The acute effects of AICAR on purine nucleotide metabolism and postischemic cardiac function

J

THORAC CARDIOVASC SURG

1988;95:286-93

The acute effects of AICAR on purine nucleotide metabolism and postischemic cardiac function The purine precursor AICAR (5-amino-4-imidazolecarboxamide) has been advocated as a substrate for myocardial adenine nucleotide repletion during postischemic reperfusion. The purpose of tbis study was to investigate the acute effects of tbis agent on adenine nucleotides, inosine monophosphate, and postischemic ventricular function in an isolated rat heart preparation. The hearts were perfused at constant flow, either continuously for 90 minutes or for a 30 minute period followed by 10 minutes of global normothermic (370 C) ischemia. The ischemic hearts were then reperfused for 15, 30, and 60 minutes. Both groups were treated with AICAR in a concentration of 100 ILmol/L throughout the perfusion protocols. In the nonischemic time control group there was no effect on the levels of adenosine nucleotides or developed pressure over 90 minutes of perfusion. In contrast, AICAR treatment increased tissue inosine monophosphate content fourfold and sevenfoldat 60 and 90 minutes, respectively (p < 0.05), but had no effect on tissue adenosine monophosphate levels. During ischemia, there was a 50 % decrease in adenosine triphosphate content in the AICAR-treated hearts and a tbirteenfold increase in adenosine monophosphate levels (p < 0.05). After 60 minutes of reperfusion, adenosine triphosphate and monophosphate levels in the AICAR-treated hearts recovered to only 52 % and 59 % of preischemic values, respectively. These findings were similar to those observed in the untreated ischemic hearts. In contrast, tissue inosine monophosphate content in the AICAR-treated hearts during reperfusion remained significantly elevated and was fivefold greater than the reperfusion values in the untreated group. Concurrently, AICAR failed to enhance the recovery of postischemic left ventricular developed pressure. These results suggest that inhibition of the conversion of inosine monophosphate to adenosine monophosphate limits the usefulness of the agent in evaluating the temporal relationships hetween postischemic adenosine triphosphate repletion and recovery of myocardial function in the acute setting.

Robert M. Mentzer, Jr., MD, Stephen W. Ely, MD, PhD, Robert D. Lasley, MS, and Robert M. Berne, MD, Charlottesville, Va.

h e association between depressed adenine triphosphate (ATP) tissue levels and delayed recovery of myocardial function during postischemic reperfusion suggests that the rate of ATP depletion may be an important determinant of the reversibility of postischemic myocardial injury. i·) Restoration of ATP levels From the Departments of Surgery and Physiology, University of Virginia School of Medicine, Charlottesville, Va. Supported by a grant from the American Heart Association, Virginia Affiliate. Dr. Robert Mentzer is the recipient of Research Career Development Award HL01299 from the National Heart, Lung, and Blood Institute. Received for publication Aug. 4, 1986. Accepted for publication Jan. 14, 1987. Address for reprints: Robert M. Mentzer, Jr., MD, 100 High St., Department of Surgery, State University of New York, Buffalo, NY 14203.

286

after ischemia occurs via the purine salvage pathway, which is dependent on adenosine, adenine, and hypoxanthine as substrate precursors,' and via the adenine nucleotide de novo synthetic pathway, which is dependent on the availability of phosphoribosyl pyrophosphate.' With regard to the latter pathway, the substrate 5-amino-4-imidazolecarboxamide riboside (AICAR) has been reported to accelerate the rate of de novo ATP synthesis." This agent is taken up by the myocardial cell, phosphorylated, and enters the de novo synthesis pathway distal to the more highly regulated control points. Mauser and associates,' using a canine preparation subjected to regional ischemia, reported that intracoronary AI CAR infusion increased the rate of myocardial de novo adenine nucleotide synthesis ninefold within the first 3 hours of reperfusion; the ATP tissue levels during the 3 hours, however, remained unchanged. In contrast,

Volume 95 Number 2 February 1988

in isolated perfused rat hearts subjected to 15 minutes of unprotected ischemia, stimulation of adenine nucleotide de novo synthesis with ribose improved both ATP levels and myocardial function over a much shorter reperfusion interval." Since the acceleration of the de novo pathway by AICAR is greater than that for ribose,' the purpose of this study was to use AICAR in the isolated perfused rat heart to determine the role of AICAR in the substrate enhancement in the postischemic myocardial nucleotide pool and elucidate further the temporal relationship between adenine nucleotide recovery and myocardial function. Methods Eighty-five male Wistar rats (250 to 300 gm) were injected with 500 units of heparin intra peritoneally and anesthetized with halothane. The chest was opened and the heart rapidly excised and placed in ice-cold Krebs-Henseleit buffer. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals." The hearts were gently blotted dry, quickly weighed, and mounted on an aortic perfusion cannula of a modified Langendorff perfusion apparatus. The perfusate consisted of a modified Krebs-Henseleit bicarbonate buffer with the following composition (in millimoles per liter): NaCI 121.3, KCI 4.6, cscr, 2.52, MgS0 4 1.25, NaHC0 3 21.9, KH 2P04 1.18, glucose 11.1, and pyruvate 1.0. The perfusate was bubbled with 95% oxygen and 5% carbon dioxide to produce an oxygen tension of 500 to 600 mm Hg, a carbon dioxide tension of 35 to 40 mm Hg, and a pH of 7.35 to 7.45. The temperature was maintained with the aid of a water jacket and a circulating bath at 37° C. During the equilibration period, a saline-filled balloon-tipped catheter was inserted in the left ventricular cavity via the left atrium to obtain a ventricular pressure tracing (arterial pressure transducer and recorder, Hewlett-Packard Company, Waltham, Mass.). Developed pressure was measured after adjusting balloon volume and recording the respective systolic and diastolic pressures. End-diastolic pressure was set at 10 mm Hg. A pacing electrode was attached to the region of the atrioventricular node and stimulation was achieved via a Grass stimulator (Grass Instrument Company, Quincy, Mass.) at 5 Hz to maintain a heart rate of 300 beats/min. Experimental design. The hearts were assigned to one of four groups: nonischemic untreated time control group, nonischemic AICAR-treated (100 /-Lmol/L) time control group, untreated hearts subjected to ischemia and reperfusion, and AICAR-treated (100 /-Lmol/L) hearts with ischemia and reperfusion. In both the untreated and treated nonischemic time control groups, the hearts were perfused continuously for 90 minutes. Developed pressure was recorded at 30, 60, and 90 minutes and tissue frozen at the same time intervals for determination of adenosine triphosphate (ATP), diphosphate (ADP), and monophosphate (AMP) and inosine monophosphate (IMP) content. In the ischemia and reperfusion studies, the untreated and treated hearts were first perfused for 30 minutes. The perfusion was stopped for 10 minutes and then restored for a total of 60 minutes. At the end of 30 minutes of equilibration, 10 minutes of ischemia. and at 15, 30, and 60

A/CAR

-

-

287

c

Cl)

c;

o

U...,.: Q) ~ "0_ OQ)

ATP

40

(5)

(4)

3.0

~ ~

E

u ::lcr>

Z ....... Q)

c


Q)

.- 0

a3

20

E

~ ::i. ~ ::l

1.0

o 110


Q) .........

cr> 0.... I ~

"0

ADP (4) _ - . . - - - -_ _...(5)

90

(4)

(5)

E

~E

o~

70

Q)

>

Q)

o

50

60

90

Time (Minutes) Fig. 1. ATP and developed pressure time controls. Effect of 90 minutes of continuous perfusion with oxygenated KrebsHenseleit solution on ATP and ADP content and developed pressure in the isolated rat heart. Numbers in parentheses are the number of hearts studied. There were no significant differences over time for ATP, ADP, or developed pressure.

minutes of reperfusion, developed pressure was recorded immediately before the hearts were frozen for measurement of adenine nucleotides and IMP. Biochemical analysis. The hearts were rapidly frozen by Wollenberger tongs precooled to the temperature of liquid nitrogen. Each frozen sample was rapidly weighed to the nearest 0.05 mg, homogenized in a solution containing 0.5 ml of ice-cold 6% perchloric acid, and extracted for 30 minutes by periodic grinding to produce a homogeneous slurry. The slurry was centrifuged at 4° C for 2 minutes, 1000 g, and the supernatant fraction was mixed intermittently in a vortex agitator at room temperature for I minute with I ml of 0.5 mol/L tri-N-octylamine in Freon fluorinated hydrocarbon to remove the acid. After centrifugation at 4° C for 2 minutes, 2000 g, the aqueous layer was filtered through a 0.45 /-Lm Millipore filter and high-pressure liquid chromatography was used for analysis of ATP, ADP, AMP, and IMP. A 100 /-Ll sample of the tissue extract was injected onto a Whatman SAX column (25 by 0.46 em, 10 /-Lm particle size; Whatman Chemical Separation, Inc., Clifton, N.J.) with a Waters Model 720 system controller and M45 and 6000 solvent delivery system (Waters Associates, Inc., Milford,

The Journal of

288 Mentzer et al.

Thoracic and Cardiovascular Surgery

! Untreated

-

2100 fJ-M AICAR

300

3: 240



(L3: 180 ~E


o E c

120

(7)

(4) _ _ ... (5)

1<5)

60

L,f' ---'------'----------'--90 30

Fig. 2. AMP time controls. Effect of AICAR on AMP content during 90 minutes of continuous perfusion with oxygenated Krebs-Henseleit solution. Numbers in parentheses indicate number of experiments. There were no significant differences over time within treatment groups and no significant differences between treatment groups.

Mass.). The nucleotides were eluted with a linear gradient of 100% buffer A (NH.H 2PO. 5 mrnol/L, pH 2.8) to 100% buffer B (NH.H 2PO. 750 mmol/L; pH 3.8) established over a period of 36 minutes at a flow rate of 2 ml/rnin. A 14-minute period was allowed for column reequilibration. IMP, AMP, ADP, and ATP were determined by absorbance at 254 nm with a Waters Model 440 absorbance detector. Various peaks were identified by comparison with retention times of known standards. Peak areas were quantified by using peaks of known concentration and nucleotide content was expressed as nanomoles or micromoles per gram wet weight of tissue. Statistics. Comparisons between the untreated and AICAR-treated groups and over time within a treatment group were made at each point in the protocol by means of a two-way analysis of variance. Data are presented as mean ± standard error of the mean. A p value of 0.05 or less was considered significant.

Results Nonischemic time control group. In the untreated hearts perfused continuously for 90 minutes with oxygenated Krebs-Henseleit solution, myocardial ATP, ADP, AMP, and IMP tissue levels and developed pressure remained constant throughout the entire time period (Figs. 1 to 3). In the nonischemic hearts perfused with AICAR, the AMP contents were similar and the IMP levels at the end of 30 minutes were unchanged. There was, however, a significant fourfold and eightfold increase in IMP content at the end of 60 and 90

minutes, respectively, of continuous perfusion in the AICAR-treated group (p < 0.05) (Fig. 3). Effect of AICAR on myocardial AMP and IMP during ischemia and reperfusion. In both the untreated and AICAR-treated hearts, there was a greater than threefold increase in the AMP content at the end of 10 minutes of normothermic ischemic arrest (Table I). On reperfusion, the AMP levels in the untreated group and AICAR-treated group fell to levels not significantly different from their respective 30 minute equilibration control values. In contrast, the IMP content increased more than thirteenfold at the end of 10 minutes of ischemia in both the untreated and treated groups. On reperfusion, however, the IMP content in the untreated hearts returned to control values within 15 minutes, whereas the IMP content in the AICAR-treated group remained significantly elevated. At the end of 30 and 60 minutes of reperfusion, the IMP content was fivefold greater than the values obtained for the untreated group at the same time points. Effect of AICAR on myocardial ATP content during ischemia and reperfusion. In the untreated group, ischemia resulted in a significant fall in tissue ATP content to 40% of the preischemic values, from 3.44 ± 0.15 to 1.40 ± 0.36 ~mol/gm wet weight (p < 0.05) (Fig. 4). On reperfusion, there was a gradual

Volume 95 Number 2 February 1988

AICAR

-

120

(J'l

Q)

*

! Untreated

~

100

289

(5)

2100 fLM AICAR

3

Q)

3

E

(J'l

<,

80

*

(4)

60

(/)

.S:.' 0

E

40

c

0..

(4)

20

~

(4)

f--<

30

90

60

Time ( Minutes) Fig. 3. IMP time controls. Effect of AICAR on IMP content in the nonischemic isolated rat heart perfused for 90 minutes. *p < 0.05 compared to untreated group at same time point; tp < 0.05 versus 3D-minute AICAR-treated time point.

Table I. Effect of ischemia and reperfusion on myocardial AMP and IMP content in hearts treated with AICAR AMP (nrnol/gm wet wt) Untreated AICAR, 100 ",moljL IMP (nmoljgm wet wt) Untreated AICAR, 100 umol/L

30 min Eq

10 min I

178 ± 60 153 ± 26 12 ± 2 16 ± 4

15 min RP

30 min RP

60 min RP

654 ± 60t 470 ± 117*t

64 ± 20 90 ± 30

114 ± 40 66 ± 10

79 ± 20 90 ± 10

216 ± 40t 210 ± 20t

IS ± 5 62 ± 9*t

16 ± 7 80 ± 5*t

15 ± 10 83 ± 10*t

Legend: Data expressed as mean ± standard error of the mean. N = 33, the total number of hearts used: Eq, equilibration: I. ischemia: RP. reperfusion.

'p < 0.05 compared to the untreated group at the same time point. tp < 0.05 compared to 30 minutes of equilibration in the same treatment group.

repletion of ATP toward control values, but overall the ATP levels remained markedly depressed, recovering to only 52% of preischemic values. In the AICAR-treated hearts, a similar fall in ATP content with ischemia (from 3.63 ± 0.18 to 1.78 ± 0.16 umoles/gm wet weight) (p < 0.05) and gradual repletion on reperfusion occurred. The group treated with AICAR showed no significant differences in repletion of ATP at any given point in the protocol versus the untreated group (Fig. 4). Effect of AICAR on developed pressure after ischemia and reperfusion. In the untreated group, hearts subjected to 10 minutes of ischemic arrest and 15 minutes of reperfusion sustained a 23% reduction in

developed pressure when compared to the mean value of 103 ± 13 mm Hg measured at the end of 30 minutes of equilibration (Fig. 5). There was no significant improvement in the developed pressure when the reperfusion time was extended to 30 and 60 minutes. The mean developed pressures at these times were 76% and 83% of the preischemic values, respectively. In the AICARtreated hearts, the developed pressure at the end of 15 minutes of reperfusion was 62% of the 30 minute equilibration value of 92 ± 10 mm Hg. There was no significant improvement in the developed pressure when the AICAR perfusion time was extended to 30 and 60 minutes (80% and 78% of the preischemic values, respectively). There was no significant difference

The Journal of Thoracic and Cardiovascular Surgery

290 Mentzer et al.

40 3.6

! Untreated

(7)

+' ~

2100 J-LM AICAR

3.2

* p<05 Compared to

Qj ~

E

~ CIl Q)

Treated Control t p<.05 Compared to Untreated Control

2.8 2.4

0

E

::L 2.0

0..

~

1.6

(N) = number of expts

1.2 0

30EQ

101

15RP

30RP

60RP

Time (Minutes) Fig. 4. Effect of AICAR on myocardial ATP content after ischemia (I) and during reperfusion (RP). EQ. equilibration. *Compared to 30 minutes of equilibration in untreated group; tcompared to 30 minutes of equilibration in treated group. There were no significant differences between treatment groups at any given time point.

between the untreated and AICAR-treated groups at any time point within the protocol. Discussion The role of AICAR in elucidating the relationship between recovery of myocardial function and adenine nucleotide metabolism after ischemia has been controversial. At the onset of ischemia, the myocardial cell experiences a variety of morphologic, metabolic, and functional abnormalities that are reversed on reperfusion.?" The recovery of ventricular function, however, may be prolonged, and this has been associated with the delayed repletion of the cardiac adenine nucleotide pool." One of the potential factors involved in the mechanism of delayed postischemic recovery of ventricular function is ATP, since it supplies the energy for muscle contraction. Several studies': 13 provide evidence that a direct linear correlation exists between tissue ATP and recovery of function during postischemic reperfusion. However, ventricular function ceases at a point where ATP cellular concentrations are much higher than that required to saturate the contractile-dependent actomyosin ATPase. 14 This suggests that there may not be a direct causal relationship between ATP levels and myocardial contractility. Part of the difficulty in testing the hypothesis that ventricular function is intimately

related to ATP concentration has been the limited ability to alter the rate of ATP repletion or de novo synthesis. At the onset of ischemia, ATP is rapidly depleted and degraded to adenosine, inosine, and hypoxanthine, which in turn diffuse into the interstitial and vascular spaces. IS, 16 On reperfusion, washout of these precursors contributes to an overall decrease in the total adenine nucleotide pool.I, 17 Repletion of the adenine nucleotides then should be enhanced by supplying precursors for purine nucleotide synthesis or enhancing the rate of de novo synthesis. We have reported previously that adenosine infusion improves ATP repletion and enhances postischemic recovery of function in the isolated perfused rat heart." With respect to the de novo synthesis pathway, other investigators have reported that the agent AICAR markedly accelerates the rate of ATP synthesis. Sabina and associates'? reported that AICAR infusion increased the adenine nucleotide pool of the normal myocardium by 10%. Swain and colleagues' reported AICAR selectively enhanced the rate of repletion of ATP and guanosine triphosphate pools in the postischemic myocardium when the agent was infused continuously for 24 hours after 12 minutes of coronary occlusion. Since the de novo pathway is known to be relatively slow, it was believed that prolonged reperfu-

Volume 95 Number 2 February 1988

A/CAR

!

Untreated

Q100 fLM AICAR

120 I

291

0-

E

100

Q) ~

t

(6)

E

(5)

80

:::J

l/l l/l Q)

~

60

0..

"0

Q)

40

Q'.i > Q) 0

20

00

0

(N) : Number of expts

30EQ

15RP

30RP

60RP

Time (Minutes) Fig. 5. Effect of AICAR on developed pressure after ischemia and during reperfusion. *p < 0.05 compared to AICAR-treated control at 30 minutes of equilibration; tp < 0.05 versus untreated control at 30 minutes of equilibration. There were no significant differences between treatment groups at any given time point.

sion was necessary to demonstrate an effect on the steady state ATP levels. In contrast, Mitsos, Jolly, and Lucchesi" reported AICAR improved the recovery of myocardial function after a relatively short reperfusion interval. These investigators suggested that the beneficial effects of AICAR during global ischemia were due to an accelerated repletion of ATP. Recently, however, Hoffmeister, Mauser, and Schaper" reported that after 3 hours ofreperfusion, not only did AICAR infusion fail to increase tissue ATP content, but postischemic segmental contractile function actually deteriorated. These investigators concluded that their findings refuted the hypothesis of a direct relationship between ATP levels and contractile function. Unlike most other workers using AICAR, however, these investigators did examine altered adenine nucleotide metabolism and recovery of postischemic ventricular function concurrently. Another issue that complicates the problem of elucidating the temporal relationship between ATP levels and function is that there may be a small pool of extramitochondrial ATP that plays an important role in the regulation of ischemic muscle contraction. Gudbjarnason, Mathes, and Ravens" reported that ischemic heart muscle stopped contracting at a relatively high ATP level (4.5 mol/gm) when only 20% of the ATP had been used, whereas nonischemic muscles survived and maintained contraction at ATP levels as low as 1.5 to 2.0 mol/gm. They also noted a rapid increase in glycogen that coincided with an early inhibition in the use of ATP stores without an interference in the use of

creatine phosphate stores and suggested that the heterogeneity of ATP and creatine phosphate depletion reflected an inhibition of transfer of intramitochondrial high-energy phosphate to the extramitochondrial compartment. They concluded that the early cessation of contractile activity in ischemic myocardial cells was the result of a reduction in the rate of regeneration of a small pool of extramitochondrial ATP available for muscle contraction. More recently, Lipasti and associates" reported that the initiation of ischemic contracture occurred at a much higher myocardial ATP level when ATP was derived from mitochondrial sources than when the ATP was generated by anaerobic glycolysis. They concluded that the differences may be due to intracellular compartmentalization of ATP providing different amounts of ATP available for contractile proteins. Soboll and Bunger" and Veech and associates" have demonstrated that there may be differences in mitochondrial and cytosolic ATP concentrations in normal myocardium, yet the technical feasibility of applying these concepts in the experimental evaluation of cytosolic fractions of ATP has yet to be realized. On the other hand, the measurement of total tissue ATP content or rate of adenine nucleotide synthesis simply may not be sufficiently sensitive to elucidate the temporal relationship between adenine nucleotide metabolism and ventricular function. Therefore, it is not surprising that considerable controversy regarding the relationship between ATP levels and changes in ventricular function persists.

The Journal of

292

Mentzer et al.

In our studies, we evaluated the effect of AICAR on ATP levels and myocardial function concurrently. We observed that AICAR was indeed readily phosphorylated and incorporated in both the normal and postischemic myocardial cell over a relatively short perfusion interval, as evidenced by the increase in IMP. Second, despite the markedly increased IMP levels in both the nonischemic and postischemic hearts, AICAR failed to augment AMP or ATP levels or to enhance recovery of function. The dose of AICAR selected for this study was based on our earlier findings that lower concentrations (10 to 50 J,LmoljL) not only failed to augment the total adenine nucleotide pool, but also failed to elevate the IMP content during perfusion intervals. This study indicates that the failure of AICAR to augment repletion of the postischemic adenine nucleotide pool is due to the inability of the myocardium to convert IMP to AMP and is consistent with a previous report that indicates that accumulation of AICAR phosphates inhibits the enzyme adenylosuccinate lyase.19 This enzyme is required to convert IMP to adenylosuccinate, the precursor of AMP. Thus, while AICAR may stimulate the rate of de novo synthesis, the enhancement of ATP production is effectively prevented. For this reason, AICAR does not provide an effective pharmacologic tool to assess the relationship between the postischemic adenine nucleotide pool and the recovery of ventricular function. Definitive elucidation of this controversy may ultimately require the use of agents that alter the rate of nucleotide repletion in conjunction with development of the technology to define subcellular adenylate distribution, particularly the cellular compartment with direct access to actomyosin ATPase. REFERENCES I. DeBoer LWV, Ingwall JS, Kloner RA, Braunwald E. Prolonged derangements of canine myocardial purine metabolism after a brief coronary artery occlusion not associated with anatomic evidence of necrosis. Proc Nat! Acad Sci USA 1980;77:5471-5. 2. Hearse OJ. Oxygen deprivation and early myocardial contractile failure: a reassessment of the possible role of adenosine triphosphate. Am J Cardiol 1979;44:1115-21. 3. Reibel OK, Rovetto MJ. Myocardial ATP synthesis and mechanical function following oxygen deficiency. Am J Physiol 1978;234:H620-4. 4. Maguire MH, Lukas MC, Rettie JF. Adenine nucleotide salvage synthesis in the rat heart: pathways of adenosine salvage. Biochim Biophys Acta 1972;262:108-15. 5. Zimmer HG, Trendelenburg C, Kammermeier H, Gerlach E. De novo synthesis of myocardial adenine nucleotides in the rat: acceleration during recovery from oxygen deficiency. Circ Res 1973;32:635-42.

Thoracic and Cardiovascular Surgery

6. Swain JL, Hines JJ, Sabina RL, Holmes EW. Accelerated repletion of ATP and GTP pools in postischemic canine myocardium using a precursor of purine de novo synthesis. 1982; Circ Res 51:102-5. 7. Mauser M, Hoffmeister HM, Nienaber C, Schaper W. Influence of ribose, adenosine and "AICAR" on the role of myocardial adenosine triphosphate synthesis during reperfusion after coronary artery occlusion in the dog. Circ Res 1985;56:220-30. 8. Pasque MK, Spray TL, Pellom GL, et al. Riboseenhanced myocardial recovery following ischemia in the isolated working rat heart. J THORAC CARDIOVASC SURG 1982;83:390-8. 9. Heydrickx GR, Millard RW, McRitchie RJ, Maroko PR, Vatner SF. Regional myocardial function and electrophysiological alterations after brief coronary artery occlusion in conscious dogs. J Clin Invest 1975;56:97885. 10. Jennings RB, Hawkins HK, Lowe JE, Hill ML, Klotman S, Reimer KA. Relation between high energy phosphate and lethal ischemia in the dog. Am J Pathol 1978;92:187214. II. Shaper J, Mulch J, Winkler B, Schaper W. Ultrastructural, functional and biochemical criteria for estimation of reversibility of ischemic injury: a study on the effects of global ischemia on the isolated dog heart. J Mol Cell Cardiol 1979;11:521-41. 12. Swain JL, Sabina RL, McHale PA, Greenfield JC, Holmes EW. Prolonged myocardial nucleotide depletion after brief ischemia in the open-chest dog. Am J Physiol 1982;H818-26. 13. Hearse DJ, Chain EB. The role of glucose in the survival and "recovery" of the anoxic isolated perfused rat heart. Biochem J 1972;128:1125-33. 14. Katz AM. Physiology of the heart. 1st ed. New York: Raven Press, 1977:140, 175-7. 15. Berne RM, Rubio R. Adenine nucleotide metabolism in the heart. Circ Res 1974;34,35 (Suppl 3):11I109-20. 16. Rubio R, Berne RM, Dobson JG. Sites of adenosine production in cardiac and skeletal muscle. Am J Physiol 1973;225:938-53. 17. Reibel OK, Rovetto MJ. Myocardial adenosine salvage rates and restoration of ATP content following ischemia. Am J Physiol 1979;237:H247-52. 18. Ely SW, Mentzer RM, Lasley RD, Lee BK, Berne RM. Functional and metabolic evidence of enhanced myocardial tolerance to ischemia and reperfusion with adenosine. J THORAC CARDIOVASC SURG 1985;90:549-56. 19. Sabina RL, Kernstine KH, Boyd RL, Holmes EW, Swain JW. Metabolism of 5-amino-4-imidazolecarboxamide riboside in cardiac and skeletal muscle. J Bioi Chern 1982;257:10178-83. 20. Mitsos SE, Jolly SR, Lucchesi BR. Recovery of myocardial function in the globally ischemic isolated cat heart with AICAriboside [Abstract]. Fed Proc 1983;42:1359. 21. Hoffmeister HM, Mauser M, Schaper W. Effect of adenosine and AICAR on ATP content and regional

Volume 95 Number 2 February 1988

contractile function in reperfused canine myocardium. Basic Res Cardiol 1985;80:445-58. 22. Gudbjarnason S, Mathes P, Ravens KG. Functional compartmentation of ATP and creatine phosphate in heart muscle. J Mol Cell Cardiol 1970;1:325-39. 23. Lipasti JA, Nevalainen TJ, Alanen KA, Tolvanen MA. Anaerobic glycolysis and the development of ischaemic contracture in isolated rat heart. Cardiovasc Res 1984; 18:145-8.

A/CAR

293

24. Soboll S, Bunger R. Compartmentation of adenine nucleotides in the isolated working guinea pig heart stimulated by noradrenaline. Hoppe-Seyler Z Physiol Chern 1981; 362:125-32. 25. Veech RL, Lawson JWR, Cornell NW, Krebs HA. Cytosolic phosphorylation potential. J Bioi Chern 1979; 254:6538-47.