Won J G, Orth D N
Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
Endocrinology. 1990 Feb;126(2):849-57. doi: 10.1210/endo-126-2-849.
We examined the effects of removing extracellular Ca2+ (Ca2+e), depleting intracellular Ca2+ (Ca2+i), inhibiting Ca2(+)-dependent calmodulin, blocking voltage-sensitive Ca2+ channels, and combining Ca2+i depletion with exposure to glucocorticoid on the secretion of ACTH by perifused dispersed rat anterior pituitary cells stimulated with ovine CRF and 8-bromo-cAMP (8-Br-cAMP). A time-course analysis of the effect of perifusing the cells for 60 min with Ca2(+)-free medium on 10 nM CRF-stimulated ACTH release revealed that inhibition required about 3 min to begin and about 40 min to reach maximal effect. Within 2 min of restoring Ca2+ to the medium, ACTH secretion rebounded for about 5 min before falling to the pre-Ca2+e removal rate. A similar pattern and time course were observed when Ca2+e was more completely removed by perifusing the cells with Ca2(+)-free medium containing 2 mM EGTA, except that greater suppression was observed. Removing Ca2+e reduced CRF- and 5 mM 8-Br-cAMP-induced ACTH release by 54% and 49%, respectively, and delayed by 1 min the response to 8-Br-cAMP, but not that to CRF. Perifusing 0.2 mM nimodipine, a dihydropyridine Ca2+ channel blocker, before and after restoration of Ca2+ to the Ca2(+)-free medium inhibited ACTH release by 40-48%, and the blockade persisted for at least 70 min after nimodipine was removed from the medium. When intracellular Ca2+ was depleted by perifusing the cells with Ca2(+)-free/EGTA medium containing the Ca2+ ionophore A23187 to facilitate the efflux of Ca2+i, CRF- and 8-Br-cAMP-stimulated ACTH release were reduced by 70% and 71%, respectively, and the responses to both agents were delayed by 1 min. Preperifusion of the cells with 5 microM penfluridol, a calmodulin inhibitor, reduced CRF- and 8-Br-cAMP-induced ACTH release by 54% and 41%, respectively. The combination of Ca2+i depletion and perifusion with 100 nM dexamethasone, a maximally inhibitory concentration, inhibited CRF- and 8-Br-cAMP-stimulated ACTH release by 82% and 83%, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
我们研究了去除细胞外钙离子（Ca2+e）、耗尽细胞内钙离子（Ca2+i）、抑制钙离子依赖性钙调蛋白、阻断电压敏感性钙离子通道以及将细胞内钙离子耗尽与暴露于糖皮质激素相结合，对经绵羊促肾上腺皮质激素释放因子（CRF）和8-溴环磷酸腺苷（8-Br-cAMP）刺激的大鼠垂体前叶分散细胞促肾上腺皮质激素（ACTH）分泌的影响。对用无钙培养基灌注细胞60分钟对10 nM CRF刺激的ACTH释放的影响进行的时间进程分析表明，抑制作用大约在3分钟开始，约40分钟达到最大效果。在培养基中恢复钙离子后2分钟内，ACTH分泌反弹约5分钟，然后降至去除细胞外钙离子前的速率。当用含有2 mM乙二醇双四乙酸（EGTA）的无钙培养基更彻底地去除细胞外钙离子时，观察到类似的模式和时间进程，只是抑制作用更强。去除细胞外钙离子分别使CRF和5 mM 8-Br-cAMP诱导的ACTH释放减少54%和49%，并使对8-Br-cAMP的反应延迟1分钟，但对CRF的反应没有延迟。在无钙培养基恢复钙离子前后，用0.2 mM尼莫地平（一种二氢吡啶钙离子通道阻滞剂）灌注细胞，可使ACTH释放减少40%-48%，并且在从培养基中去除尼莫地平后，阻断作用持续至少70分钟。当用含有钙离子载体A23187的无钙/EGTA培养基灌注细胞以促进细胞内钙离子外流来耗尽细胞内钙离子时，CRF和8-Br-cAMP刺激的ACTH释放分别减少70%和71%，并且对两种试剂的反应均延迟1分钟。用5 microM五氟利多（一种钙调蛋白抑制剂）预灌注细胞，分别使CRF和8-Br-cAMP诱导的ACTH释放减少54%和41%。细胞内钙离子耗尽与用100 nM地塞米松（最大抑制浓度）灌注相结合，分别使CRF和8-Br-cAMP刺激的ACTH释放减少82%和83%。（摘要截断于400字）
