Willems P H, Smeets R L, Bosch R R, Garner K M, Van Mackelenbergh M G, De Pont J J
Department of Biochemistry, University of Nijmegen, The Netherlands.
Cell Calcium. 1995 Dec;18(6):471-83. doi: 10.1016/0143-4160(95)90010-1.
Digital-imaging microscopy of Fura-2-loaded pancreatic acinar cells revealed that the C-terminal octapeptide of cholecystokinin (CCK8) dose-dependently recruited 94% of freshly isolated acinar cells in terms of receptor-evoked Ca2+ mobilization. Maximal and half-maximal cell-recruitment were reached with 0.1 nM and 16.8 pM CCK8, respectively. The upstroke of the dose-recruitment curve consisted of cells displaying oscillatory changes in free cytosolic Ca2+ concentration ([Ca2+]i). After having reached its maximum, the percentage oscillating cells dose-dependently decreased upon further increasing of the CCK8 concentration. Pretreatment of the acinar cells with 0.1 microM TPA caused a rightward shift of the dose-recruitment curve but did not change the maximal effect of CCK8 on the recruitment of oscillating cells. Half-maximal recruitment was obtained with 287 pM CCK8. This observation demonstrates that high levels of protein kinase C activation do not inhibit Ca2+ oscillations at a level downstream to receptor activation. Moreover, this observation demonstrates that protein kinase C-mediated inhibition of Ca2+ oscillations evoked by submaximal CCK8 concentrations occurs at the receptor level, converting it from a high-affinity state into a low-affinity state. This conclusion is supported by the observation that TPA completely inhibited the recruitment of acinar cells in response to the high-affinity receptor agonist JMV-180. The inhibitory action of TPA on CCK8-evoked cell-recruitment was paralleled by an inhibitory effect of the phorbol ester on the CCK8-evoked peak increase in average inositol trisphosphate concentration in a population of acinar cells. This observation indicates that low concentrations of CCK8 interact with the high-affinity CCK receptor to increase [Ca2+]i through the intermediation of inositol trisphosphate.
对负载Fura-2的胰腺腺泡细胞进行数字成像显微镜观察发现，就受体诱发的Ca2+动员而言，胆囊收缩素（CCK8）的C末端八肽能剂量依赖性地募集94%的新鲜分离腺泡细胞。分别用0.1 nM和16.8 pM的CCK8可达到最大和半数最大细胞募集。剂量募集曲线的上升部分由游离胞质Ca2+浓度（[Ca2+]i）呈现振荡变化的细胞组成。达到最大值后，随着CCK8浓度进一步增加，振荡细胞的百分比呈剂量依赖性下降。用0.1 microM佛波酯（TPA）预处理腺泡细胞导致剂量募集曲线右移，但未改变CCK8对振荡细胞募集的最大效应。用287 pM CCK8可获得半数最大募集。该观察结果表明，高水平的蛋白激酶C激活不会在受体激活下游水平抑制Ca2+振荡。此外，该观察结果表明，蛋白激酶C介导的对亚最大CCK8浓度诱发的Ca2+振荡的抑制发生在受体水平，将其从高亲和力状态转变为低亲和力状态。这一结论得到以下观察结果的支持：TPA完全抑制了腺泡细胞对高亲和力受体激动剂JMV-180的募集。TPA对CCK8诱发的细胞募集的抑制作用与佛波酯对一群腺泡细胞中CCK8诱发的平均肌醇三磷酸浓度峰值增加的抑制作用平行。该观察结果表明，低浓度的CCK8通过肌醇三磷酸的介导与高亲和力CCK受体相互作用以增加[Ca2+]i。
