Atsuko Fujioka

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

Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, localized in the astrocyte plasma membrane. The regulation of AQP4 is believed to be important for the homeostasis of water in the brain, but the AQP4 regulatory mechanisms are not yet known. In this study, we investigated the effect of a protein kinase C (PKC) activator on the expression of AQP4 mRNA in cultured rat astrocytes. Cultured rat astrocytes constitutively expressed AQP4 mRNA. Treatment of the cells with 0.1 μM of phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), an activator of PKC, caused a rapid 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 inhibitor, 1‐(5‐isoquinoline sulfonyl)‐2‐methylpiperazine (H7) in a dose‐dependent manner. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decrease in AQP4 mRNA by TPA. These results strongly suggest 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. Pretreatment of the cells with cycloheximide did not inhibit the decrease in AQP4 mRNA induced by TPA. To test whether the TPA‐induced decrease in AQP4 was due to a decrease in the mRNA stability, we examined the effect of actinomycin D, an inhibitor of transcription, on TPA‐treated cells. The stability of AQP4 mRNA was not decreased by the pretreatment of the cells with actinomycin D. The results suggest that AQP4 mRNA is inhibited by TPA via PKC activation without de novo protein synthesis, and that the inhibition of AQP4 mRNA could be at the transcriptional level. GLIA 25:240–246, 1999.

Differential entrainment of peripheral clocks in the rat by glucocorticoid and feeding.

The suprachiasmatic nucleus is the master circadian clock and resets the peripheral clocks via various pathways. Glucocorticoids and daily feeding are major time cues for entraining most peripheral clocks. However, recent studies have suggested that the dominant timing factor differs among organs and tissues. In our current study, we reveal differences in the entrainment properties of the peripheral clocks in the liver, kidney, and lung through restricted feeding (RF) and antiphasic corticosterone (CORT) injections in adrenalectomized rats. The peripheral clocks in the kidney and lung were found to be entrained by a daily stimulus from CORT administration, irrespective of the meal time. In contrast, the liver clock was observed to be entrained by an RF regimen, even if daily CORT injections were given at antiphase. These results indicate that glucocorticoids are a strong zeitgeber that overcomes other entrainment factors regulating the peripheral oscillators in the kidney and lung and that RF is a dominant mediator of the entrainment ability of the circadian clock in the liver.

FOS expression in orexin neurons following muscimol perfusion of preoptic area

Unilateral microdialysis-perfusion of the preoptic area with 50 μM muscimol decreased the sleep period of rats to less than 3% of the baseline value over a 90 min period before death (p = 0.018 by Wilcoxon signed-rank test). These rats showed the expression of FOS in 36% of the orexin neurons located in the perifornical/lateral hypothalamic areas on the side ipsilateral to the perfusion site, but in only 3% of the orexin neurons on the side contralateral to it (p = 0.018 by Wilcoxon signed-rank test). These results suggest that subpopulations of the preoptic neurons give an inhibitory tone to the activities of the orexin neurons in the perifornical/lateral hypothalamic areas.

A role of the C-terminus of aquaporin 4 in its membrane expression in cultured astrocytes

Background: Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, which is localized in the astrocyte plasma membrane. Membrane targeting of AQP4 is essential to perform its function. The mechanism(s) of membrane targeting is not clear in astrocytes.

Gq/11‐induced intracellular calcium mobilization mediates Per2 acute induction in Rat‐1 fibroblasts

Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up‐regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum‐induced immediate rat Per2 (rPer2) transactivation in Rat‐1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up‐regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up‐regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin‐induced acute rPer2 transactivation via Gq/11‐coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11‐PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store.

Temporal profile of circadian clock gene expression in a transplanted suprachiasmatic nucleus and peripheral tissues

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is the master oscillator that regulates the circadian rhythms of the peripheral oscillators. Previous studies have demonstrated that the transplantation of embryonic SCN tissues into SCN‐lesioned arrhythmic mice restores the behavioral circadian rhythms of these animals. In our present study, we examined the clock gene expression profiles in a transplanted SCN and peripheral tissues, and also analysed the circadian rhythm of the locomotor activity in SCN‐grafted mice. These experiments were undertaken to elucidate whether the transplanted SCN generates a dynamic circadian oscillation and maintains the phase relationships that can be detected in intact mice. The grafted SCN indeed showed dynamic circadian expression rhythms of clock genes such as mPeriod1 (mPer1) and mPeriod2 (mPer2). Furthermore, the phase differences between the expression rhythms of these genes in the grafted SCN and the locomotor activity rhythms of the transplanted animals were found to be very similar to those in intact animals. Moreover, in the liver, kidney and skeletal muscles of the transplanted animals, the phase angles between the circadian rhythm of the grafted SCN and that of the peripheral tissues were maintained as in intact animals. However, in the SCN‐grafted animals, the amplitudes of the mPer1 and mPer2 rhythms were attenuated in the peripheral tissues. Our current findings therefore indicate that a transplanted SCN has the capacity to generate a dynamic intrinsic circadian oscillation, and can also lock the normal phase angles among the SCN, locomotor activity and peripheral oscillators in a similar manner as in intact control animals.

The resetting of the circadian rhythm by Prostaglandin J2 is distinctly phase-dependent

The circadian rhythm can be reset by a variety of substances. Prostaglandin J2 (PGJ2) is one such substance and resets the circadian rhythm in fibroblasts. In our current study, we examined the phase‐dependent phase shift following PGJ2 treatment using a real‐time luciferase luminescence monitoring system. In the phase response curves, we observed 12 h differences in the times of peaks in comparison with the same analysis for forskolin. Quantification of clock gene mRNAs following PGJ2 administration additionally revealed a rapid decrease in the Per1, Rev‐erbAα and Dbp levels. Our current findings thus suggest that PGJ2 resets the peripheral circadian clock via a mechanism that is distinct from that used by forskolin (FK).

Effect of phosphodiesterase type 4 on circadian clock gene Per1 transcription

The induction of Per1 gene in the suprachiasmatic nucleus, the center of the circadian clock, is assumed to play significant roles in the adjustment of the internal clock. cAMP is one of the intracellular mediators which activates Per1 transcription. Here, we showed that the amount of the rat Per1 (rPer1) transcript induced by forskolin (FK) was significantly upregulated by the inhibition of phosphodiesterase type 4 (PDE4), a specific phosphodiesterase for cAMP, in rat-1 fibroblasts. Administration of rolipram, a specific inhibitor of PDE4, increased intracellular cAMP concentration, phosphorylation of cAMP response element binding protein (CREB) and enhanced rPer1 induction at their peaks. However, in the falling phase of rPer1 induction, the inhibition of PDE4 hardly affected the profile of rPer1 expression. These findings suggest the involvement of PDE4 for the regulation of rPer1 expression via cAMP metabolism at peak of the induction but little or no participation of PDE4 in the decreasing phase of the gene expression.

Temperature–amplitude coupling for stable biological rhythms at different temperatures

Most biological processes accelerate with temperature, for example cell division. In contrast, the circadian rhythm period is robust to temperature fluctuation, termed temperature compensation. Temperature compensation is peculiar because a system-level property (i.e., the circadian period) is stable under varying temperature while individual components of the system (i.e., biochemical reactions) are usually temperature-sensitive. To understand the mechanism for period stability, we measured the time series of circadian clock transcripts in cultured C6 glioma cells. The amplitudes of Cry1 and Dbp circadian expression increased significantly with temperature. In contrast, other clock transcripts demonstrated no significant change in amplitude. To understand these experimental results, we analyzed mathematical models with different network topologies. It was found that the geometric mean amplitude of gene expression must increase to maintain a stable period with increasing temperatures and reaction speeds for all models studied. To investigate the generality of this temperature–amplitude coupling mechanism for period stability, we revisited data on the yeast metabolic cycle (YMC) period, which is also stable under temperature variation. We confirmed that the YMC amplitude increased at higher temperatures, suggesting temperature-amplitude coupling as a common mechanism shared by circadian and 4 h-metabolic rhythms.

Profiles of Periglomerular Cells in the Olfactory Bulb of Prokineticin Type 2 Receptor-deficient Mice

Both prokineticin receptor 2 (pkr2) and prokineticin 2 (pk2) gene-deficient mice have hypoplasia of the main olfactory bulb (MOB). This hypoplasia has been attributed to disruption of the glomerulus that is caused by loss of afferent projection from olfactory sensory neurons (OSN), and to the impaired migration of granule cells, a type of interneuron. In the present study, we examined whether migration of the second type of interneuron, periglomerular cells (PGC), is dependent on the pkr2 expression by observing the localization of distinct subpopulations of PGC: calretinin (CR)-, calbindin (CB)- and tyrosine hydroxylase (TH)-expressing neurons. In the Pkr2−/− mice, the construction of the layered structure of the MOB was partially preserved, with the exception of the internal plexiform layer (IPL) and the glomerular layer (GL). In the outermost layer of the MOB, abundant CR- and CB-immunopositive neurons were observed in the hypoplastic olfactory bulb. In addition, although markedly decreased, TH-immunopositive neurons were also observed in the outermost cell-dense region in the Pkr2−/−. The findings suggest that the migration of PGC to the MOB, as well as the migration from the core to the surface region of the MOB, is not driven by the PK2-PKR2 system.