Gary A. Walter

Increases in Cerebral Cortical Perfusate Adenosine and Inosine Concentrations during Hypoxia and Ischemia

The cerebral cortical cup technique was used to monitor changes in adenosine and inosine levels in the rat cerebral cortex during periods of hypoxia, anoxia, or hemorrhagic hypotension. Basal levels of adenosine and inosine in cortical perfusates stabilized within 10 min at concentrations of 30–50 and 75–130 nM, respectively. Comparable levels were observed in normal CSF collected from the cisterna magna. Reductions in the oxygen content of the inspired air (14, 12, 8, and 5% oxygen) resulted in increases in the adenosine and inosine levels in the cortical perfusates, the magnitude of the increase being progressively more pronounced with greater reductions in the oxygen content. Cerebral anoxia/ischemia, induced by 100% nitrogen inhalation, caused a rapid increase in the adenosine and inosine contents of the cortical perfusates. Hemorrhagic hypotension (46.1 ± 1.7 mm Hg) of 5 min duration did not result in an elevated adenosine or inosine release. The results suggest that interstitial fluid adenosine levels are likely to be in the low nM range in the normoxic animal and are capable of rapid increases during hypoxic or anoxic episodes. The findings support the adenosine hypothesis of CBF regulation.

Brain Adenosine and Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of the Adenosine Deaminase Inhibitor Deoxycoformycin

Abstract: The effects of a potent adenosine deaminase inhibitor, deoxycoformycin, on purine and amino acid neuro‐transmitter release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four‐vessel occlusion. Purine and amino acid releases were compared from control ischemic animals and deoxycoformycin‐pretreated ischemic rats. Ischemia enhanced the release of glutamate, aspartate, and γ‐aminobutyric acid into cortical perfusates. The levels of adenosine, inosine, hypoxanthine, and xanthine in the same perfusates were also elevated during and following ischemia. Deoxycoformycin (500 μ/kg) enhanced ischemia‐evoked release of adenosine, indicating a marked rise in the adenosine content of the interstitial fluid of the cerebral cortex. Inosine, hypoxanthine, and xanthine levels were depressed by deoxycoformycin. Deoxycoformycin pretreatment failed to alter the pattern of amino acid neurotransmitter release from the cerebral cortex in comparison with that observed in control ischemic animals. The failure of deoxycoformycin to attenuate amino acid neurotransmitter release, even though it markedly enhanced adenosine levels in the extracellular space, implies that the amino acid release during ischemia occurs via an adenosine‐insensitive mechanism. Inhibition of excitotoxic amino acid release is unlikely to be responsible for the cerebroprotective actions of deoxycoformycin in the ischemic brain.

Effects of Deoxycoformycin on Adenosine, Inosine, Hypoxanthine, Xanthine, and Uric Acid Release from the Hypoxemic Rat Cerebral Cortex

The effects of the adenosine deaminase inhibitor, deoxycoformycin, on purine release from the rat cerebral cortex were studied with the cortical cup technique. Deoxycoformycin (5 and 500 μg/kg i.v.) enhanced the hypoxia/ischemia-evoked release of adenosine from the cerebral cortex, indicating a marked rise in the adenosine content of interstitial fluid in the cerebral cortex. Inosine and hypoxanthine release were attenuated at the higher dose of deoxycoformycin, acid release into the cortical perfusates was enhanced at the higher dose level. These results demonstrate that low doses of deoxycoformycin can be used to elevate interstitial levels of adenosine in the brain during hypoxia, and to depress the formation of some of its metabolites. The elevation of hypoxia/ischemia-evoked adenosine levels can account for the previously reported potentiation of hypoxia-evoked increases in rat cerebral blood flow after deoxycoformycin administration. The potential therapeutic utility of these findings is discussed.

Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex

The effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on adenosine, inosine and oxypurine release from the normoxic and hypoxic/ischemic rat cerebral cortex have been studied. Dipyridamole (500 micrograms/kg) enhanced adenosine release during hypoxic/ischemic challenges in comparison with saline-injected controls. It decreased the hypoxia/ischemia-elicited releases of inosine, hypoxanthine and xanthine. Both basal and hypoxia/ischemia-elicited releases of uric acid were elevated. Soluflazine, administered topically or systemically, failed to enhance adenosine release and did not consistently alter the hypoxia/ischemia-evoked releases of inosine, hypoxanthine and xanthine. Basal release of uric acid was elevated. The failure of either drug to elevate the basal or hypoxia/ischemia-evoked releases of adenosine above predrug levels illustrates one of the problems which may be inherent in the use of bidirectional nucleoside transport inhibitors for the manipulation of adenosine levels in the cerebral interstitial fluid.

The effects of hypothermia on amino acid neurotransmitter release from the cerebral cortex.

The effects of systemic hypothermia (33.5 degrees C) on the ischemia-evoked release of the neurotransmitter amino acids, glutamate, aspartate, gamma-amino-butyric acid (GABA) and glycine into rat cerebral cortical superfusates were evaluated in the rat four vessel occlusion model. Glutamate, aspartate and GABA, but not glycine, levels were enhanced during and following a 20 min period of ischemia. However, when compared with normothermic ischemic animals, no reductions in glutamate, aspartate or GABA levels in the superfusates were apparent either prior to, during or following forebrain ischemic episodes. Indeed, the superfusate levels of aspartate and GABA were transiently increased immediately following ischemia. Glycine levels were significantly depressed, both pre- and post-ischemia, in cortical superfusates from hypothermic animals in comparison with normothermic rats.

Effects of Nifedipine and Felodipine on Adenosine and Inosine Release from the Hypoxemic Rat Cerebral Cortex

The cerebral cortical cup technique has been used to study the effects of nifedipine and felodipine on adenosine and inosine release from the rat brain. After basal and hypoxia (8% 02)-evoked control levels of purine release had been established, these 1,4-dihydropyridine calcium antagonists were administered intraperitoneally (1 mg/kg). Both agents depressed basal levels of purine efflux and suppressed the hypoxia-evoked release of adenosine and inosine. An inhibition of the transporter that mediates purine efflux from brain cells is likely to account for the suppression of release from the cerebral cortex. A reduced release of adenosine into the interstitial space also explains the ability of both agents to block the increase in CBF evoked by hypoxic challenges.

Determination of rat cerebrospinal fluid concentrations of adenosine, inosine, hypoxanthine, xanthine and uric acid by high performance liquid chromatography

Abstract— Isocratic reverse‐phase high performance liquid chromatography techniques were developed to resolve and quantitate the purine nucleosides adenosine (Ado) and inosine (Ino) and their metabolites hypoxanthine (Hyp), xanthine (Xan), and uric acid (UA) in the cerebrospinal fluid of the rat. The moving phase composition for resolving hypoxanthine, xanthine and uric acid was a 0.22 M, pH 5.8 phosphate buffer. The moving phase composition for resolving adenosine and inosine was a 0.22 M, pH 6.8 phosphate buffer, 7% methanol (v/v) and 2.5 mM tetrabutylammonium phosphate. The observed cerebrospinal fluid concentrations in the rat were: Ado = 35 ± 9 nM (s.e.m.), Ino = 359 ± 85 nM, Hyp = 243 ± 77 nM, Xan = 1340 ± 423 nM and UA = 6130 ± 678 nM.

Purine concentrations in the cerebrospinal fluid of unanesthetized rats during and after hypoxia

A new technique for achieving repeated sampling of fourth ventricular cerebrospinal fluid (CSF) from the cisterna magna of unanesthetized rats is described. The sampling cannula is positioned extracranially, in contrast to previously published techniques which require insertion through the skull. CSF samples, withdrawn from unanesthetized rats before, during and after a 25 min period of inhalation of 5% oxygen in nitrogen, were analyzed for their adenosine and inosine contents by high pressure liquid chromatography. Adenosine and inosine levels increased during the hypoxic episode and were even higher 1 h later. They had declined, but were still above basal levels, 2-3 h after the hypoxic episode. Elevated CSF adenosine concentrations may be responsible for the generation of such persistent effects of hypoxia as post-hypoxic respiratory depression.

The effects of the xanthine oxidase inhibitors, allopurinol and oxypurinol on the pattern of purine release from hypoxic rat cerebral cortex.

Release of purines from the normoxic and hypoxic/ischemic rat cerebral cortex was studied with the cortical cup technique. Both allopurinol (100 mg/kg, i.v.) and its active metabolite oxypurinol (20 mg/kg, i.v.) reduced the release of uric acid to 10% of predrug levels, indicating an effective block of xanthine oxidase activity. Xanthine and hypoxanthine, substrates for xanthine oxidase, increased in the cortical perfusates following drug administration. Allopurinol treatment resulted in a decreased release of adenosine during the first postdrug hypoxic challenge, whereas oxypurinol, and allopurinol during the second postdrug challenge, increased the hypoxia-evoked level of adenosine and inosine in the perfusate. The purine sparing effects of allopurinol and oxypurinol, in addition to the prevention of xanthine oxidase-mediated free radical generation, may account for their protective action in reperfusion injury paradigms.

Effect of a brief hypoxic/hypotensive episode on the in vivo release of cerebral cortical γ-aminobutyric acid and glycine☆

gamma-Aminobutyric acid (GABA) and glycine levels in rat cerebral cortical superfusates rose during a 10-min period to reach stable concentrations of approximately 0.55 microM and approximately 12.3 microM, respectively. In cerebrospinal fluid withdrawn from the fourth ventricle, the GABA concentration was 0.1 microM, and that of glycine, 10.55 microM. GABA, and to a lesser extent glycine, concentrations increased in the cortical superfusates during and immediately following exposure of the rats to a 5-min period of 5% oxygen in nitrogen inhalation.