Jun-ichi Kitanaka

Expression of glutamate transporters in cultured glial cells

Expression of mRNAs for glutamate transporter (GLT-1) and glutamate aspartate transporter (GLAST) was investigated in three different types of purified glial cells by the reverse transcriptase-polymerase chain reaction (RT-PCR). Cultured astrocytes, oligodendrocytes, and microglia expressed mRNAs for GLAST and GLT-1; mRNA for GLAST was expressed more prominently than that for GLT-1 in astrocytes. Oligodendrocytes and microglia expressed mRNAs for both GLT-1 and GLAST equally, but the expression in microglia was not prominent, suggesting glutamate uptake is not essential in microglia. In astrocytes cultured from different brain regions, GLAST mRNA was equally expressed. GLT-1 mRNA was also detected in these astrocytes, but the expression level was lower than that of GLAST.

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.

Expression pattern of messenger RNAs for prostanoid receptors in glial cell cultures

Expression level of messenger RNAs (mRNAs) for prostanoid EP3, FP, and TP receptors was investigated in cultured rat astrocytes, oligodendrocytes, and microglia, as well as in meningeal fibroblasts, rat glioma C6 cells, rat pheochromocytoma PC12 cells, whole brain, and several peripheral tissues by reverse transcriptase-polymerase chain reaction. Cultured astrocytes and oligodendrocytes expressed mRNAs for 3 prostanoid receptors examined. In contrast, cultured microglia and pheochromocytoma PC12 cells expressed EP3 and TP receptor mRNAs, but not FP receptor mRNA. Glioma C6 cells expressed only TP receptor mRNA among 3 prostanoid receptors with the same expression level as that in astrocytes. Cultured meningeal fibroblasts expressed 3 receptor transcripts, and their expression levels were lower than those in astrocytes. Expression level of mRNA for each prostanoid receptor in cultured glial cells was higher than that in whole brain. These observations suggest that each prostanoid has its specific roles in each glial cell type of the brain.

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F2α‐induced phosphoinositide hydrolysis and elevation of intracellular Ca2+ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF2α (1 μM)‐induced phosphoinositide hydrolysis in a concentration‐dependent manner with an IC50 value of 16 μM. The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose‐response curves of PGF2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μM) inhibited the stimulation induced by PGF2α (1 μM) plus GTPγS (50 μM) without affecting that induced by GTPγS alone. PGF2α at 1 μM transiently increased astrocytic intracellular Ca2+ concentration in 39% of the cells tested. The response was completely blocked by 100 μM phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF2α receptor linked to phospholipase C in astrocytes.

Astrocytes possess prostaglandin F2α receptors coupled to phospholipase C

We examined the effect of prostaglandin (PG) F2 alpha on phosphoinositide (PI) hydrolysis in rat cultured astrocytes. PGF2 alpha stimulated the formation of [3H]inositol phosphates in [3H]inositol-labeled astrocytes with the ED50 value of 23 nM, whereas PGD2 and PGE2 were much less effective than PGF2 alpha. Transformation of astrocytes was accompanied by an increase in the stimulatory response of PGF2 alpha. Pretreatment of the astrocytes with pertussis toxin and cholera toxin did not affect the PGF2 alpha-evoked PI hydrolysis. In the digitonin-permeabilized astrocytes, PGF2 alpha significantly enhanced the GTP gamma S-evoked PI hydrolysis in the presence of Ca2+. These results indicate that rat cultured astrocytes possess PGF2 alpha receptors coupled to phospholipase C.

Desensitization of prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Desensitization of prostaglandin (PG) F2α receptor-mediated phosphoinositide (PI) hydrolysis was investigated in cultured rat astrocytes. Prolonged exposure of astrocytes differentiated by dibutyryl cyclic AMP-treatment to PGF2α caused the desensitization of subsequent PGF2α-induced PI hydrolysis. The desensitization was time- and PGF2α dose-dependent; maximal decrease in the PI hydrolysis was observed after exposure to 10 μM PGF2α for 4 h and the degree of the desensitization was 31.7±2.7% of control. Pretreatment with either PGD2 or PGE2 also induced the desensitization of subsequent PGF2α-stimulated PI hydrolysis and conversely pretreatment of PGF2α decreased the PI responses to PGD2 and PGE2. The desensitization prevented by phloretin and was reversible upon removal of the agonist. Protein synthesis inhibitors blocked the recovery of the desensitization. Treatment of the cells with phorbol 12-myristate 13-acetate had no effect on the desensitization. These results suggest that prolonged exposure of the astrocytes to PGF2α caused the desensitization of the receptors.

Cytochalasin B inhibits phosphoinositide hydrolysis in rat hippocampal slices

The effect of cytochalasin B on phosphoinositide (PI) hydrolysis was examined in rat hippocampal slices. Pretreatment of the slices with cytochalasin B caused a significant decrease in PI hydrolysis elicited by carbachol, norepinephrine, or by high K+. This effect was cytochalasin B dose- and time-dependent and was not mimicked by cytochalasin D, vinblastine, colchicine, or phloretin. In contrast, in [3H]inositol-prelabeled hippocampal membranes, cytochalasin B did not affect PI hydrolysis elicited by GTPγS and GTPγS plus carbachol. Similar result was obtained using the membranes prepared from the slices pretreated with cytochalasin B. The inhibitory effect of cytochalasin B on the carbachol-response was observed in SK-N-SH human neuroblastoma cells, but not in cultured rat astrocytes. These results indicate that cytochalasin B inhibits PI hydrolysis in neuron-specific manner and that its action may be an indirect cellular mechanism other than interaction with cytoskeleton elements.

Tunicamycin inhibits prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Effect of tunicamycin, an inhibitor of N-linked glycosylation, on prostaglandin (PG) F2α-stimulated phosphoinositide (PI) hydrolysis was examined in cultured rat astrocytes. Pretreatment of cultured astrocytes with tunicamycin (25–250 ng/ml) inhibited subsequent PGF2α (1 μM)-stimulated PI hydrolysis in concentration- and time-dependent manners. The inhibition completely recovered after removal of tunicamycin and re-incubation for 12 h. Tunicamycin pretreatment (100 ng/ml for 12 h) significantly blocked [35S]methionine incorporation into cultured astrocytes, but cell viability was not affected under the condition. Inhibitors of processing of N-linked sugar chains such as bromoconduritol, 1-deoxymannojirimycin, and swainsonine had no effect on PI response to PGF2α. These observations suggest that PGF2α receptor is N-linked glycosylated.