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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.

This study investigated the role of β-adrenoceptors in the cardioprotective and metabolic actions of adenosine A1 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), O2 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, N6-cyclohexyladenosine (CHA, 0.5 μM) improved post-ischaemic LV work (158%) and reduced glycolysis and proton production (53 and 42%, respectively). CHA+propranolol (1 μM) 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 β-adrenoceptors. Thus, the mechanism of protection by adenosine A1 receptor activation does not involve functional antagonism of β-adrenoceptors. British Journal of Pharmacology (2000) 131, 537–545; doi:10.1038/sj.bjp.0703597

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.

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. 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. 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. 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 μM) improved the recovery of LV work 2 fold. 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. 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. 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. British Journal of Pharmacology (1999) 128, 197–205; doi:10.1038/sj.bjp.0702765

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.

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 (NECAPAACHA) 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.