Michael H. O'Regan

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.

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.

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.

Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex

Elevated levels of glutamate and aspartate have been implicated in the pathogenesis of neural injury and death induced by ischemia. The mechanism(s) whereby they escape into the extracellular environment have been a subject of controversy. This study evaluated the contribution of phospholipases and protein kinases to ischemia-evoked glutamate and aspartate release from the ischemic/reperfused rat cerebral cortex. Changes in the extracellular levels of these amino acids during four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked amino acid release was compared in control vs. drug treated animals, in which selective inhibitors of phospholipases and protein kinases were applied topically onto the cerebral cortex. The phospholipase inhibitors tested included 4-bromophenacyl bromide, a non-selective inhibitor; 7,7-dimethyleicosadienoic (DEDA), an inhibitor of secretory type phospholipase A2 (PLA2); AACOCF3, an inhibitor of the Ca2(+)-dependent cytoplasmic form of PLA2, HELSS, which inhibits a Ca(2+)-independent cytoplasmic PLA2, and U73122, a selective inhibitor of phospholipase C (PLC). All five phospholipase inhibitors significantly attenuated glutamate and aspartate release into the extracellular milieu, indicating the possibility that several forms of the enzyme are likely to be involved. The protein kinase C (PKC) inhibitor, chelerythrine chloride, also reduced excitatory amino acid efflux, wheres the PKC activator phorbol 12-myristate 13-acetate (PMA) enhanced their release. The non-selective kinase inhibitor, staurosporine, and H-89, which selectively inhibits protein kinase A, did not reduce ischemia-evoked amino acid efflux. These results suggest that ischemia-evoked release of the excitatory transmitters amino acids is a result, in part, of the activation of phospholipases A2 and C, with PKC involvement in the transduction process. Destabilization and deterioration of the plasma membrane, as a consequence of phospholipid hydrolysis, may allow these transmitter amino acids to diffuse down their concentration gradients into the extracellular fluid.

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.

Deoxycoformycin antagonizes ischemia-induced neuronal degeneration

Deoxycoformycin, a potent and specific adenosine deaminase antagonist, reduced ischemic hippocampal damage and the associated hypermotility in Mongolian gerbils. Cerebral ischemia was induced by a bilateral 5 min occlusion of the carotid arteries. Deoxycoformycin (500 micrograms/kg IP), administered 15 min prior to ischemia, prevented the increase in locomotor activity normally observed with this model and significantly reduced the ischemia-induced damage to CA1 hippocampal neurons. The results suggest that deoxycoformycin may be useful in the prevention of brain damage due to cerebral ischemia.

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.

The role of adenosine in the central actions of the benzodiazepines

1. Evidence is presented which indicates that the central actions of the benzodiazepines cannot be fully accounted for by assuming an action only at the GABAA-Cl- channel supramolecular complex. 2. The hypothesis is presented, together with supporting evidence, that inhibition of adenosine uptake can account for many of the actions of the benzodiazepines. 3. New findings showing that Ro 15-1788 and Ro 5-4864 have both potentiative and antagonistic interactions with adenosine are discussed. 4. The proconvulsant beta-carbolines are shown to be adenosine antagonists. 5. The concept that benzodiazepine action may involve several mechanisms is presented.

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.

CHANGES IN EXTRACELLULAR AMINO ACID NEUROTRANSMITTERS AND PURINES DURING AND FOLLOWING ISCHEMIAS OF DIFFERENT DURATIONS IN THE RAT CEREBRAL CORTEX

The time courses of changes in extracellular levels of the transmitter amino acids, glutamate, aspartate and gamma-aminobutyric acid (GABA), and of the purines, adenosine and inosine, during 20 or 40 min periods of four vessel occlusion rat cerebral ischemia, followed by reperfusion, were investigated using a cerebral cortical cup technique. During a 20 min period of ischemia, superfusate amino acids increased by 10-30-fold and adenosine levels increased five-fold. Reperfusion was followed by the return of amino acids and purines towards pre-ischemic levels. Significantly greater increases in glutamate and aspartate levels were observed during a 40 min ischemia and, in contrast to the 20 min ischemia, the efflux of all compounds remained elevated throughout the 40 min reperfusion period. These results suggest that longer periods of ischemia are associated with increasing degrees of plasma membrane disruption allowing for a greater leakage of intracellular contents. The failure of extracellular levels of amino acids and purines to return towards pre-ischemic levels indicates that cells may be unable to effectively reconstitute their membranes after longer periods of ischemia.