Shoichi Asano

Involvement of Na+–Ca2+ Exchanger in Reperfusion‐induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca2+ depletion induces an increase in intracellular Ca2+ ([Ca2+]i) after reperfusion with Ca2+‐containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na+‐Ca2+ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca2+‐free medium for 15–60 min, a persistent increase in [Ca2+]i was observed immediately after reperfusion with Ca2+‐containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca2+]i was enhanced by low extracellular Na+ ([Na+]o) during reperfusion and blocked by the inhibitors of the Na+‐Ca2+ exchanger amiloride and 3,4‐dichlorobenzamil, but not by the Ca2+ channel antagonists nifedipine, Cd2+ and Ni2+. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na+‐Ca2+ exchanger decreased Na+–Ca2+ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion‐induced increase in [Ca2+]i and cell toxicity. The Na+‐Ca2+ exchange inhibitors 3,4‐dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8‐bromo‐cyclic GMP and low [Na+]o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca2+ entry via the Na+–Ca2+ exchanger plays an important role in reperfusion‐induced delayed glial cell death.

Increase of noradrenaline release in the hypothalamus of freely moving rat by postsynaptic 5‐hydroxytryptamine1A receptor activation

1 5‐Hydroxytryptamine (5‐HT) plays a role in the regulation of noradrenergic neurones in the brain, but the precise mechanism of regulation of noradrenaline (NA) release by 5‐HT1A receptors has not been defined. The present study describes the effect of a highly potent and selective 5‐HT1A receptor agonist, 5‐{3‐[[(2S)‐1,4‐benzodioxan‐2‐ylmethyl]amino]propoxy}‐1,3‐benzodioxole HC1 (MKC‐242), on NA release in the hypothalamus using microdialysis in the freely moving rat. 2 Subcutaneous injection of MKC‐242 (0.5mg kg_1) increased extracellular levels of NA and its metabolite, 3‐methoxy‐4‐hydroxyphenylglycol, in the hypothalamus and hippocampus. 3 The 5‐HT1a receptor agonists, 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) (0.2 mg kg−1) and buspirone (3 mg kg−1) mimicked the effect of MKC‐242 in increasing NA release in the hypothalamus. 4 The effects of MKC‐242 and 8‐OH‐DPAT in the hypothalamus were antagonized by pretreatment with WAY100135 (10 mg kg−1), a silent 5‐HT1A receptor antagonist. 5 Local administration of 8‐OH‐DPAT (10–100 μm), citalopram (1 μm), a 5‐HT reuptake inhibitor, and MDL72222 (10 μm), a 5‐HT3 receptor antagonist, into the hypothalamus, had no effect on NA release. 6 Intracerebroventricular injection with 5,7‐dihydroxytryptamine caused a marked reduction in brain 5‐HT content, but the treatment affected neither basal NA levels nor the MKC‐242‐induced increase in NA release. 7 The effect of MKC‐242 in increasing NA release was not attenuated by repeated treatment with the drug (0.5 mg kg−1, once a day for 2 weeks). 8 The present results suggest that activation of postsynaptic 5‐HT1A receptors increases NA release in the hypothalamus.

Role of Na+‐Ca2+ Exchanger in Agonist‐Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na+‐Ca2+ exchanger in the reverse mode causes Ca2+ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na+‐Ca2+ exchanger is involved in agonist‐induced Ca2+ signaling in cultured rat astrocytes. The astrocytic intracellular Ca2+ concentration ([Ca2+]i) was increased by l‐glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin‐induced increase in [Ca2+]i. The Na‐induced Ca2+ signal was also attenuated by S‐nitroso‐l‐cysteine and 8‐bromo cyclic GMP, whereas it was enhanced by 3,4‐dichlorobenzamil, an inhibitor of the Na+‐Ca2+ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na+‐Ca2+ exchanger enhanced the ionomycin‐induced increase in [Ca2+]i and blocked the effects of SNP and 8‐bromo cyclic GMP in reducing the NA‐induced Ca2+ signal. Furthermore, the ionomycin‐induced Ca2+ signal was enhanced by removal of extracellular Na+ and pretreatment with ascorbic acid. These findings indicate that the Na+‐Ca2+ exchanger is a target for NO modulation of elevated [Ca2+]i and that the exchanger plays a role in Ca2+ efflux when [Ca2+]i is raised above basal levels in astrocytes.

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca2+/calmodulin‐dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca2+‐containing medium after exposure to Ca2+‐free medium caused Ca2+ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca2+‐mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca2+ challenge‐induced injury in a dose‐dependent manner (10−10–10−8M). Cyclosporin A at 1 µM mimicked the effect of FK506. Rapamycin (1 µM) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 nM), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca2+ challenge‐induced increase in cytosolic Ca2+ level nor Na+‐Ca2+ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca2+ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.

Antidepressant-like effect by postsynaptic 5-HT1A receptor activation in mice

The antidepressant-like effect of 5-(3(-)[((2S)-1,4-benzodioxan-2-ylmethyl)amino]propoxy)-1,3- benzodioxole (MKC-242), a novel 5-HT1A receptor agonist, was studied in the forced swimming test in mice injected i.c.v. with 5,7-dihydroxytryptamine to destroy 5-HT neurons or treated with p-chlorophenylalanine to inhibit 5-HT synthesis. MKC-242 reduced immobility time of mice pretreated with vehicle and these drugs, although it did not affect their locomotor activity. The anti-immobility effect was antagonized by 5-HT1A receptor antagonists such as propranolol and N-tert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylpropana mide. These findings support the hypothesis that postsynaptic 5-HT1A receptors play an important role in the antidepressant-like effect of 5-HT1A receptor agonists.

MKC-242, a novel 5-HT1A receptor agonist, facilitates cortical acetylcholine release by a mechanism different from that of 8-OH-DPAT in awake rats

We have previously reported that 5-¿3-[((2S)-1,4-benzodioxan-2-ylmethyl)amino]propoxy¿-1,3-be nzodioxole (MKC-242), a potent and selective serotonin (5-HT)1A receptor agonist, exerts anxiolytic- and antidepressant-like effects in animal models and that the antidepressant-like effect may be mediated by postsynaptic 5-HT1A receptors. The present study, using a microdialysis technique, was undertaken to characterize in vivo the effect of MKC-242 on cholinergic neurons. Subcutaneous injection of MKC-242 (0.5-1.0 mg/kg), like the typical 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), increased extracellular acetylcholine (ACh) levels in the rat cerebral cortex. The increase in ACh release by MKC-242 was also observed in the hippocampus. The effect of MKC-242 on cortical ACh release was attenuated by pretreatment with the 5-HT1A receptor antagonists (10 mg/kg, s.c.) propranolol and N-tert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylpropana mide. The increase in cortical ACh release by MKC-242 was blocked by lesion of serotonergic neurons with 5,7-dihydroxytryptamine, whereas that by 8-OH-DPAT was not. Lesion of noradrenergic neurons with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine did not affect the MKC-242-induced increase in ACh release. These results suggest that systemic injection of MKC-242 facilitates in vivo ACh release via an activation of somadendritic 5-HT1A autoreceptors, and that MKC-242 and 8-OH-DPAT affect cholinergic neurons in the rat cerebral cortex via different mechanisms.

Isoform‐Specific Up‐Regulation by Ouabain of Na+,K+‐ATPase in Cultured Rat Astrocytes

Abstract: There are two α‐subunit isoforms (α1 and α2) and two β‐subunit isoforms (β1 and β2) of Na+,K+‐ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 mM) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain‐treated cultures were of glial origin. Low extracellular K+ and monensin (20 µM) mimicked the effect of ouabain on α1 mRNA. The ouabain‐induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µM), the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetraacetoxymethyl ester (30 µM), and the calcineurin inhibitor FK506 (1 nM). These findings indicate that chronic inhibition of Na+,K+‐ATPase up‐regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na+, Ca2+, and calcineurin may be involved in ouabain‐induced up‐regulation of the enzyme in astrocytes.

Intracellular Ascorbic Acid Inhibits the Na+‐Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca2+ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na+‐Ca2+ exchanger in the reverse mode. Ascorbic acid at 0.1–1 mM inhibited Na+‐dependent Ca2+ uptake significantly but not Na+‐dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca2+ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na+‐dependent Ca2+ uptake was also inhibited by isoascorbate at 1 mM but not by ascorbate 2‐sulfate, dehydroascorbate, and sulfhydryl‐reducing reagents such as glutathione and 2‐mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o‐phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na+‐free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na+‐dependent Ca2+ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na+‐Ca2+ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.

Heat shock protects cultured rat astrocytes in a model of reperfusion injury

We have previously found that incubation of cultured rat astrocytes in Ca(2+)-free medium caused an increase in intracellular Ca2+ ([Ca2+]i) followed by delayed cell death. Here, we examined whether thermal stress protects astrocytes from cell death in this model system of reperfusion injury. Cultured astrocytes were preincubated at 40-44 degrees C for 10-20 min in fetal calf serum-free medium, incubated at 37 degrees C for 24 h in serum-containing medium, and subjected to the in vitro reperfusion experiment. Thermal stress attenuated reperfusion-induced cell toxicity. Furthermore, the stress increased cell viability after incubation with serum-free medium containing Ca2+. These effects of heat shock required incubation in serum-containing medium for at least 12 h after heat shock, and it was blocked by the protein synthesis inhibitor cycloheximide. Thermal stress increased synthesis of several proteins, and one of the inducible proteins was identified as the 72-kDa heat shock protein by an immunoblot analysis. Neither the increase in [Ca2+]i nor the Na(+)-Ca2+ exchange activity in astrocytes induced in this model were affected by thermal stress. These findings suggest that heat shock proteins protect astrocytes from cell death in a model of reperfusion injury and they may affect processes down stream of the increase in [Ca2+]i.