Pfeiffer F, Sternfeld L, Schmid A, Schulz I
Institute of Physiology II, University of the Saarland, Homburg, Germany.
Am J Physiol. 1998 Mar;274(3 Pt 1):C663-72. doi: 10.1152/ajpcell.1998.274.3.c663.
We have investigated control mechanisms involved in the propagation of agonist-induced Ca2+ waves in isolated mouse pancreatic acinar cells. Using a confocal laser-scanning microscope, we were able to show that maximal stimulation of cells with acetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initial Ca2+ release of comparable amounts with both agonists at the luminal cell pole. Subsequent Ca2+ spreading to the basolateral membrane was faster with ACh (17.3 +/- 5.4 microns/s) than with bombesin (8.0 +/- 2.2 microns/s). The speed of bombesin-induced Ca2+ waves could be increased up to the speed of ACh-induced Ca2+ waves by inhibition of protein kinase C (PKC). Activation of PKC significantly decreased the speed of ACh-induced Ca2+ waves but had only little effect on bombesin-evoked Ca2+ waves. Within 3 s after stimulation, production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was higher in the presence of ACh compared with bombesin, whereas bombesin induced higher levels of diacylglycerol (DAG) than ACh. These data suggest that the slower propagation speed of bombesin-induced Ca2+ waves is due to higher activation of PKC in the presence of bombesin compared with ACh. The higher increase in bombesin-compared with ACh-induced DAG production is probably due to activation of phospholipase D (PLD). Inhibition of the PLD-dependent DAG production by preincubation with 0.3% butanol led to an acceleration of the bombesin-induced Ca2+ wave. In further experiments, we could show that ruthenium red (100 microM), an inhibitor of Ca(2+)-induced Ca2+ release in skeletal muscle, also decreased the speed of ACh-induced Ca2+ waves. The effect of ruthenium red was not additive to the effect of PKC activation. From the data, we conclude that, following Ins(1,4,5)P3-induced Ca2+ release in the luminal cell pole, secondary Ca2+ release from stores, which are located in series between the luminal and the basal plasma membrane, modifies Ca2+ spreading toward the basolateral cell side by Ca(2+)-induced Ca2+ release. Activation of PKC leads to a reduction in Ca2+ release from these stores and therefore could explain the slower propagation of Ca2+ waves in the presence of bombesin compared with ACh.
我们研究了分离的小鼠胰腺腺泡细胞中激动剂诱导的Ca2+波传播所涉及的调控机制。使用共聚焦激光扫描显微镜，我们能够证明，用乙酰胆碱（ACh，500 nM）或蛙皮素（1 nM）对细胞进行最大刺激时，在细胞腔面极处，两种激动剂引起的初始Ca2+释放量相当。随后，ACh（17.3±5.4微米/秒）诱导的Ca2+向基底外侧膜的扩散速度比蛙皮素（8.0±2.2微米/秒）诱导的更快。通过抑制蛋白激酶C（PKC），蛙皮素诱导的Ca2+波速度可提高至ACh诱导的Ca2+波速度。PKC的激活显著降低了ACh诱导的Ca2+波速度，但对蛙皮素诱发的Ca2+波影响很小。刺激后3秒内，与蛙皮素相比，ACh存在时肌醇1,4,5-三磷酸[Ins(1,4,5)P3]的生成量更高，而蛙皮素诱导的二酰基甘油（DAG）水平高于ACh。这些数据表明，蛙皮素诱导的Ca2+波传播速度较慢是由于与ACh相比，蛙皮素存在时PKC的激活程度更高。与ACh诱导的DAG生成相比，蛙皮素诱导的DAG生成增加幅度更大可能是由于磷脂酶D（PLD）的激活。用0.3%丁醇预孵育抑制PLD依赖性DAG生成会导致蛙皮素诱导的Ca2+波加速。在进一步的实验中，我们能够证明，钌红（100 microM），一种骨骼肌中Ca(2+)诱导的Ca2+释放抑制剂，也降低了ACh诱导的Ca2+波速度。钌红的作用与PKC激活的作用无相加性。从这些数据中，我们得出结论，在细胞腔面极处Ins(1,4,5)P3诱导的Ca2+释放后，位于腔膜和基底膜之间串联的储存库中的二次Ca2+释放通过Ca(2+)诱导的Ca2+释放改变了Ca2+向基底外侧细胞侧的扩散。PKC的激活导致这些储存库中Ca2+释放减少，因此可以解释与ACh相比，蛙皮素存在时Ca2+波传播较慢的原因。
