Adenosine's role in hypercapnia-evoked cerebral vasodilation in the rat.

Carbon dioxide induces a rapid dilation of cerebral arterioles, enabling local blood flow to match increasing metabolic requirements of tissue. Amongst the vasodilatory substances released by cortical tissue in response to CO2 are adenosine and nitric oxide. Here we report that selective adenosine A(2A) receptor antagonists, applied topically using a rat cortical window technique, significantly depressed the CO2-evoked increase in arteriolar diameter, measured using video microscopy, as well as attenuating the CO2 and pH reactivity of the cortical arterioles. Two non-selective inhibitors of nitric oxide synthase also significantly depressed the hypercapnia-evoked increase in arteriolar diameter.

Measurement of free fatty acids in cerebrospinal fluid from patients with hemorrhagic and ischemic stroke.

Free fatty acid (FFA) concentrations in cerebrospinal fluid (CSF) from patients with ischemic and hemorrhagic stroke (n=25) and in contemporary controls (n=73) were examined using HPLC. Concentrations of CSF FFAs from ischemic and hemorrhagic stroke patients obtained within 48 h of the insult were significantly greater than in control patients. Higher concentrations of polyunsaturated fatty acids (PUFAs) in CSF obtained within 48 h of insult were associated with significantly lower (P<0.05) admission Glasgow Coma Scale scores and worse outcome at the time of hospital discharge, using the Glasgow Outcome Scale (P<0.01).

Characterization of modes of release of amino acids in the ischemic/reperfused rat cerebral cortex.

Brain extracellular levels of glutamate, aspartate, GABA and glycine increase rapidly following the onset of ischemia, remain at an elevated level during the ischemia, and then decline over 20-30 min following reperfusion. The elevated levels of the excitotoxic amino acids, glutamate and aspartate, are thought to contribute to ischemia-evoked neuronal injury and death. Calcium-evoked exocytotic release appears to account for the initial (1-2 min) efflux of neurotransmitter-type amino acids following the onset of ischemia, with non-vesicular release responsible for much of the subsequent efflux of these and other amino acids, including taurine and phosphoethanolamine. Extracellular Ca(2+)-independent release is mediated, in part by Na(+)-dependent amino acid transporters in the plasma membrane operating in a reversed mode, and by the opening of swelling-induced chloride channels, which allow the passage of amino acids down their concentration gradients. Experiments on cultured neurons and astrocytes have suggested that it is the astrocytes which make the primary contribution to this amino acid efflux. Inhibition of phospholipase A(2) attenuates ischemia-evoked release of both amino and free fatty acids from the rat cerebral cortex indicating that this group of enzymes is involved in amino acid efflux, and also accounting for the consistent ischemia-evoked release of phosphoethanolamine. It is, therefore, possible that disruption of membrane integrity by phospholipases plays a role in amino acid release. Recovery of amino acid levels to preischemic levels requires their uptake by high affinity Na(+)-dependent transporters, operating in their normal mode, following restoration of energy metabolism, cell resting potentials and ionic gradients.

Effects of immunosuppressants, calcineurin inhibition, and blockade of endoplasmic reticulum calcium channels on free fatty acid efflux from the ischemic/reperfused rat cerebral cortex.

Elevated levels of free fatty acids (FFA) have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the effects of immunosuppressants agents, calcineurin inhibitors and blockade of endoplasmic reticulum (ER) calcium channels on free fatty acid formation and efflux in the ischemic/reperfused (I/R) rat brain. Changes in the extracellular levels of arachidonic, docosahexaenoic, linoleic, myristic, oleic and palmitic acids in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical cup technique. A 20-min period of ischemia elicited large increases in the efflux of all six FFAs, which were sustained during the 40 min of reperfusion. Cyclosporin A (CsA) and trifluoperazine, which reportedly inhibit the I/R elicited opening of a mitochondrial permeability transition (MPT) pore, were very effective in suppressing ischemia/reperfusion evoked release of all six FFAs. FK506, an immunosuppressant which does not directly affect the MPT, but is a calcineurin inhibitor, also suppressed the I/R-evoked efflux of FFAs, but less effectively than CsA. Rapamycin, a derivative of FK506 which does not inhibit calcineurin, did not suppress I/R-evoked FFA efflux. Gossypol, a structurally unrelated inhibitor of calcineurin, was also effective, significantly reducing the efflux of docosahexaenoic, arachidonic and oleic acids. As previous experiments had implicated elevated Ca(2+) levels in the activation of phospholipases with FFA formation, agents affecting endoplasmic reticulum stores were also evaluated. Dantrolene, which blocks the ryanodine receptor (RyR) channel of the ER, significantly inhibited I/R-evoked release of docosahexaenoic, arachidonic, linoleic and oleic acids. Ryanodine, which can either accentuate or block Ca(2+) release, significantly enhanced ischemia/reperfusion-elicited efflux of linoleic acid, with non-significant increases in the efflux of myristic, arachidonic, palmitic and oleic acids. Xestospongin C, an inhibitor of the inositol triphosphate (IP(3)R) channel, failed to affect I/R-evoked FFA efflux. Thapsigargin, an inhibitor of the Ca(2+)-ATPase ER uptake pump, elicited significant elevations in the efflux of myristic, arachidonic and linoleic acids, in the absence of ischemia. Collectively, the data suggest an involvement of both ER and mitochondrial Ca(2+) stores in the chain of events which lead to PLA(2) activation and FFA formation.

Differential effects of phospholipase inhibitors on free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury.

Free fatty acid (FFA) elevation in the brain has been shown to correlate with the severity of damage in ischemic injury. The etiology of this increase in FFA remains unclear and has been hypothesized to result from phospholipase activation. This study examines the effects of specific phospholipase inhibitors on FFA efflux during ischemia-reperfusion injury. A four-vessel occlusion model of cerebral ischemia was utilized to assess the effects of PLA(2) and PLC inhibitors on FFA efflux from rat cerebral cortex. In addition, FFA efflux from non-ischemic cortices exposed to PLA(2) and PLC was measured. Concentrations of arachidonic, docosahexaenoic, linoleic, myristic, oleic, and palmitic acids in cortical superfusates were determined using high performance liquid chromatography (HPLC). Exposure to the non-selective PLA(2) inhibitor 4-bromophenylacyl bromide (BPB) significantly inhibited FFA efflux during ischemia-reperfusion injury (P<0.01 arachidonic, oleic and palmitic; P<0.05 all others); exposure to the PLC inhibitor U73122 had no observed effect. The effects of the Ca(2+)-dependent PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)) mirrored the effects of BPB and led to reductions in all FFA levels (P<0.01 arachidonic, oleic and palmitic; P<0.05 all others). Exposure to the secretory PLA(2) inhibitor 3-(3-acetamide-1-benzyl-2-ethyl-indolyl-5-oxy) propane sulfonic acid (LY311727) and to the Ca(2+)-independent PLA(2) inhibitor bromoenol lactone (BEL) had only minimal effects on FFA efflux. Application of both PLA(2) and PLC to non-ischemic cortices resulted in significant increases in efflux of all FFA (P<0.05). The study suggests that FFA efflux during ischemia-reperfusion injury is coupled to activation of Ca(2+)-dependent PLA(2) and provides further evidence of the potential neuroprotective benefit of Ca(2+)-dependent PLA(2) inhibitors in ischemia.

Evidence for swelling-induced adenosine and adenine nucleotide release in rat cerebral cortex exposed to monocarboxylate-containing or hypotonic artificial cerebrospinal fluids.

Recent reports have described a swelling-induced release of adenosine triphosphate (ATP) from a variety of non-nervous system cell types, which may be involved in the regulatory volume decrease (RVD) response. The present study examined the effects of swelling induced by applications of hypotonic or monocarboxylic acid containing artificial cerebrospinal fluid (aCSF) on the release of adenosine nucleotides and adenosine from the in vivo rat cerebral cortex using a cortical cup technique. Hypotonic aCSF (25mM NaCl) elicited a significant increase in adenosine, but not adenine nucleotide, release. Applications of sodium L-lactate, pyruvate, or acetate (all 20mM) evoked increases in adenine nucleotides but not adenosine. D-Lactate (20mM) enhanced adenosine and ATP release. Inhibition of the plasma membrane monocarboxylate transporter with cyano-4-hydroxycinnamate (4-CIN, 2mM) blocked the effects of L-lactate on purine release. These in vivo results demonstrate that osmoregulatory processes in cortical cells evoke an efflux of adenine nucleotides and/or adenosine. In that these purines activate a variety of receptors, it is possible that they may function as autocrine or paracrine signaling agents, facilitating volume regulation and enhancing local blood flow.

Inhibition of Na(+)/Ca(2+) exchange by KB-R7943, a novel selective antagonist, attenuates phosphoethanolamine and free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury.

Reversal of the Na(+)/Ca(2+) exchanger (NCX) occurs during ischemia-reperfusion injury as a result of changes in intracellular pH and sodium concentration. Inhibition of NCXs has been shown to be neuroprotective in vitro. In this study, we evaluated the effects of KB-R7943 (50 microM), a specific inhibitor of the reverse mode of NCX, applied topically onto rat cerebral cortex prior to and during ischemia. Amino acid and free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high-performance liquid chromatography. During a 20-min period of ischemia in control animals, there were significant increases in all amino acids and in all FFAs. Following reperfusion, all FFAs remained significantly elevated. Application of KB-R7943 (50 microM) significantly inhibited effluxes of phosphoethanolamine, but had no effect on glutamate, aspartate, taurine or GABA levels. KB-R7943 also resulted in significant reductions in levels of myristic, docosahexaenoic and arachidonic acid during ischemia and in reperfusion levels of arachidonic and docosahexaenoic acids. These data indicate that inhibition of Na(+)/Ca(2+) exchange likely prevented the activation of phospholipases that usually occurs following an ischemic insult as evidenced by its attenuation of phosphoethanolamine and free fatty acid efflux. The inhibition of phospholipases may be an essential component of the neuroprotective benefits of Na(+)/Ca(2+) exchange inhibitors in ischemia-reperfusion injury and may provide a basis for their possible use in therapeutic strategies for stroke.

Inhibition of Na(+)/H(+) exchange by SM-20220 attenuates free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury.

The Na(+)/H(+) exchanger (NHE) is activated during ischemia-reperfusion in an effort to restore intracellular pH to normal levels. Inhibition of NHE with non-selective amiloride derivatives has been shown to be neuroprotective and to attenuate free fatty acid efflux during ischemia-reperfusion. We evaluated the effects of SM-20220 (20 microM), a highly selective and specific NHE inhibitor, applied topically onto rat cerebral cortex prior to and during a 20-min period of ischemia. SM-20220 application significantly reduced the ischemia-evoked efflux of myristic, palmitic, and arachidonic acids during both ischemia and reperfusion with significant decreases in linoleic and docosahexaenoic levels during reperfusion. This study confirms the importance of NHEs in eliciting free fatty acid efflux, inhibition of which may be an essential component of the neuroprotective benefits of NHE inhibitors in ischemia-reperfusion injury.

Further studies on the effects of topical lactate on amino acid efflux from the ischemic rat cortex.

A rat four-vessel cerebral occlusion model was used to examine the effects of D-lactate and oxamate, a lactate dehydrogenase inhibitor, on cortical window superfusate levels of amino acids, glucose and L-lactate. Superfusate levels of aspartate, glutamate, taurine, GABA and phosphoethanolamine rose during ischemia and then declined during reperfusion. Glycine and alanine levels tended to increase during reperfusion, whereas glutamine levels were lower. Serine levels were not altered. Glucose levels declined rapidly during ischemia and recovered during reperfusion. Lactate levels were sustained during ischemia and increased during reperfusion. Unlike L-lactate, which attenuated ischemia/reperfusion (I/R) evoked amino acid release (J.W. Phillis, D. Song, L.L. Guyot, M.H. O'Regan, Lactate reduces amino acid release and fuels recovery of function in the ischemic brain, Neurosci. Lett. 272 (1999) 195-198), topical application of D-lactate (20 mM), which is not used as an energy substrate, enhanced the I/R release of aspartate, glutamate, GABA and taurine into cortical superfusates, and also elevated L-lactate levels above those in the controls. Glucose levels were not altered. Oxamate (20 mM) application elevated the pre-ischemia levels of alanine, glycine and GABA and those of GABA during ischemia. Levels of all amino acids, with the exception of phosphoethanolamine, were elevated during reperfusion. Oxamate, an inhibitor of lactate dehydrogenases 1 and 5, did not alter the pattern of efflux of glucose and L-lactate. In the presence of oxamate, L-lactate (20 mM) failed to inhibit amino acid release. The failure of D-lactate to attenuate amino acid release confirms the inability of this isomer to act as a metabolic substrate. The oxamate data indicate that inhibition of lactate dehydrogenase is detrimental to the viability of cortical cells during I/R, even though extracellular lactate levels are elevated. The pre-ischemia increases in alanine and glycine are suggestive of elevations in pyruvate as a result of the block of its conversion to lactate, with transamination reactions converting pyruvate to form these amino acids. In summary, the results further substantiate the concept of a role for L-lactate as a cerebral energy substrate.

The effect of streptozotocin-induced diabetes on the release of excitotoxic and other amino acids from the ischemic rat cerebral cortex.

OBJECTIVE: Hyperglycemic stroke results in increased neuronal damage, the exact mechanism of which is unknown. Lactic acidosis has been implicated; however, increases in the excitotoxic amino acid glutamate, which correlate with increased neuronal damage, may be the cause for the increased damage seen in hyperglycemic stroke. METHODS: Ten Sprague-Dawley rats were treated with streptozotocin (STZ; 50 mg/kg), and 12 normoglycemic rats were used as controls. Using a four-vessel occlusion model, global ischemia was assessed at 5 to 7 days after treatment in five animals (acute STZ group) or at 4 to 6 weeks after treatment in five animals (chronic STZ group). The cortical cup model was used to collect superfusates under basal, ischemic, and reperfusion conditions and analyzed for nine different amino acids using high-performance liquid chromatography. RESULTS: Plasma glucose levels were significantly higher in the acute and chronic STZ groups as compared with the control group. Plasma lactate levels were higher in the acute STZ group as compared with the control or chronic STZ groups. Extracellular cortical glutamate levels were significantly reduced during reperfusion in the acute STZ group and during ischemia/reperfusion in the chronic STZ group as compared with the controls. Levels of extracellular gamma-aminobutyric acid were significantly reduced in the acute and chronic STZ groups as compared with the controls. CONCLUSION: A chronic state of hyperglycemia results in reduction in extracellular brain glutamate levels during ischemia/reperfusion and therefore does not appear to be responsible for the increased neuronal damage seen in diabetic stroke. Chronic hyperglycemia also causes decreased extracellular gamma-aminobutyric acid levels, which, because of the loss of the inhibitory effects of this neurotransmitter, could contribute to the increased damage observed in hyperglycemic stroke.

Real-time measurement of glutamate release from the ischemic penumbra of the rat cerebral cortex using a focal middle cerebral artery occlusion model.

Following permanent middle cerebral artery occlusion, extracellular penumbral glutamate levels, measured by a real-time glutamate electrode, increased in two different patterns. In 7/11 rats, glutamate increased from baseline levels of 19+/-4 (mean+/-SEM) to 208+/-29 microM and then declined towards baseline levels. Blood flow in the penumbral area declined to 30% of pre-ischemic levels with recovery to 60 and 70% of baseline values by 3 and 6 h, respectively. Four of 11 rats in the study also exhibited late peaks of glutamate release (120+/-40 microM ) 2 h after the onset of ischemia. There were no changes in the EEG recordings or cerebral blood flow during these late glutamate peaks.

Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex.

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with alpha-cyano-4-hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mM) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mM glutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mM) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia-evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia-evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia-evoked amino acid efflux.

Inhibition of Na(+)/H(+) exchange by 5-(N-ethyl-N-isopropyl)-amiloride reduces free fatty acid efflux from the ischemic reperfused rat cerebral cortex.

Brain tissue acidosis is considered to be a contributor to ischemic brain injury. The deleterious effects of marked acidosis may be associated with reperfusion and an excessive entry of Na(+) into cerebral neurons and glia as intracellular pH is restored by Na(+)/H(+) exchange. Normalization of pH, with activation of many calcium-dependent and other phospholipases and proteases with pH optima in the neutral or alkaline range, could account for the pronounced elevation in extracellular levels of free fatty acids which occurs during reperfusion following cerebral ischemia. In the present investigation we evaluated the effects of inhibition of Na(+)/H(+) exchange with N-(N-ethyl-N-isopropyl)-amiloride (EIPA; 25 microM) applied topically onto the rat cerebral cortex prior to and during ischemia. Free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high pressure liquid chromatography. EIPA application effectively inhibited the increases in arachidonic and linoleic acid release observed in the control rats during reperfusion, and non-significantly depressed that of palmitic and oleic acids. Superfusate levels of glucose, which decline to near zero levels during ischemia and then rebound during reperfusion, were not affected by EIPA administration. Lactate levels in cortical superfusates from EIPA-treated animals rose more rapidly during reperfusion than did those in the control rats and then significantly declined towards basal levels. The data indicate that inhibition of Na(+)/H(+) exchange prevented the activation of phospholipases that usually occurs during reperfusion following a cerebral ischemic episode. These results are the first demonstration of such an effect and may provide an explanation for the cerebroprotective effects that have been observed in stroked animals following administration of Na(+)/H(+) exchange inhibitors.

The effect of topical insulin on the release of excitotoxic and other amino acids from the rat cerebral cortex during streptozotocin-induced hyperglycemic ischemia.

Insulin has been demonstrated to be neuroprotective in brain and spinal cord ischemia. The mechanism of neuroprotection may involve alterations in metabolism, protein synthesis or uptake of GABA by astrocytes. Conversely, hyperglycemia increases the extent of neurologic damage observed during ischemia/reperfusion. Diabetic patients are 2-4 times more likely to suffer a stroke as normoglycemic patients and they also have worsened neurologic outcome. Determining if insulin, which many diabetics already use as therapy, can be neuroprotective, would be a possible means of alleviating the detrimental outcome from diabetic stroke. This study looked at the relationship between topically administered insulin (1 mIU insulin/ml and 100 mIU insulin/ml) during a four vessel occlusion model of global ischemia and the release of amino acids, especially glutamate, from the cortex in streptozotocin (STZ)-treated rats. The rats were utilized either 5-7 days (ASTZ) or 4-6 weeks (CSTZ) after a single STZ injection. In the ASTZ animals both doses of insulin increased the amount of the excitotoxic amino acids, aspartate and glutamate, released during reperfusion and the higher dose also increased the levels of taurine and GABA during reperfusion. In the CSTZ animals, both doses of insulin increased the amount of excitotoxic amino acids during reperfusion and the lower dose increased GABA levels released during reperfusion. The differences between the ACTZ and CSTZ animals may be due to metabolic differences in the utilization of glucose. Insulin may act as a neuroprotectant by increasing extracellular GABA resulting in neuroinhibition.

The effect of intravenous insulin on accumulation of excitotoxic and other amino acids in the ischemic rat cerebral cortex.

Insulin has been reported to be neuroprotective during cerebral ischemia/reperfusion. However, it may also increase the sensitivity of cultured cortical neurons to glutamate toxicity. The experiments described here utilized a rat four-vessel occlusion model with cerebral cortical windows to determine the effects of intravenous insulin, alone (I) or combined with glucose (IG) to maintain physiologic blood glucose levels, on the extracellular accumulation of amino acids in superfusates of the cerebral cortex. Aspartate, phosphoethanolamine, taurine and gamma-aminobutyric acid were increased in the I and IG groups and glutamate was increased in the IG group compared to controls during ischemia/reperfusion. Insulin treatment attenuated the rebound in cortical superfusate glucose levels in both groups of animals during reperfusion. The increases in amino acid release during reperfusion may be due to a lack of glycolytically derived energy available for amino acid uptake systems and ionic pumps.

Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with DL-threo-beta-benzyloxyaspartate.

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na(+)-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using DL-threo-beta-benzyloxyaspartate (DL-TBOA), a newly developed competitive, non-transported blocker of the EAAT 1-3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus DL-TBOA (100 microM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of DL-TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of DL-TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of DL-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na(+), and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na(+) uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na(+)-dependent, glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.

Mechanisms involved in coronary artery dilatation during respiratory acidosis in the isolated perfused rat heart.

A rat Langendorff heart preparation, perfused at constant pressure, was used to evaluate the role of K(ATP) channels in respiratory acidosis-induced coronary hyperemia. Prior administration of glibenclamide, an inhibitor of K(ATP) channels, reduced basal flow rates and eliminated the hyperemia associated with hypercapnia. These results implicate K(ATP) channels as a functional link in the respiratory acidosis-induced increase in coronary flow.

Effects of the anion channel blocker DIDS on ouabain- and high K(+)-induced release of amino acids from the rat cerebral cortex.

Amino acid release from the rat cerebral cortex was analyzed using an in vivo cortical cup perfusion model. Topical applications of ouabain or high extracellular K(+) were used to mimic two dimensions of ischemic conditions which promote cell swelling and amino acid release. Ouabain (30 microM) induced significant releases of taurine, gamma-aminobutyric acid (GABA), aspartate, glutamate and phosphoethanolamine. The anion channel blocker, 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS; 1 mM), inhibited ouabain-induced release of all these amino acids except for glutamate. Exposure to high extracellular K(+) (75 mM) induced a delayed rise in the levels of taurine in the superfusates and an immediate increase in GABA levels. There were no significant releases of other amino acids. The release of taurine and GABA was sensitive to the blocking of anion channels with DIDS. Both ouabain- and high K(+)-induced taurine release is likely to be mediated by DIDS sensitive anion channels. The extracellular accumulation of the other amino acids, where insensitive to DIDS, may be mediated by mechanisms other than swelling-induced anion channels.

Topical insulin and accumulation of excitotoxic and other amino acids in ischemic rat cerebral cortex.

Insulin plays a neuroprotectant role in the brain and spinal cord during ischemia. However, studies have shown insulin to increase the sensitivity of cultured cortical cells to glutamate toxicity. The present study looked at the relationship between topically administered insulin (1 mIU insulin/ml and 100 mIU insulin/ml) during a four-vessel model of global ischemia and the accumulation of amino acids, especially glutamate, from the ischemic rat cerebral cortex. The lower dose of insulin was found to attenuate the release of excitotoxic and other amino acids from the cortex in ischemia/reperfusion. This may occur because insulin increases glucose availability to glial cells resulting in maintenance of glycolysis and ionic pumps that can reduce glutamate release and maintain uptake during ischemia/reperfusion. The higher dose of insulin, which significantly increased the amount of aspartate, glutamate, taurine, and GABA during reperfusion, may act to stimulate the amount of glycogen stored in astrocytes, reducing the availability of glucose for metabolic purposes.