Rossi Andrea H, Sears Patrick R, Davis C William
6009 Thurston-Bowles, University of North Carolina, Chapel Hill, NC 27599-7248, USA.
J Physiol. 2004 Sep 1;559(Pt 2):555-65. doi: 10.1113/jphysiol.2004.070433. Epub 2004 Jun 24.
SPOC1 airway goblet cells secrete mucin in response to P2Y2 receptor agonists and to secretagogues, phorbol 12-myristate 13-acetate (PMA) and ionomycin, which mobilize elements of the phospholipase C pathway, PKC and Ca2+, respectively. Previous studies demonstrated that mucin secretion from SLO-permeabilized, EGTA-buffered SPOC1 cells was stimulated by PMA at low Ca2+ levels (< 0.1 microm), consistent with the notion that regulated exocytosis may occur by Ca2+-independent pathways. We tested the alternative hypothesis that PMA-induced mucin secretion is, in fact, a Ca2+-dependent process under the conditions of low bulk Ca2+, one that is permitted in the typical SLO-permeabilized cell model by the slow binding kinetics of EGTA. Both IP3 and elevated bulk Ca2+ activated mucin secretion in SPOC1 cells buffered by EGTA, suggesting that IP3 generates a local Ca2+ gradient in the vicinity of the secretory granules to the degree necessary to trigger exocytosis. BAPTA, which binds Ca2+ approximately 100-fold faster than EGTA, diminished IP3-induced mucin release over a range of concentrations by > or = 69%, yet maintained an essentially normal mucin secretory response to elevated bulk Ca2+ in permeabilized SPOC1 cells. BAPTA also diminished the mucin secretory response of permeabilized cells to PMA, relative to the EGTA-buffered control: at PMA below 30 nm, BAPTA abolished the secretory response, and at higher concentrations it was reduced significantly relative to the EGTA-buffered controls. PMA-induced secretion in EGTA was insensitive to heparin. These results suggest that Ca2+ is released locally during PMA-induced exocytosis, by an IP3-independent mechanism.
SPOC1气道杯状细胞会响应P2Y2受体激动剂以及促分泌剂（佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）和离子霉素）分泌粘蛋白，这两种促分泌剂分别激活磷脂酶C途径的相关成分、蛋白激酶C（PKC）和Ca2+。先前的研究表明，在低Ca2+水平（<0.1微摩尔）时，PMA可刺激经皂草素通透处理、EGTA缓冲的SPOC1细胞分泌粘蛋白，这与调节性胞吐作用可能通过Ca2+非依赖性途径发生的观点一致。我们检验了另一种假说，即在低总体Ca2+条件下，PMA诱导的粘蛋白分泌实际上是一个Ca2+依赖性过程，在典型的皂草素通透细胞模型中，由于EGTA的缓慢结合动力学，这种情况是可能发生的。IP3和升高的总体Ca2+均可激活经EGTA缓冲的SPOC1细胞中的粘蛋白分泌，这表明IP3在分泌颗粒附近产生局部Ca2+梯度，其程度足以触发胞吐作用。BAPTA结合Ca2+的速度比EGTA快约100倍，在一系列浓度下可使IP3诱导的粘蛋白释放减少≥69%，但在通透的SPOC1细胞中，对升高的总体Ca2+仍保持基本正常的粘蛋白分泌反应。相对于EGTA缓冲的对照，BAPTA也减弱了通透细胞对PMA的粘蛋白分泌反应：在PMA低于30纳米时，BAPTA消除了分泌反应，在较高浓度下，相对于EGTA缓冲的对照，分泌反应显著降低。在EGTA中，PMA诱导的分泌对肝素不敏感。这些结果表明，在PMA诱导的胞吐作用过程中，Ca2+通过一种不依赖IP3的机制在局部释放。
