Effect of TPA on aquaporin 4 mRNA expression in cultured rat astrocytes

Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brai ns, localized in the astrocyte plasma membrane. The regulation of AQP4 is b elieved to be important for the homeostasis of water in the brain, but the AQP4 regulatory mechanisms are not yet known. In this study, we investigate d the effect of a protein kinase C (PKC) activator on the expression of AQP 4 mRNA in cultured rat astrocytes. Cultured rat astrocytes constitutively e xpressed AQPQ mRNA. Treatment of the cells with 0.1 mM of phorbol ester 12- 0-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, caused a rapi d decrease in AQP4 mRNA. This effect was time- and dose-dependent. The TPA- induced decrease in AQP4 mRNA was inhibited by a relatively specific PKC in hibitor, l-(5-isoquinoline sulfonyl)-2-methylpiperazine (H7) in a dose-depe ndent manner. Moreover, prolonged treatment of the cells with TPA eliminate d the subsequent decrease in AQP4 mRNA by TPA. These results strongly sugge st that the TPA-induced decrease in AQP4 mRNA is mediated by PKC activation . To test whether the effect of TPA requires protein synthesis, astrocytes were pretreated with cycloheximide, an inhibitor of protein synthesis. Pret reatment of the cells with cycloheximide did not inhibit the decrease in AQ P4 mRNA induced by TPA. To test whether the TPA-induced decrease in AQP4 wa s due to a decrease in the mRNA stability we examined the effect of actinom ycin D, an inhibitor of transcription, on TPA-treated cells. The stability of AQP4 mRNA was not decreased by the pretreatment of the cells with actino mycin D. The results suggest that AQP4 mRNA is inhibited by TPA via PKC act ivation without de novo protein synthesis, and that the inhibition of AQP4 mRNA could be at the transcriptional level. (C) 1999 Wiley-Liss, Inc.