Mediation by adenosine of bradycardia in rat heart during graded global ischaemia

The role of adenosine as a mediator of the bradycardia associated with graded global ischaemia in rat heart was examined. Hearts were perfused at 37°C in the isovolumic mode with Krebs-bicarbonate medium at 12.0 ml/min/g. After equilibration, the coronary flow was reduced to 0.5, 2.5, or 5.0 ml/min/g for 20 min. Effluent was collected and assayed for adenosine and inosine by HPLC. Heart rate was measured and bipolar electrograms were obtained in severely ischaemic hearts. Basal adenosine release was 124±15 pmol/min/g. Adenosine release increased by approximately 50% in hearts perfused at 5.0 ml/min/g. In hearts perfused at 2.5 and 0.5 ml/min/g, adenosine release increased by approximately 1300 and 2300% respectively. The pattern of adenosine release at 0.5 and 2.5 ml/min/g was phasic, with adenosine release rate increasing to a maximum after about 10 min then dropping to values slightly higher than initial values. Ischaemia produced significant bradycardia and first degree AV block. Adenosine antagonism with 5 m 8-phenyltheophylline blocked up to 25% of this bradycardia and significantly reduced the conduction delay. Adenosine release rate correlated closely with that component of heart rate slowing which was inhibited by 8-phenyltheophylline. It is concluded that adenosine released during graded global ischaemia mediates up to a quarter of the associated bradycardia. The effect of adenosine is phasic. Adenosine acts primarily to depress the sinus pacemaker. First degree AV block also occurs. These effects were only apparent at coronary flow rates below 5.0 ml/min/g.     

Endogenous adenosine improves work rate to oxygen consumption ratio in catecholamine stimulated isovolumic rat heart

This study examines the possibility that endogenous adenosine modulates efficiency in isovolumic perfused rat hearts stimulated with isoproterenol or norepinephrine. Efficiency in these hearts is calculated as the rate of pressure work divided by the myocardial oxygen consumption. Within 2 min of infusion of isoproterenol (50 nM), heart rate increased by 35%, the rate pressure product by 290%, oxygen consumption by 142%, and efficiency by 67%. Infusion of adenosine deaminase (2–4 IU/ml), or 8-phenyltheophylline (5 M), into stimulated hearts augmented the increase in heart rate by 40–45%, rate-pressure product by 18–20%, and oxygen consumption by 50–55%. Efficiency was reduced by 30–35%. Adenosine release into the coronary venous effluent increased from 195±20 pmol/min/g to 2400±180 pmol/min/g after 5 min. A similar pattern of results was observed when norepinephrine (0.1 mM) was used. The results indicate that extracellular adenosine, released by catecholamine treatment, inhibits the effects of the catecholamines on rate and contractility. Consequently, adenosine reduces cardiac work (rate-pressure product), but in so doing, improves efficiency.     

Pre‐conditioning activates adenosine utilization in a cost‐effective way during myocardial ischaemia

During pre‐conditioning the interstitial concentration of adenosine, in contrast to lactate, presents a die‐away curve‐pattern for every successive episode of ischaemia. This die‐away pattern might not necessarily be attributed to diminished adenosine production. The present study was undertaken to investigate whether pre‐conditioning alters the metabolic turnover of adenosine as observed by the lactate production during ischaemia. Interstitial levels of metabolites in pre‐conditioned (n=21) and non‐preconditioned (n=21) porcine hearts were monitored with microdialysis probes inserted in both ischaemic and non‐ischaemic tissue in an open chest heart model. Three subgroups perturbated with either plain microdialysis buffer (control), buffer containing adenosine (375 μM ), or buffer containing deoxyadenosine (375 μM ) were studied. All animals were subjected to 90 min of equilibrium microdialysis before 40 min of regional myocardial ischaemia and 120 min of reperfusion. Pre‐conditioning consisted of four repetitive episodes of 10 min of ischaemia and 20 min of reperfusion. Significantly higher levels of inosine and lactate were found in the ischaemic tissue of the pre‐conditioned subgroup receiving adenosine (P < 0.05) compared with the other two subgroups receiving deoxyadenosine and plain buffer, respectively. This difference was only valid for pre‐conditioned ischaemic myocardium, and hence equal amounts of inosine and lactate were produced in the non‐preconditioned ischaemic myocardium regardless of the presence of adenosine or deoxyadenosine. In the non‐ischaemic myocardium baseline levels of metabolites were measured in all subgroups. Pre‐conditioning favoured degradation of exogenous adenosine to inosine successively ending up in enhanced lactate production. This was probably because of the involvement of the hexose monophosphate pathway in the pre‐conditioned ischaemic myocardium. This route may therefore be supplementary in energy metabolism as a metabolic flow can be started by adenosine ending up in lactate without initial adenosine 5′‐triphosphate (ATP) investment. Utilization of adenosine in this way may also explain the successive die‐away pattern of adenosine seen in consecutive pre‐conditioning cycles.     

Endogenous nitric oxide attenuates hypoxic vasoconstriction of small pulmonary arteries and veins in anaesthetized cats

Ischaemic preconditioning has cardioprotective effects. Reactive oxygen species may be possible mediators. The present study investigated whether low doses of exogenous hydrogen peroxide could mimic preconditioning in isolated, Langendorff-perfused rat hearts. Hearts were subjected to two episodes of 3 min global ischaemia and 5 min reperfusion (n = 17), or were given 10 (n=15), 20 (n=10), 30 (n=20), 40 (n=18), 80 (n=17) or 160 μM (n=10) hydrogen peroxide for 10 min, followed by 10 min recovery, before 25 min global ischaemia and 60 min reperfusion, and compared with ischaemic controls of matching perfusion time (n=17 and n=23). Cardiac performance was assessed by heart rate, left ventricular systolic, end-diastolic and developed pressures, and coronary flow. Severe reperfusion arrhythmias occurred frequently in control hearts, and was attenuated by ischaemic preconditioning. All hearts pretreated with 160 μM hydrogen peroxide had severe arrhythmias throughout reperfusion, while these were not seen in any heart perfused with 20 μM hydrogen peroxide (P< 0.01 compared to controls). Ischaemia and reperfusion induced a minor decrease in heart rate, left ventricular systolic and developed pressures, and increased end-diastolic pressure. Ischaemic preconditioning attenuated the decrease of heart rate and the increase of end-diastolic pressure, and increased coronary flow, while hydrogen peroxide did not significantly attenuate these changes. In conclusion, a low dose of exogenous hydrogen peroxide before global ischaemia inhibited severe reperfusion arrhythmias, but had no other protective effects. The present work does not suggest that reactive oxygen species are important mediators of the preconditioning effects on stunning and arrhythmias in the rat heart.     

Correlation of sinus slowing and hyperpolarization caused by adenosine in sinus node

The effect of adenosine on sinus node cells was examined in a preparation that precluded pacemaker shift. It was found that adenosine produced a dose-dependent slowing in rate. In examining the effects on the action potential parameters (n=10), adenosine caused a significant increase in the maximum diastolic potential (control =–62±2 mV, adenosine, 1×10^–4 M, =–67±3 mV) and a significant increase in the rate of rise of the action potential (control=3.3±0.6 V/s, adenosine, 1×10^–4 M, =7.2±2 V/s). There was only a slight shortening of the action potential duration and a small increase in the action potential overshoot. Adenosine caused a significant decrease in the rate of diastolic depolarization (control=100±19 mV/s, adenosine, 1×10^–4 M, =42±5 mV/s). Acetylcholine caused similar effects. The effects of adenosine were not affected by atropine or propranolol but were antagonized by aminophylline, an adenosine competitive antagonist. In another set of experiments (n=12) we sought to understand further the mechanism of sinus slowing caused by adenosine and compare with the effects of acetylcholine. The increase in cycle length due to different doses of adenosine and acetylcholine was measured. The preparation was then arrested with D-600 or NiCl₂. The cells were then exposed to the same concentrations of adenosine and acetylcholine and the amount of hyperpolarization from the resting potential ({ie75-1}=–40±4 mV) was measured. The change in cycle length and amount of hyperpolarization were linearly correlated (r=0.86). The interaction between adenosine and acetylcholine on the hyperpolarization was investigated further in another set of experiments. When acetylcholine and adenosine were added together the onset and magnitude of the hyperpolarization was greater than for adenosine alone. If atropine was given, still in presence of acetylcholine and adenosine, the hyperpolarization due to adenosine was revealed. The maximal hyperpolarization obtainable was always greater for acetylcholine than for adenosine. Furthermore, there was no additive effect of the highest dose of adenosine (2×10^–4M) on the maximal hyperpolarization caused by acetylcholine. The hyperpolarization caused by adenosine or acetylcholine was not affected by ouabain or cesium. The results suggest that adenosine and acetylcholine slow the SA node rate by a similar mechanism but via different receptors. The data are consistent with adenosine causing an increase in potassium conductance which is not blocked by cesium.Supported by Grant-in-Aid 81-911 from the American Heart Association — Virginia Affiliate, U.S. Public Health Services Grant R01-HL3111 and University of Virginia Award BRS 5S07-RR05431-22.Supported by Grant-in-Aid 81-911 from the American Heart Association — Virginia Affiliate, U.S. Public Health Services Grant R01-HL3111 and University of Virginia Award BRS 5S07-RR05431-22.     

Sinus slowing and pacemaker shift caused by adenosine in rabbit SA node

Adenosine has a negative chronotropic effect in a number of species. The studies reported here were undertaken to characterize further the effects of adenosine using isolated perfused rabbit hearts and isolated SA node tissue. In the isolated perfused hearts (n=9) the threshold doses for slowing by adenosine and 2-chloroadenosine, an adenosine analog, were 1×10^–5 M and 1×10^–7 M, respectively. In the isolated hearts adenosine, in addition to slowing sinus rate, also caused a change in the activation pattern of the right atrium. During adenosine infusion the earliest site of activation shifted from the SA node region to the right atria near the crista terminalis. The pacemaker shift was reversible upon washout of adenosine. The adenosine-induced shift in pacemaker could also be demonstrated using microelectrode recordings in the isolated SA node preparation that included the crista terminalis and some of the surrounding tissue. During control the activation of SA node preceded that of the crista terminalis (CT) by 44±4.1 ms (n=11). Adenosine infusion caused an increase in cycle length and, in addition shifted the earliest site of activation from the SA node region to CT, i.e., in the presence of adenosine CT preceded SA node activation by 26.68±3.2 ms. All the effects were reversible after washout of adenosine. Adenosine also caused conduction block within the sinus node. No effect on the action potentials or on conduction in the CT was observed. In small preparations (250×250 m) which precludes pacemaker shift (n=18), adenosine and 2-chloroadenosine slowed the rate and caused a decrease in rate of phase four depolarization. The threshold for adenosine and 2-chloro-adenosine was 1×10^–5 M and 3×10^–7, respectively. Associated with pacemaker slowing was an increase in the maximum diastolic potential with a concomitant increase in the maximum rate of rise of the action potential. Adenosine had no effect on the SA node action potential duration or amplitude. The results were similar to those observed for acetylcholine, however, the adenosine effects were blocked by aminophylline but not by atropine. Adenosine-induced sinus slowing and pacemaker shift may be of importance during periods of metabolically compromised myocardium such as hypoxia and ischemia where there is increased production of adenosine.Supported by Grant-in-Aid 81-911 from the American Heart Association — Virginia Affiliate, U.S. Public Health Services Grant R01-HL3111 and University of Virginia Award BRS 5S07-RR05431-22. Dr. Belardinelli is the recipient of NIH Research Career Development Award 1K04-HL00969-01     

Preconditioning does not attenuate cardiac dysfunction after global ischaemia in the guinea-pig

Ischaemic preconditioning reduces infarct size, but the effects on cardiac function after global ischaemia are more controversial. Additionally, species differences may exist. The present study investigates the effects of preconditioning on cardiac performance in the globally ischaemic, Langendorff-perfused guinea-pig heart. Hearts were stabilized for 25 min, and divided into the following groups: (1) (n = 8) control perfusion for 16 min before 30-min global ischaemia and 30-min reperfusion, (2) (n = 7) two episodes of 3-min ischaemia and 5-min reperfusion before global ischaemia, (3) (n = 7) 5-min ischaemia and 10-min reperfusion before ischaemia, (4) (n = 8) control perfusion before 40-min ischaemia and 30-min reperfusion, (5) (n = 8) Preconditioning as group 2 before ischaemia as group 4, (6) (n = 9) Control perfusion before 50-min ischaemia and 30-min reperfusion, (7) (n = 10) Preconditioning as group 2 before ischaemia as group 6. A dose-dependent reduction of left ventricular systolic pressure, and increase of end-diastolic pressure was observed during reperfusion after 30-, 40- and 50-min ischaemia. Preconditioning did not influence these changes, nor did it attenuate the incidence of severe reperfusion arrhythmias or reduction of coronary flow. In conclusion, ischaemic preconditioning does not improve cardiac function during reperfusion of the globally ischaemic, isolated guinea-pig heart.     

Contractile properties of skinned preparations from ischaemic canine myocardium and coronary arteries

The influence of prolonged ischaemia on the regulation of contraction in the myocardium and in the smooth muscle of coronary arteries was investigated. Chemically skinned preparations were used which enabled the contraction to be studied with the environment of the contractile filaments controlled. Myocardial ischaemia was produced in anaesthetized adult beagle dogs by occlusion of the left anterior descending artery for 3 h and followed by 30 min reperfusion. Myocardial tissue and segments from coronary arteries were obtained from the ischaemic infarcted wall region (in vivo ischaemic) and compared with control preparations from perfused coronary arteries and from the free wall of the left ventricle. Coronary and myocardial preparations were also obtained from the heart after a 3 h period in vitro under anoxic conditions at 37°C (in vitro ischaemic) simulating a state of extreme ischaemia. Control myocardial fibres were fully relaxed at pCa (-log-[Ca²⁺]) 9 and developed 24±5% (n=7) of maximum force at intermediate calcium concentration (pCa 5.5). In contrast, the in vivo and in vitro ischaemic preparations produced force at pCa 9 (28±13 and 39±8%, respectively, n=5 and 7) and showed an increased force development at pCa 5.5 (53±11 and 75±5%). The in vivo and in vitro ischaemic coronary arteries relaxed more slowly following calcium removal than control vessels. The in vitro ischaemic vascular preparations developed active force at pCa 9 and showed increased levels of myosin light chain phosphorylation and reduced phosphatase activity. This suggests a reduced rate of dephosphorylation as a cause for the changes in contracile behaviour of the smooth muscle. In conclusion, extreme ischaemia in vitro is associated with a loss of calcium regulation and an increased calcium sensitivity of the contractile system in myocardium and changes in the phosphorylation/dephosphorylation reactions of coronary arteries. The changes in myocardium appear to occur also during ischaemia in vivo, and might contribute to contracture development in cells under conditions when adenosine triphosphate synthesis is reestablished after reperfusion.     

Sulfonylureas enhance GABAA synaptic potentials in rat midbrain dopamine neurones

Long-lasting myocardial ischaemia reduces the density of sarcolemmal L-type calcium channels (LCC). Ischaemic preconditioning protects the myocardium against development of infarction. The aim of this study was to investigate if ischaemia-induced loss in LCC is affected by ischaemic preconditioning. Specific (+)-[³H]isradipine binding to LCC was compared in membranes and homogenates from control and ischaemic regions of non-preconditioned and ischaemically preconditioned hearts [two 10 min left anterior descending coronary artery (LAD) occlusions, each followed by 30 min reperfusion]. Biopsies were sampled after 60 min mid LAD occlusion from ischaemic and control (supplied by circumflex artery) regions. Sixty min ischaemia reduced binding density of specific (+)-[³H]isradipine in membranes by 23±11% (n=7, P<0.05) in the non-preconditioned group and by 20±8% (n=6, P<0.05) in the preconditioned group. Binding density in homogenates was reduced by 36±5% (n=5, P<0.05) in the non-preconditioned group and by 21±5% (n=5, P<0.05) in the preconditioned group. The reductions in the two groups and reductions in membranes and homogenates were not statistically different. The dissociation constant of binding was similar in the groups. In conclusion, 60 min of ischaemia reduced the binding density of (+)-[³H]isradipine in membranes and homogenates by 20–36%. The reduction in density of binding sites was not caused by redistribution of sarcolemmal LCC to an intracellular compartment. Ischaemic preconditioning did not affect the decline in binding density as hypothesized.     

Additive protective effects of late and early ischaemic preconditioning are mediated by the opening of KATP channels in vivo

We investigated whether a combination of ischaemic late preconditioning (LPC) and ischaemic early preconditioning (EPC) induces additive myocardial protection in vivo, and the role of ATP-sensitive K (KATP) channels in ischaemic LPC and in LPC + EPC. Sixty rabbits were divided into seven groups. Anaesthetized animals were subjected to 30 min of coronary artery occlusion and 120 min of reperfusion (I/R). Controls (CON, n = 9) were not preconditioned. LPC (n = 10) was induced in conscious rabbits by a 5-min period of myocardial ischaemia 24 h before I/R. The KATP channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg) was given 10 min before I/R with (LPC + 5-HD, n = 9) or without LPC (5-HD, n = 8). EPC (n = 8) was induced by a 5-min period of myocardial ischaemia 10 min before I/R. Animals received LPC and EPC without (LPC + EPC, n = 8) or with 5-HD (LPC + EPC + 5-HD, n = 8). LPC reduced infarct size (IS, triphenyltetrazolium staining) from 57 +/- 11% (MW +/- SD, CON) of the area at risk to 31 +/- 19% (LPC, P = 0.004). 5-HD did not affect IS (5-HD: 60 +/- 12%, P = 0.002 versus LPC), but abolished the cardioprotective effects of LPC (LPC + 5-HD: 62 +/- 18%, P = 0.001 versus LPC). EPC reduced IS to 18 +/- 8%. Additional LPC led to a further reduction to 8 +/- 4% (LPC + EPC, n = 8; P = 0.005 versus EPC; P = 0.004 versus LPC). 5-HD abolished this additional cardioprotective effect of LPC + EPC (LPC + EPC + 5-HD, n = 8; 46 +/- 11%, P < or = 0.001 versus LPC + EPC). We conclude that the combination of ischaemic LPC and EPC induces additive cardioprotection. KATP channel opening mediates the cardioprotective effects of ischaemic LPC and LPC + EPC.     

Nitric oxide mediates the anti-adrenergic effect of adenosine on calcium current in isolated rabbit atrioventricular nodal cells

The aim of this study was to determine if adenosine exerts an anti-adrenergic effect on rabbit isolated atrioventricular (AV) nodal cells and, if so, the dependence of this effect on nitric oxide (NO) production. Inward Ca current,I _Ca, was measured in AV nodal cells, enzymatically isolated from rabbit hearts. Isoprenaline (0.1 M) increasedI _Ca from 676 ± 59 to 1102 ± 86 pA (n = 25). This isoprenaline-induced increase inI _Ca, (178 ± 15 % of control) was abolished in the presence of 10 M adenosine (I _Ca 100 ± 2 % of control,n = 9, P < 0.05). This effect of adenosine was completely blocked by the A₁ receptor antagonist CPDPX (8-cyclopentyl 1, 3-dipropylxanthine, 0.1 M). In cells pre-treated with the NO synthase inhibitor,l-nitro-arginine methyl ester (l-NAME, 1 mM) the isoprenaline-induced increase inI _Ca(208 ± 39 % of control,n = 7) was not reduced by the addition of 10 M adenosine (195 ± 32% of control). Co-incubation of cells inl-NAME withl--arginine (1 mM, the endogenous substrate of NO synthase) restored the adenosine-induced attenuation ofI _Ca. In these cells, isoprenaline increasedI _Ca (157 ± 7% of control,n = 6), and, following addition of adenosine (10 M)I _Ca was reduced to 107 ± 8% (P < 0.05). The NO-releasing agent SIN-1 (3-morpholino-sydnonimine, 100 M) inhibitedI _Ca augmented by isoprenaline (n = 5). It is concluded that adenosine exerts an anti-adrenergic effect on the AV node via A, receptors to attenuate a catecholamine-stimulated increase inI _Ca and that this action involves the intracellular production of NO.     

Changes in adenosine release and blood flow in the contracting dog gracilis muscle

Ischaemic contraction of skeletal muscle increases the venous concentration of adenosine. The present investigation was undertaken to determine changes in blood flow and the release of adenosine into venous blood resulting from 5 min of free flow contractions of the isolated gracilis muscle in dogs anaesthetised with pentobarbitone sodium (42 mg·kg^–1) and artificially ventilated. Arterial and venous concentrations of adenosine were measured by high performance liquid chromatography. Five-minute-contractions (induced electrically, 6 V, 1.8 ms, 4 Hz) caused significant increases in blood flow (to 304±33% of control; mean ±SEM,n=9) and venous plasma adenosine concentration (from 126±18 nM to 293±76 nM, equivalent to an average increase in release of 7.28±1.89 nmol·min^–1 100 g^–1 wet weight of muscle). The venous oxygen tension decreased from 8.33±0.48 to 3.39±0.31 kPa (62.5±3.6 to 25.4±2.3 mm Hg). This small but significant increase in venous adenosine concentration within the vasoactive range, in the face of a concomitant increase in blood flow, suggests that an increase in the interstitial adenosine concentration during free-flow exercise may contribute to the total dilatation of the resistance vessels to increase blood flow and keep its own concentration low. A significant correlation between venous adenosine concentration and vascular conductance is therefore absent. The results suggest that adenosine may contribute to sustained active hyperaemia in skeletal muscle.     

Tissue content of adenosine,inosine and hypoxanthine in the rat kidney after ischemia and postischemic recirculation

Summary The effect of renal ischemia of 15 s to 60 min duration on the tissue levels of adenosine, inosine and hypoxanthine was investigated in Sprague Dawley rats. A sharp increase in the tissue levels of adenosine from 5.13±0.56 to 31.3±2.96 nmol/g wet weight after 1 min of ischemia was found. The tissue levels of inosine and hypoxanthine in the controls were 3.62±0.51 and 3.19±0.76 nmol/g wet weight, respectively. Maximal levels of adenosine (38.1±6.3 nmol/g wet weight) were reached after 10 min of ischemia. The hypoxanthine levels rose steadily up to 922±183 nmol/g wet weight after 60 min of ischemia. Recirculation of 15 min after 60 min ischemia resulted in a fall of adenosine and inosine levels to values comparable to controls, whereas hypoxanthine was elevated above control values. In a second experimental series with tracing of renal blood flow (RBF) by a means of an electromagnetic flow meter a transient marked reduction of RBF after occlusion of the renal artery for 30 s was observed. The 3-fold increase of adenosine tissue levels within 30 s of renal artery occlusion and the inhibition of the postocclusive RBF reduction by theophylline (3.3 mol/100 g body weight) make it likely that this phenomenon may be caused by intrarenal adenosine.Parts of this investigation were presented at the 46th Meeting of the German Physiological Society in Regensburg, March 15–20, 1976.Supported by the Deutsche Forschungsgemeinschaft Os 42/2     

Low- and high-blood flow regions in the normal pig heart are equally vulnerable to ischaemia during partial coronary stenosis

 Myocardial perfusion is heterogeneous, even in the normal heart. It is unknown whether the resting normal blood flow level predicts the severity of mismatch between local blood flow and metabolism during acute ischaemia. In the present study local blood flow (measured with radioactively labelled microspheres) and metabolic indicators of ischaemia [tissue contents of lactate and inosine (INO), a breakdown product of adenosine triphosphate (ATP)] were determined in 84–102 simultaneously frozen samples (approximately 0.9 g) of normal (n = 7) and partially ischaemic (n = 4) porcine left ventricles. Ischaemia was induced for 20 min by partially occluding the left anterior descending artery to reduce perfusion pressure from 107 ± 17 mm Hg to 39 ± 10 mm Hg (mean ± SD). Flow reduction in the ischaemic region was strongly variable, both within the subepicardium (range 6–66%, average 34%) and the subendocardium (range 33–84%, average 57%), indicating redistribution of blood flow inside transmural layers in addition to the well-known preferential decrease in subendocardial perfusion. The relative flow reduction during stenosis was not dependent on normal local perfusion level (Spearman rank correlation coefficient –0.002, P = 0.99). Samples with low or high myocardial blood flows before stenosis showed similar increases in lactate content and INO/ATP content ratio, as long as the percentage blood flow reduction was the same. It is concluded that regions with low and high resting flows in the normally perfused heart are equally susceptible to metabolism-perfusion mismatch resulting from coronary stenosis. Received: 6 December 1996 / Received after revision: 25 June 1997 / Accepted: 6 July 1997     

Natriuretic peptides increase a K+ conductance in rat mesangial cells

Mesangial cells (MC) are a main target of natriuretic peptides in the kidney and are thought to play a role in regulating glomerular filtration rate. We examined the influence of cGMP-generating (i.e. guanosine 3,5-cyclicmonophosphate) peptides on membrane voltages (V_m) of rat MC by using the fast whole-cell patch-clamp technique. The cGMP-generating peptides were tested at maximal concentrations ranging from 140 to 300 nmol/1. Whereas human CNP (C natriuretic peptide), rat guanylin and human uroguanylin had no significant effect on V_m of these cells, human BNP (brain natriuretic peptide), rat CDD/ANP-99-126 (cardiodilatin/atrial natriuretic peptide) and rat CDD/ANP-95-126 (urodilatin) hyperpolarized V_m significantly by 1.6 ± 0.4 mV (BNP,n = 8), 3.7 ± 0.3 mV (CDD/ANP-99-126,n = 25) and 2.8 ± 0.4 mV (urodilatin,n = 9), respectively. The half-maximally effective concentration (EC₅₀) for the latter two was around 400 pmol/l each. This hyperpolarization could be mimicked with 0.5 mmol/1 8-bromo-guanosine 3,5-cyclic monophosphate (8-Br-cGMP) and was blocked by 5 mmol/1 Ba²⁺. The K⁺ channel blocker 293 B (1O)) mol/l) depolarized basal V_m by 4.3 ± 0.4 mV (n = 8), but failed to inhibit the hyperpolarization induced by CDD/ANP-99-126 (160 nmol/1) (n = 8). The K⁺ channel opener cromakalim (10 mol/1) neither influenced basal V_m nor altered the hyperpolarization induced by 160 nmol/1 CDD/ANP-99-126 (n = 8). Adenosine (100 mol/1) hyperpolarized V_m by 13.4 ± 1.3 mV (n = 16). At 100 mol/1, 293 B did not inhibit the adenosine-induced hyperpolarization (n = 6). At 160 nmol/l, CDD/ANP-99-126 enhanced the adenosine-induced hyperpolarization significantly by 1.5 ± 0.6 mV (n = 10). CDD/ANP-99-126 (160 nmol/1) failed to modulate the value to which V_m depolarized in the presence of 1 nmol/l angiotensin II (n = 10), but accelerated the repolarization to basal V_m, by 49 ± 20% (n = 8). These results indicate that the natriuretic peptides CDD/ANP-99-126, CDD/ANP-95-126 and BNP hyperpolarize rat MC probably due to an increase of a K⁺ conductance. This effect modulates the voltage response induced by angiotensin II. The natriuretic-peptide-activated conductance can be blocked by Ba²⁺, but not by 293 B and cannot be activated by cromakalim. This increase in the K⁺ conductance seems to be additive to that inducable by adenosine, indicating that different K⁺ channels are activated by these hormones.     

Remote vs. local ischaemic preconditioning in the rat heart: infarct limitation,suppression of ischaemic arrhythmia and the role of reactive oxygen species

The unmet clinical need for myocardial salvage during ischaemia–reperfusion injury requires the development of new techniques for myocardial protection. In this study the protective effect of different local ischaemic preconditioning (LIPC) and remote ischaemic preconditioning (RIPC) protocols was compared in the rat model of myocardial ischaemia–reperfusion, using infarct size and ischaemic tachyarrhythmias as end‐points. In addition, the hypothesis that there is involvement of reactive oxygen species (ROS) in the protective signalling by RIPC was tested, again in comparison with LIPC. The animals were subjected to 30‐min coronary occlusion and 90‐min reperfusion. RIPC protocol included either transient infrarenal aortic occlusion (for 5, 15 and 30 min followed by 15‐min reperfusion) or 15‐min mesenteric artery occlusion with 15‐min reperfusion. Ventricular tachyarrhythmias during test ischaemia were quantified according to Lambeth Conventions. It was found that the infarct‐limiting effect of RIPC critically depends on the duration of a single episode of remote ischaemia, which fails to protect the heart from infarction when it is too short or, instead, too prolonged. It was also shown that RIPC is ineffective in reducing the incidence and severity of ischaemia‐induced ventricular tachyarrhythmias. According to our data, the infarct‐limiting effect of LIPC could be partially eliminated by the administration of ROS scavenger N‐2‐mercaptopropionylglycine (90 mg/kg), whereas the same effect of RIPC seems to be independent of ROS signalling.     

Pre-conditioning activates adenosine utilization in a cost-effective way during myocardial ischaemia.

During pre-conditioning the interstitial concentration of adenosine, in contrast to lactate, presents a die-away curve-pattern for every successive episode of ischaemia. This die-away pattern might not necessarily be attributed to diminished adenosine production. The present study was undertaken to investigate whether pre-conditioning alters the metabolic turnover of adenosine as observed by the lactate production during ischaemia. Interstitial levels of metabolites in pre-conditioned (n=21) and non-preconditioned (n=21) porcine hearts were monitored with microdialysis probes inserted in both ischaemic and non-ischaemic tissue in an open chest heart model. Three subgroups perturbated with either plain microdialysis buffer (control), buffer containing adenosine (375 microM), or buffer containing deoxyadenosine (375 microM) were studied. All animals were subjected to 90 min of equilibrium microdialysis before 40 min of regional myocardial ischaemia and 120 min of reperfusion. Pre-conditioning consisted of four repetitive episodes of 10 min of ischaemia and 20 min of reperfusion. Significantly higher levels of inosine and lactate were found in the ischaemic tissue of the pre-conditioned subgroup receiving adenosine (P < 0.05) compared with the other two subgroups receiving deoxyadenosine and plain buffer, respectively. This difference was only valid for pre-conditioned ischaemic myocardium, and hence equal amounts of inosine and lactate were produced in the non-preconditioned ischaemic myocardium regardless of the presence of adenosine or deoxyadenosine. In the non-ischaemic myocardium baseline levels of metabolites were measured in all subgroups. Pre-conditioning favoured degradation of exogenous adenosine to inosine successively ending up in enhanced lactate production. This was probably because of the involvement of the hexose monophosphate pathway in the pre-conditioned ischaemic myocardium. This route may therefore be supplementary in energy metabolism as a metabolic flow can be started by adenosine ending up in lactate without initial adenosine 5'-triphosphate (ATP) investment. Utilization of adenosine in this way may also explain the successive die-away pattern of adenosine seen in consecutive pre-conditioning cycles.     

Endogenous Adenosine Down-Modulates Mid-Trimester IntraAmniotic Tumor Necrosis Factor-α Production

Problem  To determine whether adenosine in amniotic fluid down-regulates pro-inflammatory cytokine production.
Method of study  Mid-trimester amniotic fluid from 21 women was incubated ex vivo in the presence or absence of human adenosine deaminase, the enzyme that irreversibly degrades adenosine. After 24 hr, supernatants were assayed by ELISA for tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10. Clinical parameters were obtained after completion of laboratory testing.
Results  Inclusion of adenosine deaminase resulted in a median increase in TNF-α production from 0.9 to 7.3 pg/mL ( P  = 0.0014). IL-6 production exhibited a non-significant median increase from <2.0 to 53.0 pg/mL ( P  = 0.0780). Median IL-10 production increased slightly from a median of <0.2 to 1.3 pg/mL. Adenosine deaminase-stimulated TNF-α production was proportional to parity and unrelated to gestational age, time of delivery, maternal age or indication for amniocentesis.
Conclusion  Adenosine deaminase treatment increases TNF-α production by ex vivo -cultured amniotic fluid. Adenosine contributes to immune modulation in the amniotic cavity.     

Renal denervation abolishes the protective effects of ischaemic preconditioning on function and haemodynamics in ischaemia‐reperfused rat kidneys

Studies were conducted to investigate the role of renal sympathetic nerves in the process of acquiring ischaemic tolerance in ischaemic preconditioned ischaemia‐reperfused rat kidneys. Two periods of 3‐min occlusion of bilateral renal arteries was performed prior to 30‐min bilateral ischaemia and 90‐min reperfusion in acute renal denervated or innervated kidneys. The glomerular filtration rate (GFR), fractional excretion of sodium (FENa) and lithium (FELi), and renal blood flow (RBF) were assessed in reperfused kidneys. Ischaemic preconditioning significantly improved values for all these parameters as compared with no treated ischaemia‐reperfused kidneys. Denervation caused slight increase in GFR, diuresis and natriuresis without improving RBF after reperfusion. However, protecting effects of ischaemic preconditioning on renal function were disappeared in denervated kidneys, while in innevated kidneys the effects of ischaemic preconditioning were maintained. These results clearly showed that ischaemic preconditioning pre‐treatment protects kidneys against ischaemia–reperfusion injury, and the effects are, at least in part, mediated by sympathetic nerves, as the protective effects were abolished by denervation.     

Myocardial adenosine stimulates release of cyclic adenosine monophosphate from capillary endothelial cells in guinea pig heart

Activation of coronary endothelial cell adenylate cyclase was studied in the isolated guinea pig heart by prelabelling endothelial adenine nucleotides using intracoronary infusion of [³H]-adenosine, and measuring the coronary efflux of [³H]-cyclic adenosine monophosphate (cAMP). Hypoxia (30 % O₂) caused a 4-fold increase in coronary release of [³H]-cAMP, which was decreased by 63 % by infusion of the adenosine receptor antagonist, theophylline (50 M). During normoxic control conditions, degrading adenosine to non-vasoactive inosine by intracoronary infusion of adenosine deaminase (1.7 U/ml) caused a 20 % decrease in the release of [³H]-cAMP. The effect of adenosine deaminase was reversed by a specific enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride. Coronary efflux of [³H]-cAMP during intracoronary infusion of 1 M adenosine triphosphate (ATP), adenosine diphosphate or adenosine monophosphate (AMP) (plus adenosine deaminase 8 U/ml) was only 13 % of that due to 1 M adenosine. Adenosine receptor blockers theophylline and CGS 15943A caused equivalent inhibition of the coronary vasodilator actions of adenosine and ATP. Intracoronary infusion of prostaglandin E₁ and the ₂-adrenergic agonist procaterol caused parallel, dose-dependent increases in coronary conductance and the venous release of [³H] cAMP. It is concluded that (1) under both normoxic and hypoxic conditions, adenosine formed by the heart may activate endothelial cell adenylate cyclase via membrane adenosine receptors, (2) coronary receptors for adenosine and ATP share common ligand affinities but ATP receptors are not coupled to adenylate cyclase, and (3) other vasodilators known to activate endothelial adenylate cyclase in vitro cause parallel increases in coronary conductance and adenylate cyclase activity in the beating heart.