臺灣博碩士論文加值系統

以培養的白鼠小腦星狀細胞為材料，利用鈣離子螢光指示劑 fura-2，我們發現，在 ATP 或 angiotensin II (AGII) 刺激後，在有胞外鈣存在下，胞內鈣濃度會快速增加，此增加並持續保持，但在胞外無鈣情況下，則此增加會漸漸下降至基準值，相似地，利用 thapsigargin (TG) 抑制鈣邦浦，在含鈣緩衝液下，胞內鈣濃度增加較緩慢但持續保持，在不含鈣緩衝液下，此增加不會持續。這些結果表示在白鼠小腦星狀細胞具有鈣池調控的鈣離子通道 (store-operated Ca2+ channel) (SOC)。在 TG 刺激12分鐘後，再將 TG 去除則胞內鈣濃度持續維持在增加狀態，但再將胞外鈣去除，胞內鈣濃度則快速下降至基準值，這些結果顯示 SOC 之活化不依賴本身與 TG 之直接作用，以 cyclopiazonic acid (CPA) 使鈣池排空，也可得到相同的結果。TG 刺激 SOC 之活化，作用時間有高度依賴性，在 TG 作用2分鐘 ，就可偵測到 SOC 之活化，但沒有完全活化，直到9分鐘才完全活化，這說明了鈣池排空到某程度才會活化 SOC。在不含鈉緩衝液中，SOC 會提早活化而且 TG 誘發的胞內鈣濃度增加也較高。此結果表示 SOC 是一個非選擇性陽離子通道，沒有鈉離子時，鈣離子不會與鈉離子競爭，所以胞外鈣內流就增加。然而，使用鈉離子的螢光指示劑 SBFI，細胞經 TG 作用後，無法測到胞內鈉離子濃度增加，而且鍶與鋇也無法通過 SOC，以全細胞膜電位箝制技術將電位箝制在 -70 mV，測量由 -70 mV 至 -100 mV 梯階過程中電流的改變，TG能誘發一大小在80±20 pA 間之內向電流。當胞內鉀以銫離子取代後，TG 引起的電流改變在 I-V 圖中關係呈線性，其反轉電位約在 -5 mV 到 0 mV 之間。提高胞外鈣離子，TG 活化的電流會變大，而以 Tris 取代胞外鈉離子，則 TG 活化的電流不變。所以鈣池調控的 SOC 對鈣的選擇性較高。在非興奮細胞 (nonexcitable cells)，包括老鼠的腎絲球間質細胞和人類胚胎腎293細胞 (HEK293 cells)，均具有 SOC 的活性，而在興奮細胞 (excitable cells)，包括幼鼠小腦 granule cells 和豬冠狀動脈平滑肌細胞，則不具 SOC。利用錳離子經由 SOC 進入細胞焠熄 Fura-2 螢光的方法，我們可測量磷酸化是否會調控 SOC。加入錳時會引起少許的螢光焠熄，當錳與 TG 一起加時，2分鐘後會誘發更多的螢光焠熄。細胞以絲氨酸和酥氨酸蛋白激酵素抑制劑- staurosporine 處理後，TG 誘發的螢光焠熄會增加，而以酪氨酸蛋白激酵素抑制劑- genistein 處理，則不改變螢光焠熄。所以 TG 誘發的螢光焠熄，蛋白質的絲氨酸和酥氨酸去磷酸化會增加 SOC 的活化，細胞以phorbol 12-myristate,13-acetate 或 okadaic acid 促進磷酸化，會抑制 TG 誘發的螢光焠熄。所以在白鼠小腦星狀細胞，鈣池排空會活化 SOC，且其活性由磷酸化和去磷酸化調控。

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.