牛腎上腺髓質嗜鉻細胞內鈣庫之研究

Abstract

在牛腎上腺嗜鉻細胞內鈣庫可利用不同的Ca2+釋出劑及Ca2+幫浦抑制劑來作分類。在非粒線體鈣庫中可分為對 thapsigargin (Tg)敏感及對Tg不敏感之鈣庫；對Tg敏感之鈣庫又可分為對inositol 1,4,5-trisphosphate (IP3)敏感之鈣庫，對caffeine敏感之鈣庫，及對IP3與caffeine都不敏感之鈣庫。IP3鈣庫釋出之Ca2+可刺激caffeine鈣庫，引發Ca2+-induced Ca2+ release (CICR)反應；由高K＋刺激造成之Ca2+流入則無法引發CICR反應。 利用FCCP破壞粒線體鈣庫累積Ca2+之能力或移去細胞外溶液中之Na+，皆會使高K+刺激造成之細胞內Ca2+濃度([Ca2+]i)反應之半回複期延長，若兩者同時處理此效應更加顯著，顯示粒線體鈣庫及細胞膜上之鈉/鈣交換作用(Na+/Ca2+ exchanger)對[Ca2+]i之緩衝扮演重要角色。而Tg之處理並不能有效延長[Ca2+]i反應之半回復期，顯示 Tg鈣庫之緩衝作用不明顯。 當嗜鉻細胞以bradykinin、caffeine、或Tg刺激，釋空鈣庫後可引發細胞外Ca2+流入以補充空虛之鈣庫。其中 bradykinin造成之[Ca2+]i反應有明顯振盪(oscillation)現象；caffeine造成之反應則較傾向於保持[Ca2+]i在高原狀態；Tg引發之反應較小。嗜鉻細胞膜上此一負責鈣庫釋空後補充Ca2+之特殊通道對Mn2+不通透；但Mn2+可通過對膜電位敏感之Ca2+通道與菸鹼受器。 當嗜鉻細胞以GTPγS處理後，會明顯阻礙caffeine之作用與鈣庫釋空後再補充之能力，但以GTP處理則與控制組無明顯差異，以GDPβS處理則可部分抑制鈣庫釋空後再補充之能力。上述結果暗示了G蛋白質(G-protein)可能參與在鈣庫釋空後引發細胞外Ca2+流入之過程。

In this study, the intracellular Ca2+ pools of bovine adrenal chromaffin cells were characterized in terms of their sensitivity to various Ca2+ releasing agents and inhibitors. Our results show that at least four nonmitochondrial Ca2+ pools exist in bovine adrenal chromaffin cells. The nonmitochondrial Ca2+ pools could be divided into thapsigargin (Tg) -sensitive and Tg-insensitive pools. The Tg-sensitive pools could be futher divided into three pools: the inositol 1,4,5 -trisphosphate (IP3) -sensitive, the caffeine-sensitive, and the IP3, caffeine-insensitive pools. Ca2+ released from IP3- sensitive pools triggered Ca2+ -induced Ca2+ release (CICR) from caffeine-sensitive pools. High K+ -stimulated Ca2+ influx did not trigger CICR. The half-recovery time of [Ca2+]i response triggered by high K+ stimulation was prolonged by either FCCP treament to block the Ca2+ uptake ability of mitochondria or removing Na+ from extracellular solution. Simutaneous treatments by both FCCP and removal of extracellular Na+ futher prolonged the half-recovery time of [Ca2+]i response. These results indicate that mitochondria and plasma membrane Na+/Ca2+ exchanger buffer [Ca2+]i effectively. Since Tg treatment did not change the half-recovery time of [Ca2+]i response, Tg-sensitive calcium pools may not play significant role in modulating [Ca2+]i in chrornaffin cells. Depletion of intracellular Ca2+ pools of chromaffin cells by bradykinin, caffeine or Tg induced an influx of extracellular Ca2+. Bradykinin usually induced an oscillation in [Ca2+]i of chromaffin cells, whereas caffeine usually caused an initial transient [Ca2+]i rise followed by a plateau phase. The Ca2+ influx induced by Tg was less than that induced by bradykinin or caffeine. The channels responsible for Ca2+ entry induced by depletion of Ca2+ pools were impermeable to Mn2+, which passed through voltage-gated calcium channels and nicotinic receptor-associated ion channels. In chromaffin cells perfused with GTPγS, but not GTP, the caffeine-induced [Ca2+]i rise and Ca2+ entry were blocked. GDPβS did not block the caffeine-induced [Ca2+]i response but slightly inhibit capacitative calcium entry . The results imply that G-protein is involved in caffeine-induced [Ca2+]i rise.