Akemichi Baba

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.

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.

Endothelins modulate dibutyryl cAMP-induced stellation of cultured astrocytes.

Effect of endothelin-3 (ET-3) on dibutyryl cAMP (DBcAMP)-induced stellation of rat cerebral cultured astrocytes was examined. Treatment with 1 mM DBcAMP, 10 microM forskolin, 100 microM isoproterenol and 500 nM phorbol 12-myristate 13-acetate changed protoplasmic cultured astrocytes into process-bearing ones. ET-3 (1 nM) completely prevented the astrocytic stellation induced by these agents. The effect of ET-3 showed a dose-dependence, where IC50 value and maximal effective dose were 49 pM and about 0.1 nM, respectively. ET-1 and sarafotoxin (SRTX) S6b prevented the DBcAMP-induced astrocytic stellation with potencies similar to that of ET-3. ET-3 (1 nM) did not affect the cAMP accumulation after DBcAMP treatment in cultured astrocytes. Stellate astrocytes were reversed to the protoplasmic type cells by addition of 1 nM ET-3 in the presence of DBcAMP. ET-1 and SRTX similarly reversed the astrocytic stellation. ET-3 reversed the astrocytic stellation in the absence of extracellular Ca2+. Pre-loading of BAPTA-AM, a permeable Ca2+ chelator, on stellate astrocytes had no effect on the reversal by ET-3. ET-3 did not increase intracellular free Ca2+ concentration ([Ca2+]i) of most astrocytes tested at 0.1 nM. A high concentration (100 nM) of ET-3 increased astrocytic [Ca2+]i which was negated by Ca(2+)-free and BAPTA-AM loading. These results suggest that ETs modulate morphological changes in astrocytes through cAMP- and Ca(2+)-independent mechanisms.

cDNA cloning of a thromboxane A2 receptor from rat astrocytes

A cDNA clone for rat thromboxane (TX) A2 receptor was obtained from cultured astrocytes. The cDNA encodes a protein of 341 amino acids with seven putative transmembrane domains. The receptor is homologous with mouse and human TXA2 receptors at 92.7% and 71.8%, respectively. Expression of messenger RNA for the receptor was examined by reverse transcriptase-polymerase chain reaction, showing ubiquitous expression in brain glial cells such as astrocytes, oligodendrocytes and microglia.

Cloning and expression of a cDNA for rat prostaglandin F2α receptor

Abstract We have cloned a cDNA for rat prostaglandin (PG) F 2α receptor from cultured rat astrocytes. The cDNA encodes a polypeptide of 366 amino acids with seven putative transmembrane domains. Specific binding of [ 3 H]PGF 2α in membranes of COS-7 cells transfected with the cDNA was displaced with unlabeled PGs in the order of PGF 2 α > PGD 2 > PGE 2 > PGI 2 . In the cDNA-transfected LLC-PK 1 cells, PGF 2α stimulated phosphoinositide hydrolysis. A significant 4.7-kb mRNA transcript was detected in cultured rat astrocytes and whole brain and pregnant ovary of adult rats by Northern blot analysis.

Inhibitory glutamate response on cyclic AMP formation in cultured astrocytes.

We examined the effects of glutamate receptor agonists on cyclic AMP (cAMP) formation in cultured astrocytes. L-Glutamate reduced the cAMP formation induced by either isoproterenol (IC50 7 microM) or forskolin without affecting the basal level. Glutamate agonists reduced the cAMP formation in astrocytes with the following rank order of potency: L-glutamate > trans-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) = quisqualate. Pretreatment of astrocytes with pertussis toxin resulted in a partial reduction of the glutamate response and a complete attenuation of the t-ACPD response. These results suggest that astrocytes have another type of metabotropic glutamate receptor which inhibits adenylate cyclase through pertussis toxin-sensitive G-proteins.

Endothelins are extracellular signals modulating cytoskeletal actin organization in rat cultured astrocytes

Effects of endothelin-3 on rapid morphological changes and cytoskeletal actin organization of rat cortical cultured astrocytes were examined. In serum-free medium, treatments with 1 mM dibutyryl cAMP and 5 microM cytochalasin B, an inhibitor of actin polymerization, caused astrocytic morphological changes with cytoplasmic retraction (stellation). Concurrent addition of 1 nM endothelin-3 prevented astrocytic stellation by dibutyryl cAMP and cytochalasin B. The inhibition of endothelin-3 on the astrocytic stellation was dose-dependent, where IC50 and maximal effective dose were about 50 pM and 0.1 nM, respectively. Endothelin-1 and sarafotoxin S6b prevented the cytochalasin B-induced stellation with similar potencies to endothelin-3. Endothelin-3 reversed the stellate morphology of cytochalasin B-treated cells. Sixty minutes after addition of endothelin-3, most cytochalasin B-treated astrocytes lost their apparent distinction between cell body and processes. Treatment with dibutyryl cAMP and cytochalasin B decreased actin content in a 0.5% Triton X-100-insoluble fraction (cytoskeletal fraction) of cultured astrocytes. Subsequent treatments with endothelin-3 for 2 h restored the decreased cytoskeletal actin to that of non-treated cells. Rhodamine-phalloidin staining showed that a prominent structure of organized filamentous actin in protoplasmic astrocytes is stress fibers. The astrocytic stress fibers disappeared after treatment with dibutyryl cAMP and cytochalasin B. Endothelin-3 stimulated reorganization of stress fibers both in the dibutyryl cAMP- and the cytochalasin B-treated astrocytes. These results suggest that endothelins are extracellular signals to regulate cytoskeletal actin organization of astrocytes.

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.

Involvement of Na+, K+-ATPase in the mitogenic effect of insulin-like growth factor-I on cultured rat astrocytes

Abstract: We have previously reported that insulin/insulin‐like growth factor (IGF)‐I induced the α1 isoform of Na+,K+‐ATPase in cultured astrocytes. In this study the effects of insulin/IGF‐I on Na+,K+‐ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF‐I on astrocytes. Insulin increased Na+,K+‐ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [3H]thymidine incorporation in astrocytes preincubated in fetal calf serum‐free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na+,K+‐ATPase activity and expression of the α1 mRNA preceded the mitogenic effect. 125I‐IGF‐I binding experiment showed that all the cells used here had similar binding characteristics. The insulin‐induced increase in enzyme activity was not affected by 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7), and it was observed even in Ca2+‐free medium. The stimulation by IGF‐I of [3H]thymidine incorporation was attenuated by ouabain and a low external K+ level. These findings suggest that stimulation of Na+,K+‐ATPase activity is involved in the mitogenic action of IGF‐I on cultured astrocytes.

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.