David G. L. Van Wylen

Adenosine A1 receptor mediated protection of the globally ischemic isolated rat heart

The purpose of this study was to determine if the cardioprotective effect of adenosine on the ischemic myocardium is mediated by interaction with specific adenosine receptor subtypes. Isolated rat hearts perfused at constant flow were subjected to global normothermic (37 degrees C) ischemia and the time to onset of ischemic contracture (TOIC) was used as a marker of myocardial ischemic injury. Hearts treated with adenosine and R-phenylisopropyladenosine (PIA), an adenosine A1 receptor agonist, exhibited a significantly greater TOIC than control hearts (18.60 +/- 0.40 and 16.64 +/- 1.15 min, respectively vs 9.12 +/- 0.66 min), whereas phenylaminoadenosine, an adenosine A2 receptor agonist, had no effect on TOIC (11.73 +/- 0.87 min). BW A1433U, an adenosine receptor antagonist, blocked the effects of adenosine and PIA on ischemic contracture time, and BW A1433U did not alter the ability of nifedipine or propranolol to delay the onset of ischemic contracture, thus indicating the specificity of this compound for the adenosine receptor. PIA-treated hearts exhibited significantly greater ATP levels throughout the ischemic period compared to control hearts, whereas hearts treated with BW A1433U showed a rapid decline in ATP content. These results suggest that the beneficial effects of adenosine on the ischemic myocardium are mediated by interaction with adenosine A1 receptors, and that endogenously formed adenosine plays a role in attenuating myocardial ischemic damage.

Adenosine Receptor Blockade Augments Interstitial Fluid Levels of Excitatory Amino Acids during Cerebral Ischemia

The excitotoxic hypothesis suggests that cerebral ischemic damage results in part from the accumulation of the excitatory and potentially toxic neurotransmitters glutamate and aspartate. Adenosine, which also increases during cerebral ischemia, is proposed to inhibit neurotransmitter release. The purpose of this study was to determine if adenosine receptor blockade exacerbates the accumulation of glutamate and aspartate during cerebral ischemia. Microdialysis probes, implanted bilaterally in the caudate nucleus of halothane-anesthetized rats, were used to (1) assess changes in interstitial fluid (ISF) glutamate, aspartate, adenosine, and adenosine metabolites; (2) measure local cerebral blood flow (H2 clearance); and (3) deliver 8-(p-sulfophenyl)theophylline (SPT), an adenosine receptor antagonist, locally to the brain. The probe on one side of the brain was perfused with artificial cerebrospinal fluid (CSF) containing 10−3 M SPT, while the probe on the opposite side received only artificial CSF. Animals were exposed to 20 min of ischemia (carotid occlusion + arterial blood pressure = 50 mm Hg) followed by 60 min of reperfusion. Dialysate glutamate and aspartate increased during and after cerebral ischemia, but were increased to a greater extent in the presence of adenosine receptor blockade. Likewise, the increase in dialysate adenosine and adenosine metabolites was enhanced on the side of locally administered SPT. These data suggest that endogenous adenosine attenuates the accumulation of glutamate and aspartate during cerebral ischemia.

Increases in interstitial adenosine and cerebral blood flow with inhibition of adenosine kinase and adenosine deaminase

The purpose of this study was to determine the changes in interstitial fluid (ISF) adenosine and cerebral blood flow (CBF) during inhibition of adenosine kinase or adenosine deaminase. Brain microdialysis was used to (a) measure CBF (H2 clearance), (b) sample cerebral ISF, and (c) deliver drugs locally to the brain. Microdialysis probes were implanted bilaterally in the caudate nucleus of halothane-anesthetized rats (n = 11). One probe was perfused with artificial cerebrospinal fluid (CSF) containing iodotubercidin (IODO), an adenosine kinase inhibitor, while the other probe was perfused with erythro-2-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor. Both probes were subsequently perfused with EHNA + IODO. Finally, 8-(p-sulfophenyl)theophylline (SPT), an adenosine receptor antagonist, was added to EHNA + IODO in one probe, while the other probe continued to receive only EHNA + IODO. CBF and dialysate adenosine levels increased with either EHNA or IODO; however, the increases were greater with IODO. EHNA + IODO further increased CBF and dialysate adenosine. The hyperemia observed with EHNA + IODO was abolished by adenosine receptor blockade. These data suggest that basal adenosine levels are influenced to a greater extent by adenosine kinase than by adenosine deaminase. In addition, the increased CBF observed with inhibition of adenosine metabolism and the attenuation of this vasodilatory response with adenosine receptor blockade support a role for adenosine in CBF regulation.

Neurochemical and morphological responses to acutely and chronically implanted brain microdialysis probes

The purpose of this study was to compare, in rats, brain microdialysis results obtained using microdialysis probes implanted acutely for 2 h versus probes implanted chronically for 24 h in the caudate. Specific comparisons included: (1) dialysate purine and amino acid profiles during cerebral ischemia; (2) diffusional characteristics of the microdialysis probe; and (3) tissue morphology surrounding the probe. During ischemia, the increase in dialysate levels of adenosine, inosine, and hypoxanthine was less pronounced from probes implanted chronically, while dialysate xanthine levels increased to a greater extent. An increase in dialysate amino acid neurotransmitters during cerebral ischemia was observed in the acutely implanted probes within 10 min of the onset of cerebral ischemia; in the chronically implanted probes this increase did not occur until after 50 min of severe ischemia. Both in vitro and in vivo tests revealed a diffusional barrier in chronically implanted probes. Moreover, the tissue surrounding chronically implanted probes exhibited a high degree of inflammation, and fibrin deposits were substantial. In addition, uric acid levels (an indicator of tissue injury) sampled from chronically implanted probes were 7-fold greater than levels sampled from acutely implanted probes. These data raise concerns about the use of chronically implanted microdialysis probes for the measurement of purine and amino acid profiles during cerebral ischemia.

Acadesine reduces myocardial infarct size by an adenosine mediated mechanism

OBJECTIVE 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. METHODS 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)%. CONCLUSIONS Acadesine reduces infarct size by an adenosine mediated mechanism, but this cardioprotective action is not associated with significantly augmented interstitial fluid adenosine levels.

Changes in extracellular adenosine during chemical or electrical brain stimulation

The purpose of this study was to determine the changes in adenosine and adenosine metabolites during graded electrical stimulation or kainic acid-induced activation and to assess the role of adenosine in the cerebral blood flow (CBF) response to increased brain activity. A modified brain microdialysis technique was used to sample cerebral interstitial fluid (ISF), deliver drugs locally to the brain, electrically stimulate the brain, and measure local CBF (H2 clearance). Microdialysis probes were implanted bilaterally in the caudate nuclei of ketamine-anesthetized rats. Graded electrical stimulation at 5, 15, and 30 Hz increased dialysate adenosine 1.5-fold, 2.3-fold, and 4.7-fold, respectively. Local infusion of kainic acid, an agonist of the excitatory amino acid neurotransmitter glutamate, produced a transient increase (2-fold) in dialysate adenosine and sustained increases in dialysate inosine (2-fold), hypoxanthine (4-fold) and CBF (2.4-fold). When the adenosine receptor antagonist 8(p-sulphophenyl)-theophylline (SPT, 10(-3) M) was co-administered with kainic acid, CBF increased only 1.6-fold, while the increase in dialysate adenosine was augmented by 40%. These data demonstrate that ISF adenosine increases during brain activation and suggest that adenosine contributes to active hyperemia in the brain.

Interstitial purine metabolites during regional myocardial ischemia

The purpose of this study was to determine the changes in cardiac interstitial fluid (ISF) purine metabolites during 90 min of regional myocardial ischemia. To collect ISF metabolites and measure local coronary blood flow (CBF), cardiac microdialysis probes were implanted into the left anterior descending artery (LAD) and left circumflex artery (LC) perfused myocardium of chloralose-urethane anesthetized dogs (n = 7). Regional ventricular wall thickness was measured in the LAD and LC perfused regions with sonomicrometric crystals, using systolic wall thickening (SWT) as an index of regional ventricular function. Regional myocardial ischemia, produced by occlusion of the LAD, resulted in a decrease in CBF (hydrogen clearance) from 77.3 +/- 12.4 to 10.9 +/- 4.4 ml/min/100 g (P less than 0.05), and systolic wall thinning (control SWT = 15.5 +/- 2.2%; ischemic SWT = -6.8 +/- 1.7%). ISF adenosine was transiently elevated in the ischemic region, obtaining a maximum sixfold increase after 15 min of ischemia. Inosine, hypoxanthine, and to a lesser extent xanthine, composed the majority of metabolites which accumulated in the ISF of the ischemic region, accounting for greater than 95% of the total purine metabolites in the ISF after 20 min of ischemia. Despite the marked increase in ISF inosine, hypoxanthine, and xanthine levels, ISF uric acid levels did not increase in the ischemic region. Although CBF and SWT did not change in the nonischemic LC perfused area, there were small transient increases (two- to fourfold) in ISF adenosine, inosine, and hypoxanthine levels. In summary, these data demonstrate that purine metabolites accumulate rapidly in the ISF during myocardial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Attenuation of ischemia-induced extracellular adenosine accumulation by homocysteine.

The purpose of this study was to determine the effects of homocysteine, which consumes intracellular adenosine via formation of S-adenosylhomocysteine, on interstitial fluid (ISF) adeonsine and cerebral blood flow (CBF) before, during, and after cerebral ischemia. Microdialysis probes, used to measure local CBF (H2 clearance) and to sample ISF, were implanted bilaterally into the caudate nucleus of halothane-anesthetized rats (n = 8). l-Homocysteine thiolactone was administered locally via one of the probes. Animals were exposed to 20 min of ischemia, induced by bilateral carotid occlusion plus hemorrhage to an arterial blood pressure of 50 mm Hg, followed by 60 min of reperfusion. Before ischemia, CBF and dialysate adenosine were decreased with homocysteine. During ischemia and early reperfusion, dialysate purine metabolites increased on both sides of the brain; however, the ischemia-induced increase in adenosine was attenuated on the side of local homocysteine. CBF was lower on the side of homocysteine throughout reperfusion. These data demonstrate that homocysteine (a) decreases basal ISF adenosine and CBF, (b) attenuates the increase in dialysate adenosine during ischemia, and (c) reduces hyperemia during early reperfusion.

Role of Extracellular Adenosine in Ethanol‐Induced Desensitization of Cyclic AMP Production

Abstract: The decrease in receptor‐stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol‐induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol‐induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4‐day exposure to ethanol decreased basal as well as 2‐chloroadenosine‐ and forskolin‐stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2‐day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2‐chloroadenosine‐ and forskolin‐stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum‐free defined media. The ethanol‐induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24‐h exposure of PC12 cells to 2‐chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin‐stimulated cyclic AMP levels were increased. Similar results were obtained after a 4‐day exposure of PC12 cells to 2‐chloroadenosine or 5′‐N‐ethylcarboxamido‐adenosine. The present results indicate that the ethanol‐induced decrease in receptor‐stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.

Adenosine Receptor Blockade Augments Interstitial Excitatory Amino Acids During Cerebral Ischemia

The role of excitatory amino acid neurotransmitters in the genesis of ischemic neuronal damage is of current interest. As shown by microdialysis techniques, interstitial fluid (ISF) levels of the excitatory amino acid neurotransmitters glutamate and aspartate are markedly elevated during cerebral ischemia (Benveniste et al., 1984; Hagberg et al., 1985; Hillered et al., 1989; Shimada et al., 1990). This data, in conjunction with extensive documentation of the toxic effects of glutamate on neurons in vitro (Rothman and Olney, 1986), has led to the theory that the ischemiainduced elevation of ISF excitatory amino acids overstimulate post-synaptic glutamate receptors, leading to a cascade of cellular events which ultimately cause cell death.