Adenosine formation and energy metabolism: a 31P-NMR study in isolated rat heart JOHN P. HEADRICK AND ROGER J. WILLIS Division of Science and Technology, Griffith University, HEADRICK, JOHN P., AND ROGERJ. WILLIS. Adenosine formation and energy metabolism:a 31P-NMR study in i&c&d rat heart. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H617H624, 1990.-Temporal and quantitative relations between cytosolic energy metabolism, adenosineefflux, and coronary flow were examined during 10 min of isoproterenol (ISO) infusion (60 nM) or hypoxia (5% 02) in isolated isovolumic rat heart. Myocardial metabolismwasmonitored using 3fP-nuclear magneticresonancespectroscopy,and venous effluent wascollected and assayedfor adenosine.During IS0 infusion, coronary flow increasedto ~170%, and [ATP]/[ADP] [Pi] (cytosolic phosphorylation potential) declined to ~25% of preinfusion levels, respectively (P < 0.001). During hypoxia, coronary flow increasedto 190%,and [ATP]/[ADP] [Pi] declined to ~25% of normoxic levels (P < 0.001). Releaseof adenosine into the coronary venous effluent increased X0-fold and displayed significant inverse linear correlations with log[ATP]/[ADP] [Pi] and positive linear correlations with free cytosolic [AMP] and coronary flow during IS0 infusion and hypoxia. Adenosine deaminase(ADA) treatment reducedcoronary vasodilation by ~30% during IS0 infusion and 40% during hypoxia (P c 0.001) and augmentedchronotropic and inotropic responsesto IS0 infusion (P < 0.01). Infusion of ADA potentiated changesin [ATP]/[ADP] [Pi] and [AMP] observed during IS0 infusion and hypoxia (P c 0.05). These results indicate that 1) endogenousadenosinemediatesmetabolic vasodilation in the heart, 2) adenosinemodulatesthe responseof isolated myocardium to catecholamines,3) myocardial adenosineformation appears to be linked to cytosolic metabolism via changesin [ATP]/ [ADP] [Pi] and [AMP], and 4) endogenousadenosineprovides a significant, metabolically beneficial action in isolated hearts during hypoxia and inotropic stimulation.
adenosine5’-monophosphate;cytosolic phosphorylation potential; efficiency; hypoxia; isoproterenol; metabolic vasodilation
ADENOSINE INDUCES CORONARY VASODILATION and is
released from myocardial tissue in response to alterations in the supply of or the metabolic demand for oxygen (2, 3, 27). It is proposed that adenosine is responsible for metabolic regulation of coronary blood flow (3, 23, 27). Evidence supporting this includes the ability of adenosine antagonists and adenosine deaminase to reduce metabolic coronary vasodilation (14, 19, 24, 25) and the existence of consistent parallels between coronary flow, adenosine release, and myocardial oxygen consumption (MVO~) (2, 3, 13, 16). The coronary efflux of adenosine appears to be linked to the metabolic status of the myocardium (5, 16, 23) and can be correlated with changes in the cytosolic phosphorylation potential ([ATP]/[ADP] [Pi]) (13, 27) or adenylate energy charge
Nathan
4111 QLD, Australia
([ATP] + l/2 [ADP]),‘([ATP] + [ADP] + [AMP]), where brackets designate concentration (32). Although some recent studies support a link among adenosine efflux, [ATP]/[ADP] [Pi], and coronary flow (13, 16), alternate studies indicate that coronary flow is not strictly dependent on adenosine efflux during hypoxia (19, 24) or metabolic stimulation (14), or after ischemia (25). It has also been observed that adenosine deaminase treatment has no effect on coronary vasodilation during hypoxia or during reductions in perfusion pressure (10, 12). Moreover, there is some evidence that changes in coronary flow associated with reductions in [ATP]/ [ADP] [Pi] are not mediated by adenosine, since adenosine efflux can be dissociated from the changes in [ATP] /[ADPI [pi] (21,22)* The purpose of the present study was to examine relations between adenosine release, coronary flow, [ATPl/IADPl ipi and cytosolic free [AMP] in isolated rat heart stimulated with the ,&agonist isoproterenol or subjected to hypoxic perfusion (5% 02). Furthermore, since no studies to date have examined the metabolic effects of endogenous adenosine, the effects of adenosine deaminase treatment on cytosolic metabolism were examined. 31P-nuclear magnetic resonance (31P-NMR) spectroscopy methodology was used, allowing noninvasive determination of changes in cytosolic metabolism with a time resolution of 120 s. Adenosine deaminase was employed to unmask any possible physiological and metabolic actions endogenous adenosine might have in the hypoxic and stimulated hearts. MATERIALS AND METHODS
Heart perfusion. Male Wistar rats weighing 300-350 g were anesthetized with pentobarbital sodium (100 mg/ kg ip). Hearts were rapidly excised and arrested in icecold saline. The aorta was cannulated for perfusion at a constant pressure of 100 mmHg. The perfusion medium was a Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.75 CaC12, 1.2 MgS04, 25 NaHC03, 11 glucose, and 0.5 EDTA. The buffer was equilibrated with a 95% 02-5% CO2 gas mixture and maintained at 37”C, giving a pH of 7.4. Isovolumic left ventricular developed pressure was monitored with a thin-walled latex balloon inserted into the ventricle via the mitral valve. Thebesian drainage was vented with an apical drain (polyethylene tube 1.5 mm 00). The ventricular balloon was attached to a pressure transducer (Gould Statham P23 ID) via a water-filled line, and heart rate and left ventricular de-
0363-6135/90 $1.50Copyright 0 1990the American Physiological Society
H617
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (139.080.135.089) on November 28, 2018. Copyright © 1990 the American Physiological Society. All rights reserved.
H618
ENERGY
METABOLISM
AND ADENOSINE
FORMATION
veloped pressure (LVDP) were monitored continuously on a Gilson 5/6 multichannel recorder. Coronary flow was measured volumetrically every 1 min. Perfusate and right atria1 O2 tension (~1 OJml perfusate) were monitored continuously with a Clarke O2 electrode. MVO~ was calculated as
CrP
I
a-ATP y-ATP
/sATP
Pi
MVO~ (~1 O2 min-’ l
. g-‘)
= flow rate X (perfusate O2 - effluent
02)
Coronary venous effluent was collected every 1 min and analyzed for adenosine using the high-performance liquid chromatography (HPLC) method of Sellevold et al. (26) as described previously (14, 15). Hearts were equilibrated for a period of 30 min before experimentation. Experimental protocol. Hearts were placed inside the NMR apparatus in a 20.mm glass NMR sample tube modified for perfusion work (4, 7, 8). Hearts were subjected to two experimental protocols: isoproterenol infusion with and without adenosine deaminase, and hypoxia with and without adenosine deaminase. For the isoproterenol study, equilibrated hearts were infused with a final perfusate concentration of 60 nM isoproterenol (Sigma) for a period of 10 min. Isoproterenol was prepared as a stock in perfusion fluid containing 0.4 mM ascorbic acid to prevent oxidation. The stock solution was infused into the aortic cannula at rates of 9O% of the enzyme was present as subunit size, as described previously (14, 15). 31P-NA4R spectroscopy. Isolated hearts were perfused inside a Bruker VSP-300 broad-band probe seated in the bore of a 7.05 superconducting magnet. All experiments were performed using a Bruker CXP-300 spectrometer at 121.47 MHz, as described previously (7, 8). The homogeneity of the magnetic field was controlled to maximize the heart and perfusate proton signal resolution using a 13-channel Bruker shim supply. Line width was
adenosine5’-monophosphate;cytosolic phosphorylation potential; efficiency; hypoxia; isoproterenol; metabolic vasodilation
ADENOSINE INDUCES CORONARY VASODILATION and is
released from myocardial tissue in response to alterations in the supply of or the metabolic demand for oxygen (2, 3, 27). It is proposed that adenosine is responsible for metabolic regulation of coronary blood flow (3, 23, 27). Evidence supporting this includes the ability of adenosine antagonists and adenosine deaminase to reduce metabolic coronary vasodilation (14, 19, 24, 25) and the existence of consistent parallels between coronary flow, adenosine release, and myocardial oxygen consumption (MVO~) (2, 3, 13, 16). The coronary efflux of adenosine appears to be linked to the metabolic status of the myocardium (5, 16, 23) and can be correlated with changes in the cytosolic phosphorylation potential ([ATP]/[ADP] [Pi]) (13, 27) or adenylate energy charge
Nathan
4111 QLD, Australia
([ATP] + l/2 [ADP]),‘([ATP] + [ADP] + [AMP]), where brackets designate concentration (32). Although some recent studies support a link among adenosine efflux, [ATP]/[ADP] [Pi], and coronary flow (13, 16), alternate studies indicate that coronary flow is not strictly dependent on adenosine efflux during hypoxia (19, 24) or metabolic stimulation (14), or after ischemia (25). It has also been observed that adenosine deaminase treatment has no effect on coronary vasodilation during hypoxia or during reductions in perfusion pressure (10, 12). Moreover, there is some evidence that changes in coronary flow associated with reductions in [ATP]/ [ADP] [Pi] are not mediated by adenosine, since adenosine efflux can be dissociated from the changes in [ATP] /[ADPI [pi] (21,22)* The purpose of the present study was to examine relations between adenosine release, coronary flow, [ATPl/IADPl ipi and cytosolic free [AMP] in isolated rat heart stimulated with the ,&agonist isoproterenol or subjected to hypoxic perfusion (5% 02). Furthermore, since no studies to date have examined the metabolic effects of endogenous adenosine, the effects of adenosine deaminase treatment on cytosolic metabolism were examined. 31P-nuclear magnetic resonance (31P-NMR) spectroscopy methodology was used, allowing noninvasive determination of changes in cytosolic metabolism with a time resolution of 120 s. Adenosine deaminase was employed to unmask any possible physiological and metabolic actions endogenous adenosine might have in the hypoxic and stimulated hearts. MATERIALS AND METHODS
Heart perfusion. Male Wistar rats weighing 300-350 g were anesthetized with pentobarbital sodium (100 mg/ kg ip). Hearts were rapidly excised and arrested in icecold saline. The aorta was cannulated for perfusion at a constant pressure of 100 mmHg. The perfusion medium was a Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.75 CaC12, 1.2 MgS04, 25 NaHC03, 11 glucose, and 0.5 EDTA. The buffer was equilibrated with a 95% 02-5% CO2 gas mixture and maintained at 37”C, giving a pH of 7.4. Isovolumic left ventricular developed pressure was monitored with a thin-walled latex balloon inserted into the ventricle via the mitral valve. Thebesian drainage was vented with an apical drain (polyethylene tube 1.5 mm 00). The ventricular balloon was attached to a pressure transducer (Gould Statham P23 ID) via a water-filled line, and heart rate and left ventricular de-
0363-6135/90 $1.50Copyright 0 1990the American Physiological Society
H617
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (139.080.135.089) on November 28, 2018. Copyright © 1990 the American Physiological Society. All rights reserved.
H618
ENERGY
METABOLISM
AND ADENOSINE
FORMATION
veloped pressure (LVDP) were monitored continuously on a Gilson 5/6 multichannel recorder. Coronary flow was measured volumetrically every 1 min. Perfusate and right atria1 O2 tension (~1 OJml perfusate) were monitored continuously with a Clarke O2 electrode. MVO~ was calculated as
CrP
I
a-ATP y-ATP
/sATP
Pi
MVO~ (~1 O2 min-’ l
. g-‘)
= flow rate X (perfusate O2 - effluent
02)
Coronary venous effluent was collected every 1 min and analyzed for adenosine using the high-performance liquid chromatography (HPLC) method of Sellevold et al. (26) as described previously (14, 15). Hearts were equilibrated for a period of 30 min before experimentation. Experimental protocol. Hearts were placed inside the NMR apparatus in a 20.mm glass NMR sample tube modified for perfusion work (4, 7, 8). Hearts were subjected to two experimental protocols: isoproterenol infusion with and without adenosine deaminase, and hypoxia with and without adenosine deaminase. For the isoproterenol study, equilibrated hearts were infused with a final perfusate concentration of 60 nM isoproterenol (Sigma) for a period of 10 min. Isoproterenol was prepared as a stock in perfusion fluid containing 0.4 mM ascorbic acid to prevent oxidation. The stock solution was infused into the aortic cannula at rates of 9O% of the enzyme was present as subunit size, as described previously (14, 15). 31P-NA4R spectroscopy. Isolated hearts were perfused inside a Bruker VSP-300 broad-band probe seated in the bore of a 7.05 superconducting magnet. All experiments were performed using a Bruker CXP-300 spectrometer at 121.47 MHz, as described previously (7, 8). The homogeneity of the magnetic field was controlled to maximize the heart and perfusate proton signal resolution using a 13-channel Bruker shim supply. Line width was
