Interstitial Adenosine Levels Research Articles

Estimation of skeletal muscle interstitial adenosine during forearm dynamic exercise in humans.

It has been proposed that adenosine is a metabolic signal that triggers activation of muscle afferents involved in the exercise pressor reflex. Furthermore, exogenous adenosine induces sympathetic activation that mimics the exercise pressor reflex, and blockade of adenosine receptors inhibits sympathetic activation induced by exercise. Thus, we hypothesize that adenosine is released locally by the muscle during exercise. We used microdialysis probes, placed in the flexor digitorium superficialis muscle, to estimate muscle interstitial adenosine levels in humans. We estimated resting in vivo muscle interstitial adenosine concentrations (0.292+/-0.058 micromol/L, n=4) by perfusing increasing concentrations of adenosine to determine the gradient produced in the dialysate. Muscle interstitial adenosine concentrations increased from 0.23+/-0.04 to 0.82+/-0.14 micromol/L (n=14, P<0.001) during intermittent dynamic exercise at 50% of maximal voluntary contraction. Lactate increased from 0.8+/-0.1 to 2.3+/-0.3 mmol/L (P<0.001). Lower intensity (15% maximal voluntary contraction) intermittent dynamic exercise increased adenosine concentrations from 0.104+/-0.02 to 0.42+/-0.16 micromol/L (n=7). The addition of ischemia to this low level of exercise produced a greater increase in adenosine (from 0.095+/-0.02 to 0.48+/-0.2 micromol/L) compared with nonischemic exercise (0. 095+/-0.02 to 0.25+/-0.12 micromol/L). These results indicate that microdialysis is useful in estimating adenosine concentrations and in reflecting changes in muscle interstitial adenosine during dynamic exercise in humans.

Relationship between free radicals and adenosine in the mechanism of preconditioning: are they interrelated or independent triggers?

Both free radicals (FRs) and adenosine receptor activation contribute to triggering a mechanism of preconditioning (PC) against infarction. This study examined the possibility that there is some interaction between FRs and adenosine generation during PC. In the first series of experiments, the effects of an FR scavenger, N-2-mercaptopropionyl glycine (MPG), on the interstitial adenosine level during PC and on the infarct size-limiting effect of PC were assessed in the rabbit heart in situ. PC with 5-min ischemia/5-min reperfusion limited infarct size after 30-min coronary occlusion (expressed as a percentage of area at risk, %IS/AR) from 33.2 +/- 4.7% (S.E.) to 10.8 +/- 1.1% (p < 0.05). This cardioprotection was blocked by MPG (1.5 mg/kg/min i.v.) infused before and during PC (%IS/AR = 27.4 +/- 3.6). However, the same dose of MPG did not suppress elevation of the adenosine and inosine levels in the microdialysate from the myocardium during 5-min ischemia/reperfusion. In the second series of experiments, the effect of an FR-generating system (1 mM hypoxanthine and 20 mU/ml xanthine oxidase) on the purine production was compared to that of PC in isolated rabbit hearts. Whereas PC increased the adenosine level in the coronary effluent from 0.17 +/- 0.16 microM under baseline to 1.68 +/- 0.53 microM, infusion of the FR generators over a period of 5 min did not increase the adenosine release. However, infarct size was similarly reduced by PC and by 5-min transient infusion of FR generators, and the cardioprotection by the FR generators was abolished by 300 microM MPG. These results suggest that there is no interaction between free radicals and adenosine during the trigger phase of PC in the rabbit heart.

ATP as a source of interstitial adenosine in the rat heart

The contribution of neuronal ATP to interstitial adenosine levels was investigated in isolated perfused rat hearts. Ventricular surface transudates, representing interstitial fluid, were analyzed for norepinephrine, ATP, and adenosine. Exocytotic release of norepinephrine was induced by electrical stimulation of cardiac efferents emanating from the stellate ganglion. Ganglion stimulation increased contractility, interstitial norepinephrine, ATP, and adenosine. Interstitial adenosine was 11- to 27-fold higher than interstitial ATP, suggesting that the released ATP is unlikely the only source of adenosine. In the presence of AOPCP (alpha,beta-methyleneadenosine 5'-diphosphate), an ecto-5'-nucleotidase inhibitor, the ganglion-stimulated increase in interstitial ATP and adenosine reached levels similar to those in the absence of AOPCP, also suggesting that adenosine does not derive from extracellular ATP. The perfusate Ca2+ was raised from 1 to 4 mM to determine the importance of the enhanced contractile function on the levels of norepinephrine, ATP, and adenosine. The results were increases in contractility and interstitial norepinephrine, ATP, and adenosine, which were not suppressed with atenolol, indicating a norepinephrine-independent release of ATP and adenosine. Reserpine treatment and administration of guanethidine depleted the catecholamine stores and diminished the catecholamine release, respectively. However, neither agent altered Ca2+-induced increases in ATP and adenosine. It is concluded that the amount of neuronal-derived ATP is low and most likely does not contribute significantly to interstitial levels of adenosine. Furthermore, elevations in interstitial norepinephrine, ATP, and adenosine are associated with neuronal-independent increases in contractile function.

Intravascular source of adenosine during forearm ischemia in humans: implications for reactive hyperemia.

It is believed that adenosine is released in ischemic tissues and contributes to reactive hyperemia. We tested this hypothesis in the human forearm using microdialysis to estimate interstitial and intravascular levels of adenosine and caffeine withdrawal to potentiate endogenous adenosine and determine its effect on reactive hyperemia. Forearm blood flow response to ischemia was measured by air plethysmography before and 60 hours after the last dose of caffeine (250 mg TID for 7 days, n=6). Forearm blood flow increased by 274+/-66% and 467+/-97% after 3 minutes of forearm ischemia, before and during caffeine withdrawal, respectively (P<0.05). Thus, caffeine withdrawal enhances reactive hyperemia. To determine the source of adenosine, we measured interstitial adenosine with the use of a microdialysis probe inserted into the flexor digitorum superficialis muscle of the forearm, and we measured intravascular adenosine with the use of a microdialysis probe inserted retrogradely into the medial cubital vein. Dialysate samples were collected at 15-minute intervals during resting, forearm ischemia, and recovery periods. Forearm ischemia failed to increase muscle dialysate concentrations of adenosine but did increase intravascular dialysate adenosine 2.1-fold, from 0.61+/-0.12 to 1.28+/-0.39 micromol/L (P<0.01, n=8). Intravascular dialysate concentrations of thromboxane B2 did not increase during ischemia, ruling out platelet aggregation as a source of adenosine. These results support the hypothesis that endogenous adenosine contributes to reactive hyperemia and indicate that the major source of adenosine in the human forearm is intravascular. We speculate that endothelial cells are the source of intravascular adenosine during ischemia.

Effects of adenosine and acadesine on interstitial nucleosides and myocardial stunning in the pig

5-Amino-4-imidazolecarboxamide riboside (AICAr) or acadesine has been proposed to exert cardioprotection by enhancing adenosine production in ischemic myocardium. However, there are conflicting reports on acadesine's effects in ischemic myocardium and few studies in which myocardial adenosine levels have been measured. The purpose of this study was to determine whether acadesine increases interstitial fluid adenosine levels and attenuates myocardial stunning or potentiates the effects of adenosine in the intact pig. In pentobarbital-anesthetized pigs, myocardial stunning was induced by 10 min left anterior descending coronary artery occlusion and 90 min reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening, and interstitial nucleosides were estimated by cardiac microdialysis. Control hearts were compared with hearts treated with acadesine, adenosine, and adenosine plus acadesine. Adenosine pretreatment (100 µg·kg-1·min-1, intracoronary) immediately prior to ischemia increased interstitial adenosine levels 9-fold and improved postischemic functional recovery from a control value of 17.6 ± 4.1% to 43.6 ± 3.4% of preischemic systolic wall thickening. In contrast, acadesine (20 mg/kg i.v. bolus 10 min prior to ischemia + 0.5 mg·kg-1·min-1, i.v. infusion through 60 min reperfusion) had no effect on interstitial fluid adenosine levels or the recovery of regional function (21.5 ± 5.9% recovery), nor were the functional effects of adenosine potentiated by acadesine. These findings indicate that acadesine does not enhance myocardial adenosine levels, attenuate myocardial stunning, or potentiate the cardioprotective effects of adenosine in the pig.Key words: adenosine, acadesine, interstitial fluid, microdialysis, myocardial stunning.

Endogenous interstitial adenosine in isolated myenteric neural networks varies inversely with prevailing PO2.

Isolated myenteric ganglion networks were used in a perifusion protocol to characterize the response of interstitial adenosine levels to changes in prevailing PO2. The biological activity of such adenosine was assessed using inhibition of release of substance P (SP) as a functional measure of adenosine activity, and the effect of altered O2 tension on both spontaneous and elevated extracellular K+ concentration-evoked SP release from networks was determined over a range of PO2 values from hypoxic (PO2 = 54 mmHg) to hyperoxic (PO2 = 566 mmHg). Release of SP was found to be sensitive to PO2, and a linear graded relationship was obtained. Perifusion in the additional presence of the adenosine A1-receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed considerable adenosinergic inhibition with an inverse exponential relationship and hyperoxic threshold PO2. Disinhibition of evoked SP release by DPCPX in the absence of TTX was double that observed in its presence, indicating a neural source for some of the adenosine released during hypoxia. A postulated neuroprotective role for adenosine is consistent with the demonstrated relationship between interstitial adenosine and prevailing O2 tension.

Effect of Aging on Myocardial Adenosine Production, Adenosine Uptake and Adenosine Kinase Activity in Rats

Adenosine levels present in the interstitial fluid and coronary effluent of the aged heart exceed those of the young adult heart. The present study investigated mechanisms in the Fischer 344 rat heart which may be responsible for the observed differences. (1) Total production of adenosine was determined in isolated perfused hearts by measuring coronary effluent adenosine content while inhibiting adenosine deamination and rephosphorylation with erythrohydroxy-nonyladenosine (EHNA) and iodotubercidin (ITC), respectively. Total adenosine production was similar in both young (3–4 month) and aged (20–21 month) hearts at 31.8±6.6 and 38.4±3.3 nmol/min/g dry wt, respectively. However, stimulation with theβ-adrenergic agent, isoproterenol, elicited a significantly greater increase in adenosine production in the young vs. aged heart. (2) Adenosine transport was evaluated in isolated perfused hearts by determining14C uptake by the myocardium after 20 min of14C-adenosine perfusion. Adenosine uptake in the agent-free heart was found to be decreased 17 to 25% in aged compared to young adult hearts. (3) Adenosine transport characteristics were determined with nitrobenzylthioinosine saturation-binding studies in ventricular membrane preparations. The Bmaxvalues were significantly lower in aged than young adult hearts (140.2±1.5 fmol/mg and 191.9±2.3 fmol/mg in aged and young hearts, respectively) indicating a decreased number of transporter sites in the aged heart. However, the values for Kdwere decreased with aging, suggesting an increase in the affinity of the transporter for adenosine in the aged vs. young adult heart. (4) The activities and kinetics of adenosine kinase were determined in homogenates of aged and young adult ventricular myocardium. No statistical difference was found between the two activities. Taken together these results suggest that increased interstitial adenosine levels in the aged heart result from decreased uptake of adenosine by the ventricular myocardium.

Adenosine and preconditioning revisited.

1. Myocardial tolerance against infarction is substantially increased by exposing myocytes to 3-10 min transient ischaemia. In this phenomenon, termed 'preconditioning', the adenosine receptor is one of the redundant triggers and the best characterized factor in the cardioprotective mechanism. 2. An increase in interstitial adenosine during preconditioning is thought to be derived primarily from hydrolysis of 5'-AMP in the myocyte by cytosolic 5'-nucleotidase, although a contribution of ectosolic 5'-nucleotidase remains controversial. Adenosine production during ischaemia is substantially suppressed in the preconditioned myocardium, probably due to a decrease in ATP utilization. 3. The adenosine receptor needs to be activated not only at the time of preconditioning ischemia, but also during ischaemic insult for the preconditioning to be cardioprotective. However, the extent of cardioprotection afforded by preconditioning is primarily determined by the interstitial adenosine level achieved during preconditioning ischaemia, not by the level during sustained ischaemia. These data suggest that a post-receptor mechanism downstream of the adenosine receptor may be up-regulated after preconditioning. 4. Studies in vitro suggest that the subtypes of adenosine receptor relevant to preconditioning against infarction are A1 and A3, the activation of which appears to provide additive protection. The functional interrelationship between these subtypes in vivo remains unknown. 5. An important step downstream of adenosine receptor activation is protein kinase C (PKC), which facilitates opening of ATP-sensitive potassium (KATP) channels, probably leading to enhancement of myocardial tolerance. However, activation of other protein kinases, such as tyrosine kinase, may also be important in preconditioning, depending on the animal species and preconditioning protocols. The PKC isoform and location of KATP channels (i.e. sarcolemmal vs mitochondrial KATP) that induce anti-infarct tolerance in myocytes remain to be identified.

Effects of adenosine and acadesine on interstitial nucleosides and myocardial stunning in the pig

5-Amino-4-imidazolecarboxamide riboside (AICAr) or acadesine has been proposed to exert cardioprotection by enhancing adenosine production in ischemic myocardium. However, there are conflicting reports on acadesine's effects in ischemic myocardium and few studies in which myocardial adenosine levels have been measured. The purpose of this study was to determine whether acadesine increases interstitial fluid adenosine levels and attenuates myocardial stunning or potentiates the effects of adenosine in the intact pig. In pentobarbital-anesthetized pigs, myocardial stunning was induced by 10 min left anterior descending coronary artery occlusion and 90 min reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening, and interstitial nucleosides were estimated by cardiac microdialysis. Control hearts were compared with hearts treated with acadesine, adenosine, and adenosine plus acadesine. Adenosine pretreatment (100 microg x kg(-1) x min(-1), intracoronary) immediately prior to ischemia increased interstitial adenosine levels 9-fold and improved postischemic functional recovery from a control value of 17.6 +/- 4.1% to 43.6 +/- 3.4% of preischemic systolic wall thickening. In contrast, acadesine (20 mg/kg i.v. bolus 10 min prior to ischemia + 0.5 mg x kg (-1) x min(-1), i.v. infusion through 60 min reperfusion) had no effect on interstitial fluid adenosine levels or the recovery of regional function (21.5 +/- 5.9% recovery), nor were the functional effects of adenosine potentiated by acadesine. These findings indicate that acadesine does not enhance myocardial adenosine levels, attenuate myocardial stunning, or potentiate the cardioprotective effects of adenosine in the pig.

ATP as a source of interstitial adenosine in the rat heart

The contribution of neuronal ATP to interstitial adenosine levels was investigated in isolated perfused rat hearts. Ventricular surface transudates, representing interstitial fluid, were analyzed for norepinephrine, ATP, and adenosine. Exocytotic release of norepinephrine was induced by electrical stimulation of cardiac efferents emanating from the stellate ganglion. Ganglion stimulation increased contractility, interstitial norepinephrine, ATP, and adenosine. Interstitial adenosine was 11- to 27-fold higher than interstitial ATP, suggesting that the released ATP is unlikely the only source of adenosine. In the presence of AOPCP (alpha,beta-methyleneadenosine 5'-diphosphate), an ecto-5'-nucleotidase inhibitor, the ganglion-stimulated increase in interstitial ATP and adenosine reached levels similar to those in the absence of AOPCP, also suggesting that adenosine does not derive from extracellular ATP. The perfusate Ca2+ was raised from 1 to 4 mM to determine the importance of the enhanced contractile function on the levels of norepinephrine, ATP, and adenosine. The results were increases in contractility and interstitial norepinephrine, ATP, and adenosine, which were not suppressed with atenolol, indicating a norepinephrine-independent release of ATP and adenosine. Reserpine treatment and administration of guanethidine depleted the catecholamine stores and diminished the catecholamine release, respectively. However, neither agent altered Ca2+-induced increases in ATP and adenosine. It is concluded that the amount of neuronal-derived ATP is low and most likely does not contribute significantly to interstitial levels of adenosine. Furthermore, elevations in interstitial norepinephrine, ATP, and adenosine are associated with neuronal-independent increases in contractile function.

Adenosine mediates sustained adrenergic desensitization in the rat heart via activation of protein kinase C.

Adenosine attenuates the myocardial metabolic and contractile responses induced by ss-adrenergic stimulation. Our study was conducted to investigate the longevity of this antiadrenergic action after adenosine exposure. Adenosine (33 micromol/L) was infused into isolated perfused rat hearts for 1, 5, 30, or 60 minutes, and the adrenergic responsiveness (AR) to isoproterenol (10(-8) mol/L) was determined at the end of each infusion period and during a 45-minute adenosine washout period. Interstitial levels of adenosine, as determined from epicardial surface transudates, returned to preinfusion levels within 10 minutes of washout. The duration of adenosine infusion had no effect on the extent of attenuation of AR at the end of the infusion. Whereas AR returned to preadenosine levels with washout of shorter adenosine infusions (1 and 5 minutes), there was a slow and incomplete recovery of AR after the longer exposures (30 and 60 minutes) to adenosine. The magnitude of this persistent antiadrenergic effect (PAE) of adenosine at 15 minutes of washout was proportional to the epicardial concentration of adenosine during infusion of the nucleoside. Infusion of adenosine either with the nonselective adenosine receptor antagonist 8-p-sulfophenyl theophylline or with the selective A1-receptor antagonist 1,3-dipropyl, 8-cyclopentylxanthine, abolished the PAE during the washout period. In addition, the PAE could be demonstrated only with the selective A1-receptor agonist 2-chloro-N6-cyclopentyladenosine and not with the selective A3-receptor agonist 4-aminobenzyl-5'-N methylcarboxamido-adenosine. When the protein kinase C (PKC) inhibitor chelerythrine was coadministered with adenosine, the PAE of adenosine was not apparent during adenosine washout. A 30-minute infusion of phenylephrine, an alpha-adrenergic agonist that enhances PKC activity, produced a PAE that lasted for up to 30 minutes of washout. This effect was prevented by the coinfusion of chelerythrine. Thus, it is concluded that the PAE of adenosine is determined by the myocardial concentration of this nucleoside and is manifested when myocardial concentrations of adenosine returned to baseline levels. Moreover, a 5-minute duration of adenosine exposure is required for the expression of the PAE. This latter effect seems to be dependent on adenosine-induced PKC activation via A1-receptors.

Infarct-size limitation by preconditioning is enhanced by dipyridamole administered before but not after preconditioning: evidence for the role of interstitial adenosine level during preconditioning as a primary determinant of cardioprotection.

Although the importance of adenosine (Ado)-receptor activation in preconditioning (PC) has been established, it is unclear whether cardioprotection afforded by PC is determined by the Ado level during PC ischemia or by that during sustained ischemia. Accordingly, we tested whether the PC effect is modified by augmenting the increase in the interstitial Ado level during PC or by that during sustained ischemia. In the first series of experiments, the effect of 0.25 mg/kg dipyridamole (DIP) on the interstitial Ado level was assessed by in vivo microdialysis in the rabbit heart. Dialysate Ado during 2-min ischemia was 70% higher in the heart pretreated with 0.25 mg/kg of DIP than in the untreated controls, indicating that DIP was capable of enhancing an ischemia-induced increase of interstitial Ado. In the second series of experiments, myocardial infarction was induced in the rabbit by 30-min coronary artery occlusion and 3-h reperfusion. Infarct size was determined by tetrazolium staining and expressed as percentage of area at risk (%IS/AR). Rabbits were subjected to one of nine treatments before the 30-min ischemia: no treatment, DIP (0.25 mg/kg, i.v.), PC with 2-min ischemia, DIP before 2-min PC, DIP after 2-min PC, PC with 3-min ischemia, DIP before 3-min PC, DIP after 3-min PC, or 8-sulfophenyltheophylline (SPT) after 3-min PC. DIP alone did not modify %IS/AR (38.8 +/- 5.8% vs. 41.2 +/- 4.7%), but administration of DIP before 2-min PC significantly enhanced the infarct size-limiting effect (14.6 +/- 2.1% with DIP vs. 32.1 +/- 4.7% without DIP). Although the 3-min PC per se could achieve significant infarct limitation, the effect of DIP on 3-min PC was not significant (14.7 +/- 1.9% with DIP vs. 20.5 +/- 1.8% without DIP). On the other hand, the effect of DIP administered after PC was very slight (only 7% reduction of %IS/AR) and statistically insignificant, regardless of the duration of PC ischemia. However, infarct limitation by 3-min PC was inhibited by SPT given after the PC (%IS/AR = 34.5 +/- 3.2), as reported previously. These results suggest that the interstitial Ado level during PC ischemia, not the level during sustained ischemia, is a primary determinant of the extent of cardioprotection by PC and that the threshold for Ado-receptor activation required during sustained ischemia is much lower than that for triggering PC.

Effects of ischemia, preconditioning, and adenosine deaminase inhibition on interstitial adenosine levels and infarct size

Effects of ischemia, preconditioning, and adenosine deaminase inhibition on interstitial adenosine levels and infarct size

Effects of ischemia, preconditioning, and adenosine deaminase inhibition on interstitial adenosine levels and infarct size.

In order to examine the relationship between local adenosine concentrations before, during, and after ischemia and the extent of ischemic myocardial damage, measurements of interstitial fluid (ISF) nucleosides were made using microdialysis probes implanted in the ischemic region of isoflurane anesthetized Micropigs undergoing 60' coronary artery occlusion (CAO) and 3 h of reperfusion (REP). Nucleoside concentrations in the dialysate collected from the microdialysis probes were used as an index of ISF levels. Dialysate nucleoside concentrations (ADO, inosine and hypoxanthine), myocardial infarct size, and myocardial blood flow (MBF) were determined in control animals (n = 6), animals preconditioned with a single 10' cycle of CAO and REP (PC, n = 6), and those treated with the adenosine deaminase inhibitor pentostatin (n = 6, 0.2 mg/Kg i.v. 30' prior to CAO). The brief PC occlusion resulted in a transient but significant increase in dialysate ADO (6.7 +/- 1.8 microM vs. 0.67 +/- 0.1 microM at baseline). Pentostatin administration had no significant effect on either dialysate nucleosides or MBF at baseline. During the 60' CAO, dialysate ADO increased in control animals. In PC animals, however, dialysate ADO during CAO was lower than control. Pretreatment with pentostatin resulted in a six-fold augmentation in dialysate ADO during the 60 min CAO when compared to the control values (110.62 +/- 30.2 microM vs. 16.31 +/- 2.1 microM at 60 min of ischemia). Pentostatin also resulted in a significant reduction in the accumulation of inosine and hypoxanthine, indicating inhibition of adenosine deaminase activity. There were no significant differences in MBF between groups at any time point. Following 3 h REP, infarct size was 35.4 +/- 5.5%, 8.1 +/- 1.5% and 8.3 +/- 1.8% of the region at risk in control, PC, and pentostatin groups, respectively. These data suggest that marked increase in ISF ADO during CAO, may be as effective in reducing INF as a modest increase in ISF ADO prior to prolonged CAO.

Adenosine transport inhibition ameliorates postischemic hypoperfusion in pigs

Cerebral ischemia is often followed by a period of delayed hypoperfusion that may contribute to tissue injury. We tested the hypothesis that augmentation of interstitial adenosine can improve tissue perfusion under this condition. 10 min global ischemia was produced in two groups of isoflurane-anesthetized newborn pigs by occlusion of subclavian and brachiocephalic arteries, and changes in local cortical blood flow and cortical interstitial purine metabolites were measured using the combined hydrogen clearance-microdialysis technique. In one group, the dialysis probe was perfused with artificial cerebrospinal fluid buffer containing nitrobenzyl-thioinosine (NBTI, 100 μmol/1), a competitive inhibitor of adenosine transport. In the untreated group (n = 9), baseline cortical blood flow (39 ± 3 ml/min/100 g) was depressed by 51 ± 5% and 42 ± 6% at 40 and 60 min, respectively, of postischemic reperfusion. NBTI increased baseline interstitial adenosine levels 2.4-fold which increased baseline cortical blood flow 1.5-fold to 60 ± 4 ml/min/ 100 g, and increased both absolute adenosine levels as well as adenosine as a percentage of total purine metabolites throughout ischemia and reperfusion. As a result, the extent of postischemic hypoperfusion was significantly lessened in NBTI-treated animals (n = 9), with reductions in cortical blood flow of only 28 ± 3% and 24 ± 5% at 40 and 60 min of reperfusion, respectively. These results indicate that inhibition of adenosine transport by NBTI elevates interstitial adenosine concentration during and following cerebral ischemia, and concomitantly improves cortical perfusion in the post-ischemic period. The latter effect may contribute to the documented neuroprotective efficacy of adenosinergic therapy in cerebral ischemia.

Adenosine transport inhibition ameliorates postischemic hypoperfusion in pigs

Cerebral ischemia is often followed by a period of delayed hypoperfusion that may contribute to tissue injury. We tested the hypothesis that augmentation of interstitial adenosine can improve tissue perfusion under this condition. 10 min global ischemia was produced in two groups of isoflurane-anesthetized newborn pigs by occlusion of subclavian and brachiocephalic arteries, and changes in local cortical blood flow and cortical interstitial purine metabolites were measured using the combined hydrogen clearance-microdialysis technique. In one group, the dialysis probe was perfused with artificial cerebrospinal fluid buffer containing nitrobenzyl-thioinosine (NBTI, 100 μmol/1), a competitive inhibitor of adenosine transport. In the untreated group (n = 9), baseline cortical blood flow (39 ± 3 ml/min/100 g) was depressed by 51 ± 5% and 42 ± 6% at 40 and 60 min, respectively, of postischemic reperfusion. NBTI increased baseline interstitial adenosine levels 2.4-fold which increased baseline cortical blood flow 1.5-fold to 60 ± 4 ml/min/ 100 g, and increased both absolute adenosine levels as well as adenosine as a percentage of total purine metabolites throughout ischemia and reperfusion. As a result, the extent of postischemic hypoperfusion was significantly lessened in NBTI-treated animals (n = 9), with reductions in cortical blood flow of only 28 ± 3% and 24 ± 5% at 40 and 60 min of reperfusion, respectively. These results indicate that inhibition of adenosine transport by NBTI elevates interstitial adenosine concentration during and following cerebral ischemia, and concomitantly improves cortical perfusion in the post-ischemic period. The latter effect may contribute to the documented neuroprotective efficacy of adenosinergic therapy in cerebral ischemia.

INTERSTITIAL ADENOSINE AND FUNCTION IN RAT HEART IN VIVO: EFFECTS OF ADRENALINE AND 8‐CYCLOPENTYL‐1,3‐DIMETHYLXANTHINE

1. Left ventricular interstitial adenosine and cardiac function were studied in open chest rats during adrenaline stimulation and P1-purinoceptor antagonism with 8-cyclopentyl-1,3-dimethylxanthine (8-CPT). 2. Cardiac microdialysate adenosine concentration was 0.10 +/- 0.01 mumol/L (n = 24) under basal conditions, giving an estimated interstitial adenosine concentration of 0.27 mumol/L. Stimulation with 3.2 and 8.0 micrograms/kg per min adrenaline increased the rate-pressure product (heart rate x systolic blood pressure) by 72 and 157%, respectively, and increased dialysate adenosine to 0.26 +/- 0.04 and 0.65 +/- 0.11 mumol/L (n = 12), respectively (interstitial concentrations of approximately 0.70 and 1.76 mumol/L). 3. Treatment with 60 micrograms/kg per min 8-CPT did not alter basal adenosine concentrations, but potentiated elevations in dialysate adenosine during infusion of 3.2 and 8.0 micrograms/kg per min adrenaline to 0.54 +/- 0.10 and 1.30 +/- 0.22 mumol/L, respectively (n = 12). Basal function and the response to 8.0 micrograms/kg per min adrenaline were unaltered by 8-CPT, whereas elevations in heart rate and rate-pressure product during stimulation with 3.2 micrograms/kg per min adrenaline were enhanced by 8-CPT (by up to 30%). 4. Studies in isolated hearts confirmed the inhibitory potency of 8-CPT at A1 vs A2 P1-purinoceptors (e.g. pK(B) of 7.7 +/- 0.2 and 6.4 +/- 0.1 for 5'-N-ethyl carboxamidoadenosine-mediated bradycardia and vasodilatation, respectively; n = 6). Studies in intact animals verified effective A1 blockade by 60 micrograms/kg per min 8-CPT, but also revealed some inhibition of A2-mediated responses. 5. In conclusion, the data show that cardiac interstitial adenosine levels exist within a physiologically active range in vivo and increase dose-dependently during graded adrenaline stimulation. Adenosine receptor antagonism enhances elevations in interstitial adenosine and modifies functional responses to moderate, but not high, doses of adrenaline. Whether 8-CPT-dependent elevations in interstitial adenosine are due to A1 inhibition vs inhibition of A2-mediated vasodilatation requires further investigation.

Ischemic preconditioning against infarction: its mechanism and clinical implications.

Exposing the myocardium to brief ischemia followed by reperfusion enhances myocardial resistance to infarction from a subsequent sustained ischemia. This phenomenon, termed preconditioning, is most likely to be triggered by adenosine A1 receptor activation, and the dependence of the preconditioning effect on the duration of preconditioning ischemia and the number of its repetition is probably through the interstitial adenosine level achieved by each preconditioning protocol. Our studies support the theory that activation of protein kinase C subsequent to stimulation of the A1 receptor enhances myocardial ischemic tolerance. The ATP-sensitive potassium channel may be involved in preconditioning, but its relation with protein kinase C is unclear, and the relative importance of this channel might be species dependent. The mechanism of preconditioning needs to be further elucidated in animal models and preconditioning in the human heart needs to be further characterized before we can adapt its biochemical basis to clinical therapy.

Acadesine reduces myocardial infarct size by an adenosine mediated mechanism

The aim was to test the hypotheses that acadesine (1) augments endogenous interstitial fluid (ISF) adenosine during ischaemia, and (2) reduces infarct size by adenosine receptor mediated mechanisms. To test these hypotheses, the left coronary artery of anaesthetised rabbits (n = 33) was occluded for 30 min and reperfused for 120 min. Acadesine (1 mg.kg-1.min-1 for 5 min, then 0.2 mg.kg-1.min-1) was infused intravenously beginning 30 min before coronary occlusion and ending 30 min after reperfusion. The area at risk was comparable in all groups, averaging 34.7 (SEM 2.2%) of the left ventricle. In separate studies (n = 22), estimates of ISF adenosine and adenosine metabolites were obtained by cardiac microdialysis. Although dialysate adenosine levels increased significantly in the area at risk during ischaemia in the untreated group [from 0.044(0.008) to 0.339(0.146) microM], acadesine did not significantly augment dialysate adenosine levels before or during ischaemia [preischaemia = 0.094(0.032) microM; ischaemia = 0.542(0.262) microM]. In addition, there was no significant difference in dialysate adenosine concentrations during the first 10 min of reperfusion, after which adenosine levels returned to baseline levels. A 2.5-fold large dose failed to increase interstitial fluid adenosine. However, the adenosine receptor blocker 8-p-sulphophenyltheophylline (SPT) in the presence of acadesine increased ISF adenosine fourfold. Acadesine significantly (P < 0.05) reduced infarct size [n = 8, 19.7(2.9)% of risk area] compared with the untreated group [n = 8, 29.4(1.3)%]. This infarct size reduction with acadesine was antagonised by SPT given during ischaemia-reperfusion [n = 8, 46.2(3.0)%] or only during reperfusion [n = 9, 42.7(2.6)%. Acadesine reduces infarct size by an adenosine mediated mechanism, but this cardioprotective action is not associated with significantly augmented interstitial fluid adenosine levels.

Effects of anesthesia and K+ ATP channel blockade on interstitial adenosine accumulation in ischemic rabbit myocardium.

Glibenclamide, a K+ ATP channel antagonist, blocks the anti-infarct effect of ischemic preconditioning in rabbits, but only when the latter are anesthetized with ketamine-xylazine. Furthermore, the protection triggered by pinacidil, a K+ ATP channel opener, can be aborted by treatment with the adenosine antagonist 8-(P-sulfophenyl)theophylline. This study tests whether either the anesthetic regimen or glibenclamide affects infarct size by modulating interstitial adenosine levels. Interstitial adenosine and total purine concentrations were assessed in open-chest rabbits by the microdialysis technique. Dialysis fibers were inserted into myocardium served by a coronary artery branch surrounded by a snare. All animals sustained a 30-min coronary occlusion and then 120-min reperfusion. Rabbits were anesthetized with either sodium pentobarbital or a ketamine-xylazine mixture. Half of the latter animals also received glibenclamide. The control levels of adenosine in the dialysate were comparable in the three groups, as were those of total purines, and the infusion of glibenclamide caused no change. Ischemia led to 10- to 20-fold increases in interstitial adenosine and 10- to 40-fold rises in total purine concentrations. These increases were equivalent in all groups. Further-more, infarct size as a percentage of the myocardium at risk was also comparable in the three groups. Neither the anesthetic agent nor glibenclamide appears to modulate interstitial adenosine release from ischemic tissue.