Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50 of 32 microM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.

Summary: This study used the brain dialysis technique to test the hypothesis that the adenosine concentration of cerebral interstitial fluid increases during situations in which cerebral oxygen supply is inadequate for oxygen demand, Sealed 300-ILm hollow dialysis fibers were im planted in the caudate nucleus of pentobarbital-anesthe tized rats and perfused at 2 ILl/min with artificial cerebro spinal fluid, In vitro tests indicated the recovery of aden osine, inosine, and hypoxanthine from the external medium to be �20% at 2 ILl/min and close to 100% at 0, I ILl/min, Three in vivo interventions were tested: hypoxia/ hypotension (PaOZ = 41.9 mm Hg; MABP = 42.8 mm Metabolic regulation of CBF is widely accepted (Kuschinsky and Wahl, 1978; Berne et aI., 1981h; Kontos, 1981;Wahl, 1985), although the mediators involved are still controversial (Wahl, 1985). The purine nucleoside adenosine, a by-product of ATP metabolism and known cerebral blood vessel di lator (Berne et aI. , 1974;Wahl and Kuschinsky, 1976; Gregory et aI. , 1980; Berne et aI. , 1981a) 522Hg; n = 9), local potassium infusion (n = 4), and cere bral anoxia/ischemia (n = 10). These interventions pro duced 10-, 4-, and 30-fold increases in perfusate adeno sine concentration, respectively, as well as increases in perfusate concentrations of inosine and hypoxanthine. A separate group of rats (n = 9) perfused at 0. 1 ILl/min yielded estimates of cerebral interstitial fluid adenosine, inosine, and hypoxanthine concentrations of 1. 26, 3.30, and 7. 19 ILM, respectively. These results are consistent with the adenosine hypothesis for the regulation of CBF.

The purpose of this study was to determine the effect of ischemic preconditioning on the changes in interstitial fluid (ISF) purine metabolites and lactate during prolonged regional myocardial ischemia. The study consisted of two groups of anesthetized dogs: a control group (n = 13) that was exposed to 60 minutes of regional myocardial ischemia and a preconditioned group (n = 10). In the preconditioned group, regional myocardial ischemia was induced for two 5-minute episodes, each followed by 10 minutes of reperfusion. These preconditioning episodes were followed by 60 minutes of sustained regional ischemia. Cardiac ISF was sampled by microdialysis probes implanted in the left ventricular myocardium; dialysate levels served as indices of ISF concentrations. In the preconditioned group, dialysate concentrations of adenosine, inosine, hypoxanthine, total purines, and lactate increased during each of the 5-minute ischemia episodes, with further increases occurring during the first 5 minutes of reperfusion. However, the increases in dialysate purine metabolites during the first period of ischemia/reperfusion were greater than those that occurred during the second ischemia/reperfusion. In addition, preconditioning reduced the rate of ISF purine metabolite and lactate accumulation during the prolonged ischemia when compared with nonpreconditioned animals. These data suggest that preconditioning reduces the energy imbalance that occurs during subsequent myocardial ischemia and thereby diminishes the rate of net adenine nucleotide degradation and cellular production and release of purine metabolites and lactate. In addition, the increase in ISF adenosine seen during the preconditioning episode is consistent with the notion that preconditioning-induced cardioprotection is mediated in part by the action of adenosine at extracellular A1 adenosine receptors.

The purpose of this study was twofold: 1) to investigate the feasibility and usefulness of cardiac microdialysis for the simultaneous estimation of regional cardiac interstitial fluid (ISF) adenosine (ADO) concentration and coronary blood flow (CBF); and 2) to determine the changes in the ISF levels of ADO and CBF during cardiac stimulation or regional myocardial ischemia. Cardiac microdialysis probes were implanted in the left ventricular myocardium of chloralose-urethan-anesthetized dogs and perfused with Krebs-Henseleit buffer. The concentration of ADO in the effluent dialysate was used as an index of intramyocardial ISF ADO concentration while local CBF was measured by H2 clearance via a platinum wire within the dialysis fiber. Dialysate ADO was elevated immediately after insertion of the microdialysis probe, declined rapidly in the first 20 min, stabilized by 60 min, and remained constant for 2 h. Based on the relationship in vitro and in vivo between microdialysis probe perfusion rate and dialysate ADO concentration, ISF ADO concentration within the left ventricular myocardium was estimated to be 0.9-1.3 microM. Dobutamine (10 micrograms.kg-1.min-1) infusion resulted in a 36% increase in CBF and a 2.5-fold increase in dialysate ADO (n = 9; P less than 0.05). Regional myocardial ischemia, induced by occlusion of the left anterior descending artery (LAD), caused a 13-fold increase in dialysate ADO in the LAD perfused myocardium (n = 9; P less than 0.05). These results are consistent with the ADO hypothesis and suggest that cardiac microdialysis provides a reliable technique for the sampling of regional intramyocardial ISF.

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.