATP)(+) channels are not required for the increase in coronary blood flow during exercise. Furthermore, interstitial adenosine concentration does not increase to compensate for the loss of K(ATP)(+) channel function.

Alteration of cardiovascular and neuronal function in M1 knockout mice.

We used gene targeting to generate mice lacking the M1 muscarinic acetylcholine receptor. These mice exhibit a decreased susceptibility to pilocarpine-induced seizures, loss of regulation of M-current potassium channel activity and of a specific calcium channel pathway in sympathetic neurons, a loss of the positive chronotropic and inotropic responses to the novel muscarinic agonist McN-A-343, and impaired learning in a hippocampal-dependent test of spatial memory.

Adenosine coronary vasodilation during hypoxia depends on adrenergic receptor activation.

The adenosine hypothesis of coronary control was investigated during steady-state hypoxia by making measurements of coronary venous and epicardial well adenosine concentrations in adrenergically intact dogs and animals with alpha - and beta-receptor blockade. The unexpected result was that a role for adenosine coronary vasodilation during hypoxia could only be found when adrenergic receptors were intact, but not during adrenergic blockade.

Coronary autoregulation.

Autoregulation of coronary blood flow is complicated because the heart provides the blood flow and pressure for its own perfusion. Aortic pressure is not only the perfusion pressure for the coronary circulation, but is also the afterload for the left ventricle. Coronary autoregulation has therefore been studied when the coronary circulation is cannulated and perfused separately from the aorta. Even then, changes in coronary artery pressure result in alterations in myocardial metabolism due to the Gregg effect. Local metabolic vascular control appears to be the dominant factor in coronary autoregulation. If myocardial metabolism is enhanced, coronary autoregulation occurs at a higher level of flow. The balance between myocardial oxygen supply and demand is critical for coronary autoregulation, since good autoregulation is only observed when the coronary venous oxygen tension is near the normal value of about 20 mmHg. At present there is little evidence for a myogenic mechanism of coronary autoregulation, and adenosine also does not seem to be involved. It is concluded that coronary autoregulation is predominantly due to a local metabolic mechanism, but the substance that mediates the control is unknown.

Adrenergic control of transmural coronary blood flow.

Tachycardia and an increase in myocardial metabolism result from the sympathetic activation that occurs during baroreceptor reflexes, emotion, and exercise. Paradoxically, a concomitant adrenergic alpha-receptor-mediated coronary vasoconstriction competes with the local metabolic coronary vasodilation that occurs during these conditions, and thereby limits metabolic hyperemia. Measurements of transmural blood flow in alpha-receptor blocked and alpha-receptor intact regions of the left ventricle during exercise demonstrate that adrenergic vasoconstriction helps maintain blood flow to the vulnerable subendocardium during tachycardia. This may be the explanation as to why paradoxical adrenergic coronary vasoconstriction has evolved. During controlled conditions of constant coronary flow, an anti-transmural steal effect due to adrenergic vasoconstriction in the subepicardium can be demonstrated during ischemic conditions. These observations demonstrate unexpected beneficial effects of adrenergic coronary vasoconstriction during tachycardia and cardiovascular stress.

Parasympathetic control of coronary blood flow.

Active parasympathetic coronary vasodilation in excess of any changes in myocardial metabolism has been observed in a number of circumstances. Electrical stimulation of the cardiac end of the cut vagus nerve produces a cholinergic coronary vasodilation that is blocked by atropine. Activation of carotid body chemoreceptors, carotid sinus baroreceptors, or left ventricular receptors elicits reflex parasympathetic coronary vasodilation. The coronary vasodilation produced by these reflexes can be prevented by vagotomy or atropine. The relative importance of parasympathetic coronary control in relation to sympathetic and local metabolic coronary control awaits further research.

Reflex parasympathetic coronary vasodilation elicited from cardiac receptors in the dog.

Veratrum alkaloids injected into the coronary circulation stimulate myocardial receptors to produce reflex bradycardia and arterial hypotension (the Bezold-Jarisch reflex). This study investigated the hypothesis that parasympathetic coronary vasodilation occurs as part of the Bezold-Jarisch reflex. Blood flow in the circumflex coronary artery was measured in chloralose-anesthetized, closed-chest dogs with a newly developed cannula-tip flow transducer. Alpha-receptor blockade with Dibozane (2 mg/kg) was used to prevent peripheral vasodilation, and beta-receptor blockade with propranolol (1 mg/kg) was used to prevent adrenergic cardiac effects. Electrical pacing was used to maintain a constant heart rate. Under these conditions, veratridine injected into the anterior descending coronary artery but not into the circumflex coronary artery produced a 63% increase in circumflex coronary blood flow and an 88% increase in diastolic coronary conductance. The effect was abolished when the reflex arc was interrupted by either vagotomy or atropine administration. It is concluded that a cardiocoronary reflex parasympathetic coronary vasodilation can be elicited by stimulating cardiac receptors with veratridine.

Control of myocardial oxygen tension by sympathetic coronary vasoconstriction in the dog.

The effect of sympathetic alpha-receptor coronary vasoconstriction on myocardial oxygen tension was studied in open- and closed-chest, chloralose-anesthetized dogs. Blood oxygen tension in the coronary sinus and blood flow in the circumflex coronary artery were continuously measured in a three-part experiment. With stimulation of the left stellate ganglion (15 Hz, 3 msec, 4-7 v, 90-second train) under vagotomy control conditions (part 1), heart rate, blood pressure, and coronary blood flow increased, but coronary sinus oxygen tension decreased from 19 mm Hg to 15 mm Hg. In part 2, beta-receptor blockade with propranolol (2.0 mg/kg. iv) in the same dogs blunted the positive inotropic and chronotropic effects of sympathetic stimulation; coronary alpha-receptor vasoconstriction was unmasked, and coronary sinus blood oxygen tension fell from 17 mm Hg to 11 mm Hg. Since increases in oxygen metabolism were blunted, it appeared that the decrease in coronary sinus oxygen tension was caused by alpha-receptor coronary artery vasoconstriction. This hypothesis was tested in part 3 by the addition of alpha-receptor blockade with Dibozane (3.0 mg/kg, iv). Sympathetic stimulation no longer resulted in a change in either coronary vascular resistance or coronary sinus oxygen tension. These results indicate that the fall in oxygen tension during beta-receptor blockade in part 2 was due to alpha-receptor coronary vasoconstriction. Thus, myocardial oxygen tension may be regulated by coronary sympathetic vasomotion as well as by myocardial oxygen metabolism and local vascular control mechanisms.

Neural control of coronary blood flow.

Parasympathetic control of coronary blood flow has been extensively studied in dogs, and a clear vasodilator effect not dependent on changes in myocardial metabolism was observed. Parasympathetic vasodilatation is mediated via nitric oxide (EDRF) and is activated during carotid baroreceptor and chemoreceptor reflexes. Intracoronary infusions of acetylcholine in humans results in increased coronary blood flow and epicardial coronary artery dilatation except in atherosclerotic epicardial coronary vessels, which show a paradoxical vasoconstriction. Sympathetic alpha-adrenoceptor-mediated coronary vasoconstriction has been repeatedly demonstrated whenever there is adrenergic activation of the heart, as during exercise or a carotid sinus baroreceptor reflex in dogs or during a cold pressor reflex in humans. Recent evidence indicates that there is a beneficial effect of this paradoxical vasoconstrictor influence in that it helps preserve flow to the vulnerable inner layer of the left ventricle, but only when both heart rate and coronary flow are high. Beta-adrenoceptor-mediated coronary vasodilatation also occurs during adrenergic activation of the heart. The dominant site for beta-vasodilatation is in small arterioles, while the dominant site for alpha-vasoconstriction is in microvessels larger than approximately 100 microm diameter. The beta-adrenoceptor coronary vasodilatation is an example of feedforward open-loop control that complements the closed-loop negative feedback control by local metabolic factors. The combined feedback and feedforward control mechanism has the advantage of an excellent match between coronary blood flow and myocardial oxygen consumption with a rapid response time but without the instability inherent in high gain feedback systems.

Coronary physiology.

The major areas of normal coronary physiological research since Berne's 1964 review have been the measurement of ventricular transmural blood flow distribution with microspheres, the adenosine hypothesis of local metabolic control of coronary blood flow, and the autonomic control of coronary blood flow. There is an improved understanding of intramyocardial tissue pressure and extravascular compressive forces on coronary vessels. However, the unexpected finding of zero flow during a prolonged diastole with a coronary artery pressure of 40 mmHg (PZF) is a reminder that the physical forces, including vascular smooth muscle contraction, that determine coronary vascular resistance are incompletely understood. During normal circumstances, the left ventricular subendocardium probably receives more blood flow than the subepicardium does, but the difference is small. When the coronary circulation is compromised by stenosis or aortic valve lesions, the subendocardium is much more vulnerable to underperfusion than is the subepicardium. The coronary vasodilating effect of arterial hypoxia has been confirmed in many studies, but the role of tissue oxygen tension in local metabolic control of coronary blood flow during normoxia is unknown. The coronary vasodilating action of carbon dioxide has received renewed attention, but its role in local control is also unknown. The adenosine hypothesis has passed several critical tests, but despite much research the importance of adenosine in normal coronary regulation is not established. Local metabolic control of coronary blood flow probably involves more than just one factor, but a unified hypothesis has not been put forward. Sympathetic alpha-receptor-mediated coronary vasoconstriction has been demonstrated by nerve stimulation and during a carotid sinus baroreceptor reflex. Sympathetic coronary vasoconstriction is capable of competing with local metabolic control to lower coronary venous oxygen tension under experimental circumstances, but its importance during normal resting conditions is not established. Parasympathetic muscarinic coronary vasodilation has been shown by vagal nerve stimulation, but a role for it during normal blood flow regulation has yet to be demonstrated. There have been elegant descriptive studies of the coronary blood flow response during excitement and exercise, where coronary blood flow increases pari passu with myocardial metabolism; however, there are also data that indicate a concomitant sympathetic vasoconstrictor effect during strenuous exercise. Overall there has been encouraging progress in coronary physiology. Inevitably new knowledge has focused old questions and presented new ones.

Adrenergic vasoconstriction lessens transmural steal during coronary hypoperfusion.

The hypothesis that alpha-receptor-mediated coronary vasoconstriction can lessen transmural steal during hypoperfusion was tested in 14 open-chest, chloralose-anesthetized dogs. Coronary flow to two regions of the left ventricle was controlled by separately cannulating and pump perfusing the anterior descending and circumflex coronary arteries. An intracoronary infusion of the alpha-receptor blocking agent phenoxybenzamine (0.25 mg/kg) was administered to one region while coronary sinus blood flow was trapped to minimize recirculation. All animals received atropine (0.5 mg/kg) and propranolol (2 mg/kg) intravenously. alpha-Receptor activation in both regions of the left ventricle was achieved with an intracoronary infusion of norepinephrine. Radioactive microspheres were used to measure the transmural distribution of myocardial blood flow as coronary flow in the paired regions was reduced from 100 to 80, 70, 60, and 50% of normal. When total coronary flow was reduced sufficiently to cause ischemia and maldistribution of flow across the myocardial wall, the subendocardial blood flow was greater in the alpha-receptor-intact region than in the alpha-blocked region. This indicates that alpha-receptor-mediated coronary vasoconstriction has an unexpected beneficial effect that lessens transmural steal during hypoperfusion.

Technique for producing heart block in closed-chest dogs without electrical recording.

An instrument and technique have been devised for the production of complete atrioventricular heart block in anesthetized, closed-chest animals. The instrument is inserted into the right atrium via the right external jugular vein. Localization of the atrioventricular node region by the visualization of intracardiac landmarks with fluoroscopy makes electrical recording unnecessary. Discrete injections of a radiopaque necrosing solution produces the heart block. Procedure time is under 30 min a success rate of 90%.

Effect of coronary artery pressure on transmural distribution of adrenergic coronary vasoconstriction in the dog.

The transmural distribution of alpha-receptor coronary vasoconstriction was studied in 14 closed-chest, morphine- and chloralose-anesthesized dogs. The alpha-receptor-blocking agent, phenoxybenzamine (0.25 mg/kg), was infused into either the anterior or circumflex coronary artery while collection of coronary sinus blood minimized recirculation. The left main coronary artery was cannulated and perfused at constant pressure. Myocardial lactate extraction and blood flow (9 micron radioactive microspheres) were measured during adrenergic activation with intracoronary norepinephrine (2 micrograms/min) at coronary artery pressures of 100, 70, 50, and 38 mm Hg. Flow was 15-25% less in the control region (alpha-receptors intact) than in the alpha-receptor-blocked region, in all layers of the left ventricular wall, at coronary pressures of 100 and 70 mm Hg. When coronary pressure was 50 mm Hg, lactate production resulted (-4% extraction) and significant alpha-receptor vasoconstriction was observed in the outer layer, but was marginal (P = 0.043) in the inner layer, of the left ventricle. Flows were not different in control and alpha-receptor-blocked regions at a coronary pressure of 38 mm Hg (lactate extraction -49%). These data indicate a uniform transmural alpha-receptor vasoconstriction at normal coronary artery pressures that diminished as the heart was progressively underperfused.

Adenosine is unimportant in controlling coronary blood flow in unstressed dog hearts.

The adenosine hypothesis of local metabolic control of coronary blood flow was tested in the unstressed heart with adenosine deaminase, which converts adenosine to nonvasoactive inosine. If adenosine is normally an important physiological regulator, then adenosine deaminase should lower coronary blood flow. The left main coronary artery was perfused at constant pressure in anesthetized, closed-chest dogs. Adenosine deaminase was deposited in one region of the left ventricle by selective infusion into a branch of the left coronary artery. Coronary blood flow measured with radioactive microspheres was not lower in the region treated with adenosine deaminase than flow measured simultaneously in an untreated control region of the same heart. This finding is contrary to the prediction of the adenosine hypothesis. Coronary vasodilation elicited by intracoronary adenosine infusion was inhibited in the adenosine deaminase-treated region compared with the control region, indicating that adenosine deaminase lowered adenosine concentration at the vascular adenosine receptor. Inhibition of exogenous adenosine vasodilation was fully reversed by intracoronary infusion of a specific inhibitor of adenosine deaminase. Measurement of adenosine deaminase activity in cardiac lymph provided evidence that adenosine deaminase reached the myocardial interstitial space. These results demonstrate that introducing adenosine deaminase into the interstitial space of the unstressed heart did not lower coronary blood flow. This finding indicates that adenosine is normally below the vasoactive threshold and therefore is not important in mediating local metabolic control of blood flow in the unstressed heart.

Carotid baroreceptor reflex coronary vasodilation in the dog.

The hypothesis that neurally mediated coronary vasodilation occurs as part of the carotid baroreceptor reflex was investigated. The left main coronary artery was cannulated and perfused at constant pressure (100 mm Hg) in closed-chest, chloralose-anesthetized dogs. The heart was paced at a constant rate between 60 and 70 beats/min after atrioventricular heart block. Propranolol (1 mg/kg) was given to prevent beta-receptor-mediated alterations in contractility. Aortic blood pressure was stabilized with a blood reservoir. The aortic depressor nerves were cut bilaterally to prevent the buffering influence of aortic arch baroreceptors on the carotid baroreceptor reflex. The carotid sinuses were vascularly isolated and perfused with arterial blood at controlled pressures. Under these conditions, a step change in carotid sinus pressure from 70 to 210 mm Hg produced a 0.29 ml/min per g increase in coronary flow above control and a 10 mm Hg increase in coronary sinus blood oxygen tension. A step in carotid sinus pressure from 70 to 150 mm Hg resulted in a flow increase of 0.13 ml/min per g and a coronary sinus oxygen tension increase of 5.3 mm Hg relative to prestimulation values. Atropine (0.5 mg/kg, iv) blocked most of the reflex coronary vasodilation, indicating a parasympathetic component, and the addition of adrenergic alpha-receptor blockade with phenoxybenzamine (0.25 mg/kg, ic) abolished the remaining response, demonstrating sympathetic participation. The reflex nature of the coronary response was confirmed with carotid sinus denervation and vagotomy. It is concluded that carotid sinus hypertension results in a graded reflex neural coronary vasodilation independent of myocardial metabolic factors. The major component is due to activation of parasympathetic coronary vasodilator fibers, but there is also inhibition of sympathetic vasoconstrictor fibers.