SUMMARY This study was done to compare rates of high energy phosphate (HEP) utilization and depletion, as well as the production and distribution of catabolic products of adenine nucleo tides in dog heart during total ischemia in vitro and severe ischemia in vivo. Both HEP production from anaerobic glycolysis and HEP utilization occurred much more quickly during the fir»t 15 mmtuet of severe ischemia in vivo than in total ischemia in vitro. HEP utilization exceeded production in both types of ischemia and tissue HEP decreased progressively. Much of the creatine phosphate (CP) was lost within the first 1-3 minutes; adenosine triphosphate (ATP) depletion occurred more slowly than CP and more slowly in vitro than in vivo. ATP was reduced from control contents of 5-6 /imol/g to 1.0 jirool/g by 75 minutes of total ischemia in vitro, but reached a similar level within only 30 minutes of severe ischemia in vivo. HEP utilization and production during ischemia were estimated from the rate of accumulation of myocardial lactate and essentially ceased when the ATP reached 0.4 jimol/g wet weight. At this time, more than 80% of the HEP that had been utilized in ischemia in vivo or in total ischemia in vitro had been derived from anaerobic glycolysis. ATP depletion was paralleled by dephosphorylation of adenine nucleottdeg. The lost nucleotides were recovered stoichiometrically as adenosine, inosine, hypoxantbine, and xanthine in both models of ischemia, a finding which demonstrates that the low collateral flow of severe ischemia allows little washout of nucleosides, bases, or lactate to the systemic circulation. These results Indicate that total ischemia in vitro can be used as a model of severe ischemia in vivo in that the pathways of energy production and depletion and of adenine nucleotide degradation generally are similar. Moreover, the larger quantities of uniformly ischemic tissue and the slower time course of changes make total ischemia well suited to the study of relationships between the metabolic, ftmctional, and structural consequences of ischemic injury, drc Res 49: 892-900, 1981 WITHIN seconds of the onset of severe ischemia induced by coronary occlusion in vivo, myocardial HEP begins to decrease, and by the time a minute has passed, 80% or more of the creatine phosphate (CP) is lost (Braasch et al., 1968;Dunn and Griggs, 1975). Within 15 minutes, 65% of the tissue adenosine triphosphate (ATP) and 55% of the total adenine nucleotides (£Ad) have disappeared (Jennings et al., 1978). After 40 minutes of ischemia, the HEP supply is approaching zero and most of the severely ischemic cells have become irreversibly injured and do not recover even if blood flow is restored (Jennings et al., 1978).The present report compares the rates of HEP production from anaerobic glycolysis as weD as the rates of utilization and depletion of HEP and the distribution of catabolic products of adenine nu- cleo tides in total ischemia in vitro and severe ischemia in vivo. Both the rate of high energy phosphate production and of ATP deg...

The relationship between the effects of isoproterenol and prostaglandin El (PGE1) on contractile state, cyclic AMP accumulation, and the activation states of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37), phosphorylase kinase, glycogen synthase, and glycogen phosphorylase have been studied in the isolated perfused rat heart. Perfusion of hearts with isoproterenol (10 or 80 nM) caused enhancement of left ventricular dP/dt (P, pressure), increased intracellular cyclic AMP, increased the activation states of protein kinase, phosphorylase kinase, glycogen phosphorylase, and conversion of glycogen synthase to a less active form. PGE1 (2 or 30 ;&M) increased cyclic AMP accumulation and activated protein kinase, but caused no detectable changes in dP/dt or the activation states of the protein kinase substrates involved in glycogen metabolism. Perfusion of hearts with either 10 nM isoproterenol or 30 1sM PGE1 produced comparable increases in cyclic AMP accumulation and protein kinase activity. Exposure of hearts to a combination of these agents caused additive effects on cyclic AMP content and protein kinase activity. However, values for phosphorylase kinase, glycogen phosphorylase, glycogen synthase, and dP/dt did not differ from those observed in the presence of 10 nM isoproterenol alone. The failure of PGE1 to stimulate phosphorylation of protein kinase substrates was not due to an increase in phosphorylase phosphatase activity. We conclude that an increase in intracellular cyclic AMP and the subsequent activation of protein kinase are insufficient to change either the activities of phosphorylase kinase, glycogen phosphorylase, and glycogen synthase or the inotropic state of eart muscle. Kuo and Greengard (1) have advanced the idea that ubiquitous cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) mediate most, if not all, of the effects of cAMP in eukaryotes (1-3). According to this hypothesis the ability of a hormone to elicit a response mediated by cAMP would be determined by the presence of the appropriate adenylate cyclase-linked receptor on the surface of the cell and by the presence within the cell of appropriate substrates for protein kinase. Thus, receptors specify the spectrum of hormonal sensitivity of a cell and available substrates specify the precise response. Much evidence supports the universality of Kuo and Greengard's hypothesis. It is apparent that cAMP is the only ligand necessary for the activation of protein kinase (4, 5). Numerous events, notably glycogenolysis (2) and lipolysis (6), result from phosphorylation of proteins by cAMP-dependent protein kinases subsequent to elevation of intracellular cAMP. Other than phosphodiesterase, the regulatory subunit of protein kinase appears to be the major high-affinity binding protein for cAMP (1). As noted by Rall, however, the protein kinase hypothesis presents us with "the unsatisfying picture of the catalytic subunit of protein kinase swimming about, happily phosphorylating a variety of cellula...

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The effects of ischemia and anoxia on cardiac adenosine 3',5'-monophosphate (cyclic AMP) concentration, glycogen phosphorylase activity ratio (-5'-AMP: + 5'-AMP), phosphorylase kinase activity ratio (pH 6.8:8.2), and myocardial contractility (left ventricular dP/dt) were studied in an open-chest rat heart preparation. Ischemia produced by termination of coronary blood flow increased cyclic AMP from 0.55 to 0.77 yu.moles/kg in 5 seconds and phosphorylase from 0.14 to 0.57 in 20 seconds. Anoxia induced by breathing N., increased cyclic AMP from 0.50 to 0.62 ju.moles/kg in 10 seconds and phosphorylase from 0.14 to 0.65 in 30 seconds. Phosphorylase kinase increased with ischemia but did not change with anoxia. Beta-receptor blockade with practolol prevented the rise in cyclic AMP and phosphorylase kinase but blocked the increase in phosphorylase only in ischemia. Myocardial contractility declined precipitously during the first 20 seconds of anoxia. Epinephrine (0.1 |ixg/kg) caused an increase in cyclic AMP comparable to that elicited by anoxia, and it produced an increase in dP/dt during N a breathing. These results suggest that in the intact working heart ischemia induces phosphorylase a formation through a cyclic AMP-dependent transformation of phosphorylase kinase; however, in anoxia phosphorylase a formation depends only on the regulation of the catalytic activity of phosphorylase kinase without conversion of this enzyme to its activated form. An increase in cyclic AMP during anoxia is not associated with a positive inotropic response even though such a response is obtained with epinephrine. Factors other than the elevation of myocardial cyclic AMP may be limiting in the control of both cardiac glycogenolysis and inotropic state. KEY WORDS adenosine 3',5'-monophosphate phosphorylase kinase practolol glycogenolysis contractility epinephrine rat heart• The activation of glycogenolysis that occurs with ischemia and anoxia in the myocardium results from an increase in glycogen phosphorylase activity (1-4). The increase in enzyme activity occurs very rapidly, within approximately 10 seconds, and appears to be largely due to the conversion of the enzyme from the b to the a form (4-6). Epinephrine elicits activation of cardiac phosphorylase by a sequence of reactions beginning with the production of adenosine 3',5'-monophosphate (cyclic AMP) through the activation of adenylate cyclase (7-9). The mechanism of action of cyclic AMP appears to be the activation of protein kinase (10). This enzyme catalyzes the adenosine triphosphate-dependent transformation of phosphory-

The purpose of this investigation was to contrast the effect of glucagon and that of epinephrine on the concentration of cyclic adenosine 3',5'-monophosphate (cyclic AMP), the activity of phosphorylase a and the contractile amplitude of isolated perfused rat hearts. The two drugs were about equally effective except that the maximal augmentation of contractility by epinephrine (5 x 10 -9 moles) was twice that produced by an equivalent dose of glucagon with a fourfold greater increase in cyclic AMP concentration. Combination of large doses of the two drugs caused increases in the cyclic nucleotide considerably greater than those required for maximal phosphorylase activation or associated with a maximal inotropic response. The effects of glucagon also developed more slowly than those of epinephrine. An increase in cyclic AMP was not detectable until after phosphorylase a and contractile amplitude had increased. The beta-receptor-blocking agents dichloroisoproterenol and pronethalol did not block the biochemical responses to glucagon in doses which abolished the epinephrine-induced increases in cyclic AMP and phosphorylase a . These results, along with those obtained by other investigators, indicate that glucagon can elicit the same biochemical responses in intact heart as have been obtained with epinephrine, but by action at a different receptor site.