L.M. Perkins

Excitatory Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of Adenosine Receptor Agonists and Antagonists

Abstract: The effects of selective adenosine receptor agonists [N6‐cyclopentyladenosine (CPA) and N‐ethylcarboxamidoadenosine (NECA)] and antagonists {8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) and 9‐chloro‐2‐(2‐furanyl)‐5,6‐dihydro‐1,2,4‐triazolo[1,5‐c]quinazoline‐5‐imine (CGS‐15943A)} on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four‐vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug‐treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10−10M) and NECA (10−9M) significantly inhibited the ischemia‐evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia‐evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10−6M) or NECA (10−5M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia‐evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS‐15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia‐evoked release of excitatory neurotransmitter amino acids via high‐affinity A1 receptors, whereas coactivation of lower‐affinity A2 receptors may block (or reverse) the A1‐mediated response.

Characterization of glutamate, aspartate, and GABA release from ischemic rat cerebral cortex

The purpose of this study was to evaluate potential mechanisms of ischemia-evoked amino acid transmitter release. Changes in extracellular levels of transmitter amino acids and lactic acid dehydrogenase (LDH) in rat cerebral cortex during and following four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked release of glutamate, aspartate and gamma-amino-butyric acid (GABA) was compared in control vs. drug-treated animals. Tetrodotoxin and antagonists of glutamate receptors (DNQX, MK-801, and AP-3) depressed the initial rate of increase in extracellular glutamate and aspartate without altering the total amount of these amino acids collected in the cortical superfusates. Cobalt, a calcium channel antagonist, failed to alter efflux. Acidic amino acid transport inhibitors (dihydrokainate, L-trans-PDC) depressed the rate of onset of glutamate and aspartate release and dihydrokainate depressed total release by 44%. PD 81723, an allosteric enhancer at the A1 adenosine receptor, depressed glutamate efflux, as did L-NAME, an inhibitor of nitric oxide synthase. Extracellular increases in GABA levels were depressed by tetrodotoxin and L-trans-PDC. The GABA transport inhibitor, nipecotic acid, increased the initial rate of onset of GABA release. Increases in LDH levels in the extracellular fluid became apparent during the period of ischemia and continued to increase during the subsequent 90 min of reperfusion. These results suggest that ischemia evokes a release of neurotransmitter amino acids that is only partially dependent upon Ca2+ influx activation or the reversal of amino acid transporters. Nonselective mechanisms, resulting from the disruption of plasma membrane integrity, may contribute significantly to the total ischemia-evoked release of excitatory amino acids.

A possible role for phospholipases in the release of neurotransmitter amino acids from ischemic rat cerebral cortex.

The involvement of phospholipases in ischemia-evoked release of aspartate, glutamate, glycine, and GABA from the cerebral cortex was studied in a four vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with the control group, the phospholipase A2 inhibitor mepacrine significantly decreased the ischemia-evoked efflux of transmitter amino acids into cortical superfusates. Direct application of phospholipases A2 or C to the cerebral cortex of non-ischemic animals resulted in a significant increase in amino acid levels. These results suggest that neurotransmitter release following cerebral ischemia may involve phospholipase induced plasma membrane disruption.

Adenosine receptor agonists inhibit the release of γ-aminobutyric acid (GABA) from the ischemic rat cerebral cortex

The effects of CPA (a selective A1 receptor agonist), NECA (a mixed A1 and A2 receptor agonist), and CGS 21680 (a selective A2 receptor agonist) on the ischemia-evoked release of gamma-aminobutyric acid (GABA) from rat cerebral cortex was investigated with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four vessel occlusion. In control animals, superfusate GABA increased from a basal level of 206 +/- 26 nM (mean +/- S.E.M., n = 18) to 10,748 +/- 3,876 nM during the reperfusion period. Pretreatment with adenosine receptor agonists failed to affect basal levels of GABA release. However, CPA (10(-10) M), NECA (10(-9) M), and CGS 21680 (10(-8) M) significantly suppressed the ischemia-evoked release of GABA. The ability to block the ischemia-evoked release of GABA was not evident when the adenosine receptor agonists were administered at higher concentrations. Thus, the selective activation of either A1 or high-affinity A2a adenosine receptors results in an inhibition of ischemia-evoked GABA release.

Adenosine 5′-triphosphate release from the normoxic and hypoxic in vivo rat cerebral cortex

The release of adenine nucleotides and adenosine from the in vivo rat cerebral cortex has been measured in the presence of DL-alpha-glycerophosphate (10(-2) M), an inhibitor of non-specific phosphatases. In normoxic brains, the superfusate concentrations of adenosine and its nucleotides (in nM) were: adenosine 38.9 +/- 10.9; AMP 13.9 +/- 1.6; ADP 12.4 +/- 2.3; ATP 17.7 +/- 2.1. During 10 min episodes of hypoxia (8% oxygen inhalation) superfusate levels of adenosine doubled, but the release of nucleotides was unaltered. These findings indicate that extracellular levels of ATP in the cerebral cortex are in the range at which this compound dilates cerebral blood vessels, and that ATP may be an endogenous regulator of regional cerebral blood flow. The absence of any increase in nucleotide levels during hypoxic episodes implies that adenosine is released into the extracellular space as the nucleoside, and not as a nucleotide which would then be hydrolyzed to adenosine.

Amino Acid and Purine Release in Rat Brain Following Temporary Middle Cerebral Artery Occlusion

Excitatory amino acid release and neurotoxicity in the ischemic brain may be reduced by endogenously released adenosine which can modulate both glutamate or aspartate release and depress neuronal excitability. The present study reports on the patterns of release of glutamate and aspartate; the inhibitory amino acids GABA and glycine; and of the purine catabolites adenosine and inosine from the rat parietal cerebral cortex during 20 and 60 min periods of middle cerebral artery (MCA) occlusion followed by reperfusion. Aspartate and glutamate efflux into cortical superfusates rose steadily during the period of ischemia and tended to increase even further during the subsequent 40 min of reperfusion. GABA release rose during ischemia and declined during reperfusion, whereas glycine efflux was relatively unchanged during both ischemia and reperfusion. Adenosine levels in cortical superfusates rose rapidly at the onset of ischemia and then declined even though MCA occlusion was continued. Recovery to pre-occulusion levels was rapid following reperfusion. Inosine efflux also increased rapidly, but its decline during reperfusion was slower than that of adenosine.

Oxypurinol‐Enhanced Postischemic Recovery of the Rat Brain Involves Preservation of Adenine Nucleotides

Abstract: The present study investigated the effect of the administration of oxypurinol (40 mg/kg), an inhibitor of xanthine oxidase, on adenosine and adenine nucleotide levels in the rat brain during ischemia and reperfusion. The brains of the animals were microwaved before, at the end of a 20‐min period of cerebral ischemia, and after 5, 10, 45, and 90 min of reperfusion. Cerebral ischemia was elicited by four‐vessel occlusion with arterial hypotension to 45–50 mm Hg. Adenosine and adenine nucleotide levels in the oxypurinol‐pretreated (administered intravenously 20 min before ischemia) rats were compared with those in nontreated animals exposed to the same periods of ischemia and reperfusion. Oxypurinol administration resulted in significantly elevated ATP levels at the end of ischemia and 5 min after ischemia, but not at 10 min after ischemia. ADP levels were also elevated, in comparison with those in the control rats, at the end of the ischemic period. Conversely, AMP levels were significantly reduced at the end of ischemia and during the initial (5 min) period of reperfusion. Adenosine levels were lower in oxypurinol‐treated rats, during ischemia, and in the initial reperfusion phase. Oxypurinol administration resulted in a significant increase in the energy charge both during ischemia and after 5 min of reperfusion. Physiological indices, namely, time to recovery of mean arterial blood pressure and time to onset of respiration, were also shortened in the oxypurinol‐treated animals. These beneficial effects of oxypurinol may have been a result of its purine‐sparing (salvage) effects and of its ability to inhibit free radical formation by the enzyme xanthine oxidase. Preservation of high‐energy phosphates during ischemia likely contributes to the cerebroprotective potency of oxypurinol.

Arachidonic acid and lysophosphatidylcholine modulate excitatory transmitter amino acid release from the rat cerebral cortex

The involvement of phospholipase A2 (PLA2) products in ischemia-evoked release of excitatory neurotransmitter amino acids (EAAs) from the cerebral cortex was studied in a four vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with untreated animals, arachidonic acid (AA; 5 x 10(-7) M to 5 x 10(-5) M) significantly reduced the ischemia-evoked efflux of glutamate and aspartate into cortical superfusates. Direct application of lysophosphatidylcholine (LysoPC; 55.4 micrograms/ml) to the cerebral cortex of non-ischemic animals resulted in a significant increase in glutamate levels. These results indicate that the immediate products of PLA2 action on plasma membrane phospholipids can either enhance or inhibit excitotoxic amino acid release following cerebral ischemia. The effect of AA is likely to be a result of its ability to inhibit PLA2; that of LysoPC, a consequence of its detergent action.

Indomethacin modulates ischemia-evoked release of glutamate and adenosine from the rat cerebral cortex

The effects of indomethacin (10 mg/kg) on the release of the transmitter amino acids, glutamate, aspartate, GABA, and of the purines, adenosine and inosine, from the cerebral cortex was studied in a four-vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with the control group, indomethacin significantly attenuated the ischemia-evoked release of glutamate and aspartate, but not of GABA. Adenosine levels in the cortical superfusates were significantly elevated following indomethacin administration. As indomethacin is a potent inhibitor of adenosine uptake, these results suggest that, by blocking adenosine uptake, indomethacin could elevate extracellular adenosine levels and depress glutamate and aspartate efflux as a consequence of the activation of adenosine A1 receptors.

Transmitter amino acid release from rat neocortex: Complete versus incomplete ischemia models

Release of the excitotoxic amino acids, glutamate and aspartate, from the ischemic rat cerebral cortex was compared in two models; the seven vessel occlusion model (7VO) of complete cerebral ischemia and the four vessel occlusion model (4VO) of incomplete cerebral ischemia. Amino acid efflux into cortical superfusates was measured using cortical cups placed on both hemispheres. Whereas a 20 min period of ischemia causes a pronounced release of glutamate and aspartate from the 4VO model, efflux was significantly reduced in the 7VO model. Release of the inhibitory transmitter GABA, was similar in the two models. This result suggests that excitotoxic amino acid efflux into the extracellular spaces of the cerebral cortex may be enhanced by the residual blood flow in an incomplete ischemia.