Enhanced glucose uptake via GLUT4 fuels recovery from calcium overload after ischaemia-reperfusion injury in sevoflurane- but not propofol-treated hearts.

BACKGROUND: So far, no study has explored the effects of sevoflurane, propofol, and Intralipid on metabolic flux rates of fatty acid oxidation (FOX) and glucose oxidation (GOX) in hearts exposed to ischaemia-reperfusion. METHODS: Isolated paced working rat hearts were exposed to 20 min of ischaemia and 30 min of reperfusion. Peri-ischaemic sevoflurane (2 vol%) and propofol (100 µM) in the formulation of 1% Diprivan(®) were assessed for their effects on oxidative energy metabolism and intracellular diastolic and systolic Ca(2+) concentrations. Substrate flux was measured using [(3)H]palmitate and [(14)C]glucose and [Ca(2+)] using indo-1AM. Western blotting was used to determine the expression of the sarcolemmal glucose transporter GLUT4 in lipid rafts. Biochemical analyses of nucleotides, ceramides, and 32 acylcarnitines were also performed. RESULTS: Sevoflurane, but not propofol, improved the recovery of left ventricular work (P=0.008) and myocardial efficiency (P=0.008) compared with untreated ischaemic hearts. This functional improvement was accompanied by reduced increases in post-ischaemic diastolic and systolic intracellular Ca(2+) concentrations (P=0.008). Sevoflurane, but not propofol, increased GOX (P=0.009) and decreased FOX (P=0.019) in hearts exposed to ischaemia-reperfusion. GLUT4 expression was markedly increased in lipid rafts of sevoflurane-treated hearts (P=0.016). Increased GOX closely correlated with reduced Ca(2+) overload. Intralipid alone decreased energy charge and increased long-chain and hydroxyacylcarnitine tissue levels, whereas sevoflurane decreased toxic ceramide formation. CONCLUSIONS: Enhanced glucose uptake via GLUT4 fuels recovery from Ca(2+) overload after ischaemia-reperfusion in sevoflurane- but not propofol-treated hearts. The use of a high propofol concentration (100 µM) did not result in similar protection.

Characterization of 5'AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia.

Despite the high expression of 5'AMP activated protein kinase (AMPK) in heart, the activity and function of this enzyme in heart muscle has not been characterized. We demonstrate that rat hearts have a high AMPK activity, comparable to that found in liver, which could be stimulated up to 3-fold by 5'AMP. Cardiac AMPK is also under phosphorylation control, since in vitro incubation of cardiac AMPK with protein phosphatase 2A completely abolished activity, while incubation with ATP/Mg(2+) resulted in over a 2-fold increase in activity. To investigate the function of AMPK in heart muscle, isolated working rat hearts were subjected to 30 min of global no-flow ischemia, followed by 60 min of aerobic reperfusion. AMPK activity was increased in heart at the end of reperfusion compared to aerobic controls (379 +/- 53 (n=5) vs. 139 +/- 19 (n=5) pmol x min(-1) x mg protein(-1), P<0.05, respectively). Treatment of AMPK in vitro with protein phosphatase 2A reversed this activation. Since AMPK can phosphorylate and inactivate acetyl-CoA carboxylase (ACC) in other tissues, and heart ACC has an important role in regulating fatty acid oxidation, we measured ACC activity in hearts reperfused post-ischemia. ACC activity was decreased at the end of reperfusion compared to aerobic controls (3.64 +/- 0.36 (n=9) vs. 10.93 +/- 0.60 (n=11) nmol x min(-1) x mg protein(-1), respectively, P<0.05). A significant negative correlation (r= -0.78) was observed between AMPK activity and ACC activity measured in aerobic and reperfused ischemic hearts. Low ACC activity could be reversed if ACC was extracted from hearts in the absence of phosphatase inhibitors, suggesting that phosphorylation of ACC decreased enzyme activity. This suggests that following ischemia AMPK is phosphorylated and activated (possibly by an AMPK kinase). AMPK then phosphorylates and inactivates ACC. The resultant decrease in malonyl-CoA levels could explain the acceleration of fatty acid oxidation that is observed during reperfusion of ischemic hearts.

Activation of Ca(2+)-independent nitric oxide synthase by 17beta-estradiol in post-ischemic rat heart.

BACKGROUND: Nitric oxide (NO) donors or facilitation of endogenous NO production is cardioprotective. This study sought to determine whether enhanced myocardial NO production might contribute to estrogen-induced cardioprotection. METHODS: Ca(2+)-dependent and Ca(2+)-independent NOS activities (pmol min(-1) mg(-1) protein), NOS protein expression (quantitative immunoblot), cGMP content (pmol mg(-1) protein) and LV work (Joules) were measured in hearts isolated from ovariectomized rats that were either untreated or treated chronically with 17beta-estradiol (0.25 mg, 21 day release formulation). RESULTS: After 14 days, serum levels of 17beta-estradiol were 6+/-1 and 135+/-16 pg ml(-1) in untreated and 17beta-estradiol-treated animals, respectively. After 60 min aerobic working mode perfusion, Ca(2+)-dependent NOS (untreated, 1.47+/-0.36; 17beta-estradiol 1.13+/-0.25) and Ca(2+)-independent NOS (untreated, 0.45+/-0.24; 17beta-estradiol, 0.41+/-0.21) activities, eNOS and iNOS proteins and cGMP content (untreated, 0.64+/-0.08; 17beta-estradiol, 0.76+/-0.12) were not different in the two groups. After 60 min low-flow (0.5 ml min(-1)) ischemia and 30 min reperfusion, Ca(2+)-dependent NOS activities were again similar (untreated, 1.25+/-0.23; 17beta-estradiol, 0.78+/-0.27). However, after reperfusion, Ca(2+)-independent NOS activity (untreated, 0. 39+/-0.10; 17beta-estradiol, 1.36+/-0.36) was 3.5-fold higher (P=0. 008) and cGMP content (untreated, 0.30+/-0.03; 17beta-estradiol, 0. 49+/-0.07) was 1.6-fold higher (P=0.017) in hearts from 17beta-estradiol-treated animals. Although pre-ischemic function was similar, recovery of post-ischemic LV work was 2-fold greater (P=0.024) in the 17beta-estradiol group. CONCLUSION: The ability of ischemia and reperfusion in combination with chronic 17beta-estradiol to increase Ca(2+)-independent NOS activity and cGMP content supports a role for enhanced myocardial NO signaling in 17beta-estradiol-induced cardioprotection.

Heterogeneity of nucleoside transport inhibitory sites in heart: a quantitative autoradiographical analysis.

1. The distribution of nucleoside transport inhibitory sites in rat and guinea-pig cardiac sections was investigated by use of [3H]-nitrobenzylthioinosine ([3H]-NBMPR) autoradiography. The distribution of these sites was heterogeneous in guinea-pig sections and homogeneous in rat sections. 2. The areas of high density of nucleoside transport inhibitory sites found in guinea-pig cardiac sections were compared to the distribution of an endothelial cell marker, von Willebrand Factor, determined by radioimmunocytochemistry. These two markers were co-localized suggesting that coronary endothelial cells from guinea-pig have a high density of NBMPR binding sites and thus may have a high nucleoside transport capacity. 3. Nucleoside transporter subtypes with differing affinity for NBMPR or dipyridamole were investigated by quantitative autoradiography. Sites in rat tissues had high affinity for NBMPR (KD = 0.6 nM) but were of low sensitivity to dipyridamole (Ki = 3.1 microM). In guinea-pig sections, areas of high and low [3H]-NBMPR binding site density were analyzed separately. In both areas, sites had high affinity for NBMPR (KD = 1.4 nM, 4.5 nM, respectively) and dipyridamole (Ki = 108 nM, 245 nM, respectively). 4. While differences in density of nucleoside transport inhibitory sites are detectable between distinct regions of the heart, subtypes differing in affinity for NBMPR or dipyridamole were not evident. Therefore, more detailed structure activity studies are required to determine if subtypes of these sites exist within a single heart.

Potentiation of the effects of adenosine on isolated cardiac and smooth muscle by diazepam.

1. Adenosine (10(-7) to 3 x 10(-4) M) or 2-chloroadenosine (10(-8) to 10(-5) M) produced concentration-dependent inhibition of the responses of the rat isolated vas deferens to electrical field stimulation. In electrically driven (2 Hz) guinea-pig isolated left atria, adenosine (10(-6) to 10(-3) M) or 2-chloroadenosine (10(-8) to 10(-7) M) produced concentration-dependent decreases in isometric tension. 2. Diazepam (10(-6) and 10(-5) M) had no direct effect per se, but significantly potentiated the inhibitory action of adenosine on both tissues without altering the inhibitory effect of 2-chloroadenosine. 3. The adenosine uptake inhibitors, hydroxynitrobenzylthioguanosine (HNBTG, 10(-5) M) and dipyridamole (10(-5) M) also potentiated the inhibitory actions of adenosine in rat vas deferens, but not hose of 2-chloroadenosine. 4. Following adenosine uptake inhibition in rat vas deferens by HNBTG (10(-5) M), diazepam (10(-5) M) failed to produce any significant further potentiation of the inhibitory action of adenosine. 5. It is concluded that the potentiation of adenosine by diazepam is possibly due to an inhibition of adenosine uptake.

Effects of end-tidal concentrations of cyclopropane, halothane and diethyl ether on peripheral autonomic neuroeffector systems in the rat.

The effects of the inhalation anaesthetics, cyclopropane, halothane and diethyl ether were examined on peripheral neuroeffector systems in the pithed and in the conscious rat. 2 In the absence of a suitable means of accurately quantifying doses of inhalation anaesthetics given to small animals, an apparatus was constructed whereby end-tidal gas samples were collected semi-automatically from the mechanically ventilated rat. 3 Cyclopropane (15.3 and 29.3% end-tidal), halothane (0.20, 0.52 and 0.83% end-tidal) and diethyl ether (2% and 4% end-tidal) lowered the arterial pressure of the pithed rat. Heart rate was increased by diethyl ether 4%, decreased by halothane and unchanged by cyclopropane. 4 While each anaesthetic depressed the pressor responses to sympathetic nerve stimulation, cyclopropane increased and halothane and diethyl ether depressed the pressor responses to exogenous noradrenaline. 5 Each anaesthetic reduced the motor responses of the smooth muscle of the colon to parasympathetic stimulation. 6 The significance of the effects on peripheral neuroeffector systems is discussed in relation to the overall circulatory changes produced by these anaesthetics in the whole animal.

Adenosine receptors and nucleoside transport sites in cardiac cells.

1. Potential mechanisms responsible for the prominent depression of atrioventricular conduction by adenosine have been investigated in guinea-pig heart. 2. Adenosine A1 receptors and nucleoside transport (NT) sites were identified and enumerated in cardiac myocytes, atrioventricular conduction cells and coronary endothelial cells in 10 microns sections by autoradiographical analysis of the binding of the A1 selective antagonist 8-cyclopentyl-1,3-[3H]-dipropylxanthine ([3H]-DPCPX) and the NT ligand [3H]-nitrobenzylthioinosine ([3H]-NBMPR), respectively. 3. Atrioventricular conduction cells were identified by acetylcholinesterase histochemistry and endothelial cells by von Willebrand factor immunohistochemistry. 4. Site-specific binding of [3H]-DPCPX, when expressed as grains per cell nucleus was significantly higher (30 fold) in conduction cells than in surrounding myocytes. [3H]-DPCPX site density on endothelial cells in adjacent coronary vessels was not significantly different from myocytes. 5. In contrast, autoradiography of [3H]-NBMPR sites in these areas indicated that, relative to myocytes, conduction cells and endothelial cells were significantly enriched (2 fold and 4.5 fold, respectively) in NT sites. 6. The pronounced dromotropic effect of adenosine in guinea-pig heart is correlated with a higher density of adenosine A1 receptors in atrioventricular conduction cells than in myocytes. The NT capacity of these cells, as estimated by [3H]-NBMPR binding site density, is not increased in proportion to A1 receptors.

Comparison of the haemodynamic effects of adenosine monophosphate with sodium nitroprusside in a canine model of acute global left ventricular dysfunction.

1. The haemodynamic effects of adenosine 5'-monophosphate (AMP) and sodium nitroprusside (SNP) were compared in anaesthetized dogs following the induction of acute left ventricular (LV) dysfunction. 2. LV dysfunction was induced by the intracoronary administration of glass microbeads until left ventricular end diastolic pressure (LVEDP) was increased from 5 to 15 mmHg. This was associated with a decrease in LV dP/dt and cardiac index (CI) of 30% and 27%, respectively, and an increase in systemic vascular resistance index (SVRI) of 37%. 3. Graded doses of AMP (100 to 1000 micrograms kg-1 min-1) or SNP (1 to 10 micrograms kg-1 min-1) reduced SVRI and increased CI in a dose-related manner. Heart rate was not altered by either agent. At doses that caused similar reductions in SVRI, CI was increased more by AMP than by SNP. 4. The mechanisms responsible for the greater elevation of CI by AMP relative to SNP may be related to its more selective arterial vasodilator activity. SNP reduced cardiac preload that limited the expected increase in CI. 5. The haemodynamic profile of AMP suggests that it may be useful in the pharmacological management of acute cardiac failure, either when used alone or in combination with positive inotropic agents and/or selective venodilators.

Effects of ketamine on the peripheral autonomic nervous system of the rat.

The effects of ketamine (2-(o-chlorophenyl) 2-methylaminocyclohexanone) (2-50 mg/kg) on the responses of the pithed rat arterial pressure, anococcygeus muscle and colon to selective stimulation of the spinal autonomic outflows were examined. Ketamine depressed the vasopressor response produced by stimulation of the lumbar sympathetic outflow in a dose-dependent manner but did not significantly affect the pressor response to intravenous noradrenaline (NA) administration. Ketamine depressed the motor responses of the anococcygeus to stimulation of the pre-ganglionic lumbar sympathetic outflow or to stimulation of post-ganglionic fibres in the sacral region in a dose-dependent manner, the response to preganglionic stimulation being relatively more sensitive to such depression. The anococcygeus response to NA was significantly potentiated with doses of ketamine of 20 mg/kg and 50 mg/kg. Ketamine depressed the motor response of the smooth muscle of the colon to stimulation of the sacral parasympathetic outflow in a dose-dependent manner and at lower doses than were required to produce an equivalent depression of the sympathetic responses in the other tissues. A comparison was made of the effects of ketamine and cocaine on the motor responses of the anococcygeus muscle in vitro to NA, carbachol and field stimulation. Both ketamine and cocaine produced a non-specific depression of all responses at high doses whereas cocaine but not ketamine produced a large potentiation of NA and motor nerve responses at lower doses. The results are discussed in relation to the hypothesis that ketamine might elevate blood pressure in conscious animals and man by potentiating vascular adrenergic responses.

A technique for the study of muscle relaxants by stimulating the spinal motor nerve outflow in the pithed rat.

The motor nerve outflow in the pithed rat was stimulated from the spinal column and contractions of individual skeletal muscles recorded. The preparation is anaesthetic-free and well suited to a study of muscle relaxants.

Alteration of glycogen and glucose metabolism in ischaemic and post-ischaemic working rat hearts by adenosine A1 receptor stimulation.

1. Cardioprotection by adenosine A1 receptor activation limits infarct size and improves post-ischaemic mechanical function. The mechanisms responsible are unclear but may involve alterations in myocardial glucose metabolism. 2. Since glycogen is an important source of glucose during ischaemia, we examined the effects of N6-cyclohexyladenosine (CHA), an A1 receptor agonist, on glycogen and glucose metabolism during ischaemia as well as reperfusion. 3. Isolated working rat hearts were perfused with Krebs-Henseleit solution containing dual-labelled 5-3H and 14C glucose and palmitate as energy substrates. Rates of glycolysis and glucose oxidation were measured directly from the production of 3H2O and 14CO2. Glycogen turnover was measured from the rate of change of [5-3H and 14C]glucosyl units in total myocardial glycogen. 4. Following low-flow (0.5 ml min-1) ischaemia (60 min) and reperfusion (30 min), left ventricular minute work (LV work) recovered to 22% of pre-ischaemic values. CHA (0.5 microM) improved the recovery of LV work 2 fold. 5. CHA altered glycogen turnover in post-ischaemic hearts by stimulating glycogen synthesis while having no effects on glycogen degradation. CHA also partially inhibited glycolysis. These changes accelerated the recovery of glycogen in CHA-treated hearts and reduced proton production. 6. During ischaemia, CHA had no measurable effect on glycogen turnover or glucose metabolism. Glycogen phosphorylase activity, which was elevated after ischaemia, was inhibited by CHA, possibly in response to CHA-induced inhibition of AMP-activated protein kinase activity. 7. These results indicate that CHA-induced cardioprotection is associated with alterations of glycogen turnover during reperfusion as well as improved metabolic coupling of glycolysis to glucose oxidation.

Inhibition of glycolysis and enhanced mechanical function of working rat hearts as a result of adenosine A1 receptor stimulation during reperfusion following ischaemia.

1. This study examined effects of adenosine and selective adenosine A1 and A2 receptor agonists on glucose metabolism in rat isolated working hearts perfused under aerobic conditions and during reperfusion after 35 min of global no-flow ischaemia. 2. Hearts were perfused with a modified Krebs-Henseleit buffer containing 1.25 mM Ca2+, 11 mM glucose, 1.2 mM palmitate and insulin (100 muu ml-1), and paced at 280 beats min-1. Rates of glycolysis and glucose oxidation were measured from the quantitative production of 3H2O and 14CO2, respectively, from [5-3H/U-14C]-glucose. 3. Under aerobic conditions, adenosine (100 microM) and the adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA, 0.05 microM), inhibited glycolysis but had no effect on either glucose oxidation or mechanical function (as assessed by heart rate systolic pressure product). The improved coupling of glycolysis to glucose oxidation reduced the calculated rate of proton production from glucose metabolism. The adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 0.3 microM) did not alter glycolysis or glucose oxidation per se but completely antagonized the adenosine- and CHA-induced inhibition of glycolysis and proton production. 4. During aerobic reperfusion following ischaemia, CHA (0.05 microM) again inhibited glycolysis and proton production from glucose metabolism and had no effect on glucose oxidation. CHA also significantly enhanced the recovery of mechanical function. In contrast, the selective adenosine A2a receptor agonist, CGS-21680 (1.0 microM), exerted no metabolic or mechanical effects. Similar profiles of action were seen if these agonists were present during ischaemia and throughout reperfusion or when they were present only during reperfusion. 5. DPCPX (0.3 microM), added at reperfusion, antagonized the CHA-induced improvement in mechanical function. It also significantly depressed the recovery of mechanical function per se during reperfusion. Both the metabolic and mechanical effects of adenosine (100 microM) were antagonized by the nonselective A1/A2 antagonist, 8-sulphophenyltheophylline (100 microM). 6. These data demonstrate that inhibition of glycolysis and improved recovery of mechanical function during reperfusion of rat isolated hearts are mediated by an adenosine A1 receptor mechanism. Improved coupling of glycolysis and glucose oxidation during reperfusion may contribute to the enhanced recovery of mechanical function by decreasing proton production from glucose metabolism and the potential for intracellular Ca2+ accumulation, which if not corrected leads to mechanical dysfunction of the postischaemic myocardium.

Stimulatory and inhibitory histamine receptors in canine cystic duct.

1 The effects of histamine receptor stimulation were assessed on the resistance of the canine cystic duct in vivo and on the contractility of circular muscle preparations of canine cystic duct in vitro. 2 In anaesthetized dogs, the H1-receptor agonist, 2-pyridylethylamine (0.05 to 15 mumol, i.a.), elicited dose-dependent increases in cystic duct resistance, whereas the H2-receptor agonist, 4-methylhistamine (0.05 to 15 mumol, i.a.) decreased cystic duct resistance. These responses were antagonized by the H1-receptor antagonist, diphenhydramine, and the H2-receptor antagonist, cimetidine, respectively. 3 Histamine (0.1 to 3000 nmol, i.a.) also increased cystic duct resistance in vivo. In the presence of diphenhydramine, the stimulatory effect of histamine was antagonized and slight decreases in cystic duct resistance became apparent. Cimetidine or prazosin also antagonized the stimulatory effects of histamine. 4 Histamine (1 to 100 microM) or 2-pyridylethylamine (1 to 100 microM) contracted, whereas 4-methylhistamine (1 to 100 microM) relaxed, circular muscle preparations of cystic duct. These excitatory and inhibitory responses were antagonized by diphenhydramine and cimetidine, respectively. 5 These results indicate that the canine cystic duct possesses excitatory H1- and inhibitory H2-receptors. The predominant effect of histamine is an H1-receptor-mediated increase in cystic duct resistance. Histamine, which may be released in association with cholecystitis, may exert significant effects on the regulation of bile flow in and out of the gallbladder and may contribute to gallbladder stasis during biliary disease.

Cardiovascular selectivity of adenosine receptor agonists in anaesthetized dogs.

1. In order to determine the relevance of adenosine (Ado) receptor classification obtained from in vitro methods to the cardiovascular actions of Ado agonists in vivo, the cardiovascular effects of adenosine 5'-monophosphate (AMP), N6-cyclohexyladenosine (CHA, 400 fold A1-selective), 5'-N-ethyl-carboxamidoadenosine (NECA, A1 approximately A2) and 2-phenylaminoadenosine (PAA, 5 fold A2-selective) were compared in open-chest, fentanyl-pentobarbitone anaesthetized dogs. 2. Graded doses of CHA (10 to 1000 micrograms kg-1), NECA (0.5 to 100 micrograms kg-1) or PAA (0.1 to 20 micrograms kg-1) were administered intravenously and changes in haemodynamics and myocardial contractility were assessed 10 min following each dose. The effects of graded infusions of AMP (200 to 1000 micrograms kg-1 min-1) were also evaluated. 3. AMP and each of the Ado analogues (NECA > PAA > CHA) increased the systemic vascular conductance index (SVCI) in a dose-dependent manner and reduced mean arterial pressure (MAP). At doses causing similar increases in SVCI, these agonists caused (i) similar reflex increases in heart rate (HR) and cardiac index (CI) and decreases in AV conduction interval (AVi) and (ii) similar increases in coronary vascular conductance (CVC). 4. After cardiac autonomic blockade with atropine (0.2 mg kg-1) and propranolol (1 mg kg-1), AMP, CHA and PAA still increased SVCI and CVC and decreased MAP. CHA and PAA had no marked effects on HR, CI or AVi. As in the absence of cardiac autonomic blockade, equieffective vasodilator doses of CHA and PAA had identical effects on CVC, CI and AVi. 5.Myocardial contractility, as assessed by E,,,,, measurements, was stimulated by AMP in control animals. Following cardiac autonomic blockade, PAA increased contractility while AMP and CHA had no significant effects.6. Despite marked.differences in receptor selectivity in vitro, no marked differences between the actions of these Al- and A2-selective Ado receptor agonists on the cardiovascular system in vivo were apparent.Difficulties therefore exist in the application of in vitro Ado receptor selectivity data to the prediction of the cardiovascular effects of Ado agonists in vivo.

K(ATP)-channel activation: effects on myocardial recovery from ischaemia and role in the cardioprotective response to adenosine A1-receptor stimulation.

1. Optimization of myocardial energy substrate metabolism improves the recovery of mechanical function of the post-ischaemic heart. This study investigated the role of K(ATP)-channels in the regulation of the metabolic and mechanical function of the aerobic and post-ischaemic heart by measuring the effects of the selective K(ATP)-channel activator, cromakalim, and the effects of the K(ATP)-channel antagonist, glibenclamide, in rat fatty acid perfused, working hearts in vitro. The role of K(ATP) channels in the cardioprotective actions of the adenosine A1-receptor agonist, N6-cyclohexyladenosine (CHA) was also investigated. 2. Myocardial glucose metabolism, mechanical function and efficiency were measured simultaneously in hearts perfused with modified Krebs-Henseleit solution containing 2.5 mM Ca2+, 11 mM glucose, 1.2 mM palmitate and 100 mu l(-1) insulin, and paced at 300 beats min(-1). Rates of glycolysis and glucose oxidation were measured from the quantitative production of 3H20 and 14CO2, respectively, from [5-3H/ U-14C]-glucose. 3. In hearts perfused under aerobic conditions, cromakalim (10 microM), CHA (0.5 microM) or glibenclamide (30 microM) had no effect on mechanical function. Cromakalim did not affect glycolysis or glucose oxidation, whereas glibenclamide significantly increased rates of glycolysis and proton production. CHA significantly reduced rates of glycolysis and proton production but had no effect on glucose oxidation. Glibenclamide did not alter CHA-induced inhibition of glycolysis and proton production. 4. In hearts reperfused for 30 min following 30 min of ischaemia, left ventricular minute work (LV work) recovered to 24% of aerobic baseline values. Cromakalim (10 microM), administered 5 min before ischaemia, had no significant effect on mechanical recovery or glucose metabolism. CHA (0.5 microM) significantly increased the recovery of LV work to 67% of aerobic baseline values and also significantly inhibited rates of glycolysis and proton production. Glibenclamide (30 microM) significantly depressed the recovery of mechanical function to < 1% of aerobic baseline values and stimulated glycolysis and proton production. 5. Despite the deleterious actions of glibenclamide per se in post-ischaemic hearts, the beneficial effects of CHA (0.5 microM) on the recovery of mechanical function and proton production were not affected by glibenclamide. 6. The data indicate that the cardioprotective mechanism of adenosine A1-receptor stimulation does not involve the activation of K(ATP)-channels. Furthermore, in rat fatty acid perfused, working hearts, stimulation of K(ATP)-channels is not cardioprotective and has no significant effects on myocardial glucose metabolism.

Influence of beta-adrenoceptor tone on the cardioprotective efficacy of adenosine A(1) receptor activation in isolated working rat hearts.

This study investigated the role of beta-adrenoceptors in the cardioprotective and metabolic actions of adenosine A(1) receptor stimulation. Isolated paced (300 beats min(-1)) working rat hearts were perfused with Krebs-Henseleit solution containing 1.2 mM palmitate. Left ventricular minute work (LV work), O(2) consumption and rates of glycolysis and glucose oxidation were measured during reperfusion (30 min) following global ischaemia (30 min) as well as during aerobic conditions. Relative to untreated hearts, N(6)-cyclohexyladenosine (CHA, 0.5 microM) improved post-ischaemic LV work (158%) and reduced glycolysis and proton production (53 and 42%, respectively). CHA+propranolol (1 microM) had similar beneficial effects, while propranolol alone did not affect post-ischaemic LV work or glucose metabolism. Isoprenaline (10 nM) impaired post-ischaemic function and after 25 min ischaemia recovery was comparable with 30 min ischaemia in untreated hearts (41 and 53%, respectively). Relative to isoprenaline alone, CHA+isoprenaline improved recovery of LV work (181%) and reduced glycolysis and proton production (64 and 60%, respectively). In aerobic hearts, CHA, propranolol or CHA+propranolol had no effect on LV work or glucose oxidation. Glycolysis was inhibited by CHA, propranolol and CHA+propranolol (50, 53 and 52%, respectively). Isoprenaline-induced increases in heart rate, glycolysis and proton production were attenuated by CHA (85, 57 and 53%, respectively). The cardioprotective efficacy of CHA was unaffected by antagonism or activation of beta-adrenoceptors. Thus, the mechanism of protection by adenosine A(1) receptor activation does not involve functional antagonism of beta-adrenoceptors.

Angiotensin II reduces infarct size and has no effect on post-ischaemic contractile dysfunction in isolated rat hearts.

1. In order to test the hypothesis that angiotensin II exacerbates myocardial ischaemia-reperfusion (IR) injury, we examined the effects of graded angiotension II concentrations of angiotensin II on IR injury in both working and non-working (Langendorff) isolated rat hearts. 2. Non-working hearts were subjected to 30 min aerobic perfusion (baseline) then 25 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=7) or presence of 1 (n=6) or 10 nM (n=5) angiotensin II). Recoveries of LV developed pressure and coronary flow after 30 min reperfusion in control hearts (58+/-9 and 40+/-8% of baseline levels, respectively) were no different from hearts treated with 1 or 10 nM angiotensin II. Infarct size (determined at the end of reperfusion by triphenyltetrazolium chloride staining) was reduced by angiotensin II in a concentration-dependent manner (from a control value of 27+/-3 to 18+/-4% and 9+/-3% of the LV, respectively). 3. Working hearts were subjected to 50 min pre-ischaemic (pre-I) aerobic perfusion then 30 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=14) or presence of 1 (n=8), 10 (n=7) or 100 nM (n=7) angiotensin II). In controls, post-ischaemic (post-I) left ventricular (LV) work and efficiency of oxygen consumption were depressed (43+/-9 and 42+/-10% of pre-I levels, respectively). The presence of angiotensin II throughout IR had no effect on LV work compared with control. 4. Thus, angiotensin II reduces infarct size in a concentration-dependent manner but has no effect on contractile stunning associated with IR in isolated rat hearts.

Deficiency in myocardial NO biosignalling after cardioplegic arrest: mechanisms and contribution to post-storage mechanical dysfunction.

1 In order to understand mechanisms that limit the safe ischaemic time of donor hearts, this study evaluated NO/cyclic GMP biosignalling in the recovery of function after cardioplegia and hypothermic storage. 2 Hearts removed from anaesthetized rats were either perfused in working mode (Fresh) or arrested (St. Thomas' II cardioplegia) and stored at 3 degrees C for 8 h (CPL) prior to working mode perfusion. LV work and indices of the production of NO (Ca2+-dependent and Ca2+-independent NOS), cyclic GMP (soluble guanylyl cyclase (sGC) and GTP) and superoxide (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)) were measured. 3 Relative to Fresh hearts, CPL hearts were deficient in cyclic GMP and had poor function. Correction of cyclic GMP deficiency (SNP, 200 microM) improved LV work and LV compliance. SNP effects were prevented by inhibition of sGC (ODQ, 3 microM), and potentiated by inhibition of cyclic GMP-dependent phosphodiesterase (zaprinast, 20 microM). SNP (200 microM) had no effect on function of Fresh hearts. 4 NOS activities (pH = 7.2) were similar in CPL and Fresh hearts, but at end-ischaemic pH (6.3), Ca2+-dependent NOS activity was reduced. The sensitivity of sGC to SNP was greater, and activities of XO and XDH were higher, in CPL than in Fresh hearts. 5 The deficiency in NO biosignalling in CPL hearts may arise due to acidosis-induced inhibition of NOS activity, reduced availability of GTP and/or enhanced inactivation of NO by superoxide. These findings provide rationales for novel strategies to prevent the deficiency in NO biosignalling and so improve the function of the transplanted heart.

Nucleoside transport in guinea pig myocytes. Comparison of the affinities and transport velocities for adenosine and 2-chloroadenosine.

The affinities of adenosine and 2-chloroadenosine for the nucleoside transport system of guinea pig myocytes were evaluated indirectly by studying the inhibition of the binding of [3H]nitrobenzylthioinosine and directly by measuring the influx of [3H]radiolabeled substrates. Maximal transport velocities of the two nucleosides were also obtained. [3H]Nitrobenzylthioinosine bound to a single class of high-affinity sites (KD of 0.8 nM) which possessed a maximal binding capacity (Bmax) of 870,000 sites/cell. Adenosine, 2-chloroadenosine or the nucleoside transport inhibitor, dipyridamole, competitively inhibited the site-specific binding of [3H]nitrobenzylthioinosine with Ki values of 318 microM, 22 microM and 75 nM respectively. Both [3H]adenosine and [3H]2-chloroadenosine entered myocytes in a saturable and inhibitible manner. Observed transport kinetic constants (Km and Vmax) were 146 microM and 24.2 pmoles/10(6) cells/sec, respectively, for adenosine and 36 microM and 11.7 pmoles/10(6) cells/sec, respectively for 2-chloroadenosine. Affinities of adenosine, 2-chloroadenosine, nitrobenzylthioinosine and dipyridamole for the nucleoside transport system derived from binding and influx methodologies were equivalent which confirms that [3H]nitrobenzylthioinosine binding sites are closely associated with the nucleoside transporter.

5'-Deoxy-5'-methylthioadenosine: a nucleoside which differentiates between adenosine receptor types.

The activities of an endogenous nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine A1 and A2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8-p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [3H]nitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A1 receptor but an antagonist at the A2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A2 receptor which activates adenylate cyclase.