Straub Stephen V, Giovannucci David R, Bruce Jason I E, Yule David I
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642, USA.
J Biol Chem. 2002 Aug 30;277(35):31949-56. doi: 10.1074/jbc.M204318200. Epub 2002 Jun 13.
Stimulation of pancreatic acinar cells with acetylcholine (ACh) and cholecystokinin (CCK) results in an elevation of cytosolic calcium (Ca(2+)) through activation of inositol 1,4,5-trisphosphate receptors (InsP(3)R). The global temporal pattern of the Ca(2+) changes produced by ACh or CCK stimulation differs significantly. The hypothesis was tested that CCK stimulation results in a protein kinase A (PKA)-mediated phosphorylation of InsP(3)R and this event contributes to the generation of agonist-specific Ca(2+) signals. Physiological concentrations of CCK evoked phosphorylation of the type III InsP(3)R, which was blocked by pharmacological inhibition of PKA. Imaging of fura-2-loaded acinar cells revealed that the rate of Ca(2+) rise during CCK-evoked oscillations slows with each subsequent oscillation, consistent with a developing modulation of release, whereas the kinetics of ACh-evoked oscillations remain constant. Stimulation of cells with ACh following activation of PKA resulted in a slowing of the ACh-evoked Ca(2+) rise, which now resembled a time-matched CCK response. PKA activation also resulted in a slowing of Ca(2+) increases elicited by photolysis of caged InsP(3). Targeted, PKA-mediated phosphorylation of type III InsP(3)R is involved in a physiological CCK response, as disruption of the targeting of PKA with the peptide HT31 resulted in marked changes in the CCK-evoked Ca(2+) signal but had no effect on ACh-evoked responses. Stimulation of cells with bombesin, which evokes Ca(2+) oscillations indistinguishable from those produced by CCK, also results in PKA-mediated phosphorylation of type III InsP(3)R. Thus, we conclude that PKA-mediated phosphorylation of type III InsP(3)R is a general mechanism by which the patterns of Ca(2+) oscillations are shaped in pancreatic acinar cells.
用乙酰胆碱（ACh）和胆囊收缩素（CCK）刺激胰腺腺泡细胞，会通过激活肌醇1,4,5 - 三磷酸受体（InsP(3)R）导致胞质钙（Ca(2+)）升高。ACh或CCK刺激所产生的Ca(2+)变化的整体时间模式有显著差异。本研究对“CCK刺激导致蛋白激酶A（PKA）介导的InsP(3)R磷酸化，且该事件有助于产生激动剂特异性的Ca(2+)信号”这一假说进行了验证。生理浓度的CCK可诱发III型InsP(3)R的磷酸化，而PKA的药理学抑制可阻断这一过程。对负载fura - 2的腺泡细胞进行成像显示，在CCK诱发的振荡过程中，Ca(2+)升高的速率随着后续每次振荡而减慢，这与释放的逐渐调节一致，而ACh诱发振荡的动力学保持不变。在PKA激活后用ACh刺激细胞，会导致ACh诱发的Ca(2+)升高减慢，此时类似于与CCK时间匹配的反应。PKA激活还会导致由笼锁InsP(3)光解引发的Ca(2+)增加减慢。III型InsP(3)R的靶向性PKA介导的磷酸化参与了生理性CCK反应，因为用肽HT31破坏PKA的靶向性会导致CCK诱发的Ca(2+)信号发生显著变化，但对ACh诱发的反应没有影响。用蛙皮素刺激细胞，其诱发的Ca(2+)振荡与CCK产生的振荡无法区分，这也会导致III型InsP(3)R的PKA介导的磷酸化。因此，我们得出结论，III型InsP(3)R的PKA介导的磷酸化是一种普遍机制，通过该机制可塑造胰腺腺泡细胞中Ca(2+)振荡的模式。
