| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 86 === Using the fluorescent Ca2+ indicator fura-2, we found that, in cultured rat cerebellar astrocytes, a sustained increase of cytosolic Ca2+ concentration ([Ca2+]i) was induced by ATP or angiotensin II (AGII) following a transient increase of [Ca2+]i in the presence of extracellular Ca2+, while it was not seen in the absence of extracellular Ca2+, [Ca2+]i being declined to the basal level. Similarly, a slow and sustained [Ca2+]i increase, or a slow but not sustained [Ca2+]i increase was observed after intracellular Ca2+ pump was inhibited by thapsigargin (TG) in the presence or absence of extracellular Ca2+, respectively. These results suggest the existence of store-operated Ca2+ channel (SOC) in rat cerebellar astrocytes. This is further evidenced by the fact that 12 min after TG-induced stores depletion had occurred, [Ca2+]i remained in the sustained phase upon removal of TG, while it rapidly declined to the basal level after extracellular Ca2+ was removed. These results also indicate that activation of SOC was not dependent on its direct interaction with TG. Similar results were observed after Ca2+ stores were depleted by cyclopiazonic acid (CPA). This TG-induced, extracellular Ca2+-sensitive [Ca2+]i increase was highly dependent on the time period of TG exposure. Thus, Ca2+ influx could be detected as early as 2 min after TG exposure, while it was not fully activated until 12 min further suggesting that SOC was not activated until significant degree of Ca2+ stores were depleted. SOC could be detected earlier and TG-induced [Ca2+]i increase was greater in cells bathed in Na+ free buffer than those bathed in normal Na+ containing buffer indicating that SOC is a nonselective cation channel and Ca2+ influx was enhanced in the absence of Na+ since the competetion of Ca2+ with Na+ was no longer existing. However, using the fluorescent Na+ indicator, SBFI, [Na+]i increase was not detected after cells had been exposed to TG. Furthermore, the TG-activated cation channel was not permeable to Sr2+ or Ba2+, either. Using whole cell voltage clamp technique and step-pulse recordings from a holding potential of -70 mV to a test potential of -100 mV, a 60 - 100 pA inward current was activated by TG-induced Ca2+ stores depletion. The current-voltage curve of TG-activated current showed a linear relationship with the reversal poitential ranged from -5 to 0 mV in the presence of high internal Cs+ and external Na+. The amplitude of TG-activated current remained the same in Na+-free external solution, while it was larger at higher concentrations of external Ca2+. Taken together, our results suggest that SOC in rat cerebellar astrocytes is preferentially permeable to Ca2+. Similar SOC activity was seen in human embryonic kidney 293 cells (HEK293 cells) which is nonexcitable cells, while excitable cells, including rat cerebellar granule cells and procine aortic smooth muscle cells, did not display SOC. We next examined whether the activity of SOC was regulated by phosporylation by measuring the fura-2 fluorescence quench induced by Mn2+ influx via SOC. Addition of Mn2+ to the bathing buffer caused a slight but significant fluorescence quench and further fluorescence quench was induced 2 min after coadministration of Mn2+ and TG simultaneously. Pretreatment of cells with staurosporine, a Ser/Thr protein kinase inhibitor, enhanced the TG-induced fluorescence quench, while genistein, a tyrosine kinase inhibitor, had no effect on it indicating that dephosphorylation of Ser/Thr residues of proteins enhanced the SOC activity. This is further supported by the fact that promotion phosphorylation by phorbol 12-myristate 13-acetate or okadaic acid abolished TG-induced fluorescence quench. In conclusion, in rat cerebellar astrocytes, SOC is activated by depletion of Ca2+ store and its activity is regulated by phosporylation and dephosphorylation.
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