Kazuya Sobue

Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors.

The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.

Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated Protein Kinase-dependent Pathway in Rat Astrocytes*

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.

Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.

Interleukin‐1β induces the expression of aquaporin‐4 through a nuclear factor‐κB pathway in rat astrocytes

Interleukin (IL)‐1β is known to play a role in the formation of brain edema after various types of injury. Aquaporin (AQP)4 is also reported to be involved in the progression of brain edema. We tested the hypothesis that AQP4 is induced in response to IL‐1β. We found that expression of AQP4 mRNA and protein was significantly up‐regulated by IL‐1β in cultured rat astrocytes, and that intracerebroventricular administration of IL‐1β increased the expression of AQP4 protein in rat brain. The effects of IL‐1β on induction of AQP4 were concentration and time dependent. The effects of IL‐1β on AQP4 were mediated through IL‐1β receptors because they were abolished by co‐incubation with IL‐1 receptor antagonist. It appeared that IL‐1β increased the level of AQP4 mRNA without involvement of de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, did not inhibit the effects of IL‐1β. Inhibition of the nuclear factor‐κB (NF‐κB) pathway blocked the induction of AQP4 by IL‐1β in a concentration‐dependent manner. These findings show that IL‐1β induces expression of AQP4 through a NF‐κB pathway without involvement of de novo protein synthesis in rat astrocytes.

Differential regulation of aquaporin expression in astrocytes by protein kinase C

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In astrocytes of the central nervous system (CNS), using the reverse transcription-polymerase chain reaction (RT-PCR), we previously detected AQP3, 5 and 8 mRNAs in addition to the reported AQP4 and 9. However the mechanisms regulating the expression of these AQPs are not known. In this study, we investigated the effects of a protein kinase C (PKC) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with TPA caused decreases in AQP4 and 9 mRNAs and proteins in time- and concentration-dependent manners. The TPA-induced decreases in AQP4 and 9 mRNAs were inhibited by PKC inhibitors. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decreases in AQP4 and 9 mRNAs caused by TPA. Pretreatment of cells with an inhibitor of protein synthesis, cycloheximide, did not inhibit the decreases in AQP4 and 9 mRNAs induced by TPA. These results suggest that signal transduction via PKC may play important roles in regulating the expression of AQP4 and 9.

Experimental implication of celiac ganglionotropic invasion of pancreatic-cancer cells bearing c-ret proto-oncogene with reference to glial-cell-line-derived neurotrophic factor (GDNF)

Perineural invasion is a prominent clinical feature of pancreatic cancer which causes difficulty in curative resection. In the present study, the human pancreatic cancer cell lines, PaCa‐2, AsPC‐1, SW1990 and Capan‐2, were all found to express abundant c‐ret proto‐oncogene mRNA and RET protein, a member of the receptor‐tyrosine‐kinase superfamily, identified as being a receptor for glial‐cell‐line‐derived neurotrophic factor (GDNF). In an invasion assay, the migration of pancreatic cancer cells was markedly induced by co‐cultivation with human glioma cells, T98G or A172, capable of producing and secreting GDNF. Anti‐GDNF antibody in conditioned media of glioma cells suppressed much of the migratory activity. Checkerboard analysis of the migration showed both chemotactic and chemokinetic activity of GDNF. There was no detectable expression of another GDNF receptor component, a glycosyl‐phosphatidylinositol‐linked receptor (GFRα‐1), in pancreatic‐cancer cell lines, suggesting that the neural invasion of pancreatic‐cancer cells spreads along a concentration gradient of GDNF produced from peripheral ganglions through direct interaction of GDNF with its receptor, the c‐ret proto‐oncogene product. Immunochemical localization of GDNF in human celiac ganglionic tissue supported this contention. Int. J. Cancer 81:67–73, 1999.

Lactic acid increases aquaporin 4 expression on the cell membrane of cultured rat astrocytes

The water channel protein aquaporin (AQP) may play roles in the homeostasis of water content in the brain and brain edema. One possible mechanism of brain edema is glial swelling due to lactic acidosis associated with ischemia. Here, we investigated the effect of lactic acid on the expression and cellular distribution of AQP 4 in cultured rat astrocytes. After 24h of incubation, the AQP4 expression level increased maximally with 35mM lactic acid. The AQP4 expression levels also increased with hydrochloric acid or acetic acid. In contrast, with sodium lactate, the AQP4 levels did not increase. The increase in AQP4 expression level occurred without a significant increase in AQP4 mRNA expression level by lactic acid. Under the conditions of de novo protein synthesis inhibition with cycloheximide, lactic acid increased the AQP4 expression level. Furthermore, lactic acid increased the AQP4 expression level on the cell surface of the astrocytes, as determined by a cell surface biotinylation assay and immunocytochemical examination. The increase in AQP4 expression level on the cell membrane of astrocytes induced by lactic acid may be a new regulation mechanism of AQP4 in the brain.

Donepezil Improves Cognitive Function in Mice by Increasing the Production of Insulin-Like Growth Factor-I in the Hippocampus

Insulin-like growth factor-I (IGF-I) exerts beneficial effects on cognitive function. The selective acetylcholinesterase inhibitor donepezil increases serum IGF-I levels in elderly subjects. Because stimulation of sensory neurons induces IGF-I production by releasing calcitonin gene-related peptide (CGRP) in the mouse brain, we hypothesized that donepezil increases IGF-I production by sensory neuron stimulation to improve the cognitive function in mice. Donepezil, but not tacrine, increased the CGRP release from dorsal root ganglion neurons isolated from wild-type (WT) mice. Pretreatment with the protein kinase A inhibitor KT5720 [(9S,10S,12R)-2,3,9,10,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]-benzo-diazocine-10-carboxylic acid hexyl ester] reversed the effects induced by donepezil. Increase in tissue levels of CGRP, IGF-I, and IGF-I mRNA in the hippocampus was observed at 4 weeks after oral administration of donepezil in WT mice. In these animals, c-fos expression in spinal dorsal horns, parabrachial nuclei, the solitary tract nucleus, and the hippocampus was increased. Enhancement in angiogenesis and neurogenesis was observed in the dentate gyrus of the hippocampus of WT mice after donepezil administration. Improvement of spatial learning was observed in WT mice after donepezil administration. Oral administration of tacrine for 4 weeks produced none of the aforementioned effects induced by donepezil in WT mice. However, none of the effects observed in WT mice was seen after donepezil administration in CGRP-knockout mice and WT mice subjected to functional denervation. These observations suggest that donepezil may improve cognitive function in mice by increasing the hippocampal production of IGF-I through sensory neuron stimulation. These effects of donepezil may not be dependent on its acetylcholinesterase inhibitory activity.

Differential regulation of aquaporin-5 and -9 expression in astrocytes by protein kinase A.

Aquaporins (AQPs) transport water through the membranes of numerous tissues, but the molecular mechanisms for regulating water balance in brain are unknown. In this study, we investigated the effects of a protein kinase A (PKA) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with dbcAMP caused decreases in AQP5 mRNA and protein and increases in AQP9 mRNA and protein in time- and concentration-dependent manners. However, AQP4 mRNA and protein were not changed by treatment with dbcAMP. The dbcAMP-induced effects on AQP5 and AQP9 mRNAs were inhibited by PKA inhibitors. In addition, pretreating the cells with an inhibitor of protein synthesis, cycloheximide, inhibited the increase in AQP9 mRNA induced by dbcAMP, but not the decrease in AQP5 mRNA. These results suggest that signal transduction via PKA may play important roles in regulating the expression of AQP5 and AQP9, and the effect on AQP9 may be mediated by some factors induced by dbcAMP.

Regulation of aquaporin-4 expression in astrocytes

Aquaporin-4 (AQP4), a mercury-insensitive water channel protein, is abundant in the central nervous system and is localized in astrocytes and ependymal cells. AQP4 is speculated to maintain the homeostasis of intracellular and extracellular water in the brain, but little is known about the mechanism of induction of its expression. To investigate the expressional regulation of AQP4, we analyzed changes in its expression during chemically induced differentiation of embryonal carcinoma cells (P19) to neuronal and astrocytic cells, and during the cell cycle of glioma cells. After exposure to retinoic acid for 4 days AQP4 mRNA expression started at the initiation of astrocytic differentiation of P19 cells at 6 days, and increased markedly by 21 days. AQP4 expression was parallel to that of GFAP, a marker intermediate filament of astrocytes. In glioma cell lines, AQP4 mRNA was not detected in the growing phase, but was induced when the cell cycle was arrested at G0/G1 by transient expression of p21. Although quiescent astrocytes in the G0/G1-phase cultured under the serum-free condition exhibited a high expression of AQP4, serum supplement moved them to the S-phase and markedly decreased the AQP expression. These results suggest that AQP4 expression may be induced not only at the initiation of astrocytic differentiation of neural stem cells, but also at the G0/G1-phase during the cell cycle of astrocytes.