Takano Takahiro, Franchini Luca, Wagner Larry E, Cooley Michelle, Groblewski Guy E, Orlandi Cesare, Yule David I
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York, USA.
Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin, USA.
J Physiol. 2025 Jun 25. doi: 10.1113/JP288957.
Elevated cytoplasmic [Ca⁺] and protein kinase C (PKC) activation are key signalling events driving secretion in pancreatic acinar cells after stimulation by the secretagogues cholecystokinin (CCK) and acetylcholine (ACh). Although both ACh and CCK binding to their cognate receptors activates G proteins, leading to inositol 1,4,5-trisphosphate (IP₃)-mediated Ca⁺ release and diacylglycerol (DAG)-dependent PKC activation, it has been proposed that physiological CCK stimulation bypasses this canonical pathway, instead mobilizing Ca⁺ via production of nicotinic acid adenine dinucleotide phosphate (NAADP). We reassessed the role of G signalling in CCK-induced responses using a bioluminescence resonance energy transfer (BRET) assay, demonstrating that both CCK1 (CCK1R) and muscarinic M3 receptors (M3R) engage G along with other Gα subunits. Importantly YM-254890, a G antagonist, inhibited coupling through G but did not alter the interactions of CCK1R or M3R with other G protein families. YM-254890 eliminated CCK1R- and M3R-induced Ca⁺ signals in isolated acinar cells. Consistent with the in vitro data, systemic CCK injection, intrinsic neural stimulation or feeding failed to elicit Ca⁺ responses in vivo in mice pre-treated with YM-254890, indicating that physiological stimulation of Ca signalling events requires G activation. Additionally YM-254890 suppressed Ca⁺-activated Cl⁻ currents, a key event underlying fluid secretion, and amylase secretion in acini after CCK or ACh stimulation. These findings establish that CCK- and ACh-induced exocrine pancreatic secretion strictly requires G activation, leading to IP₃ generation, DAG production and downstream signalling that is essential for physiological function. KEY POINTS: An increase in cytoplasmic Ca and PKC activity after CCK and ACh stimulation following feeding is a pivotal event in the activation of fluid secretion and exocytosis from pancreatic acinar cells. In contrast to ACh, it has been suggested that at physiological concentrations, CCK stimulation results in the production of nicotinic acid dinucleotide adenine phosphate, without activating the canonical G pathway, and the production of inositol 1,4,5,-trisphosphate (IP) and diacylglycerol (DAG). After having established that YM-254890 is an exquisitely selective G inhibitor, we show that Ca signals stimulated in vitro and in vivo in response to both M3R and CCK1R stimulation are completely inhibited by YM-254890. YM-254890 completely abrogates Ca-activated Cl current activation, pivotal for fluid secretion together with amylase secretion stimulated by both M3R and CCK1R activation. We conclude that ACh and CCK stimulation results in Gq/11 activation, an increase in IP and DAG, and this event is fundamentally important for exocrine function.
细胞质中钙离子浓度升高和蛋白激酶C（PKC）激活是促分泌剂胆囊收缩素（CCK）和乙酰胆碱（ACh）刺激后驱动胰腺腺泡细胞分泌的关键信号事件。尽管ACh和CCK与它们各自的受体结合都会激活G蛋白，导致肌醇1,4,5 - 三磷酸（IP₃）介导的钙离子释放和二酰基甘油（DAG）依赖性PKC激活，但有人提出生理性CCK刺激绕过了这条经典途径，而是通过产生烟酰胺腺嘌呤二核苷酸磷酸（NAADP）来动员钙离子。我们使用生物发光共振能量转移（BRET）测定法重新评估了G信号在CCK诱导反应中的作用，证明CCK1（CCK1R）和毒蕈碱M3受体（M3R）与G以及其他Gα亚基相互作用。重要的是，G拮抗剂YM - 254890抑制了通过G的偶联，但没有改变CCK1R或M3R与其他G蛋白家族的相互作用。YM - 254890消除了分离的腺泡细胞中CCK1R和M3R诱导的钙离子信号。与体外数据一致，在预先用YM - 254890处理的小鼠中，全身注射CCK、内在神经刺激或进食在体内均未能引发钙离子反应，表明钙离子信号事件的生理性刺激需要G激活。此外，YM - 254890抑制了钙离子激活的氯离子电流，这是液体分泌的关键事件，以及CCK或ACh刺激后腺泡中的淀粉酶分泌。这些发现表明，CCK和ACh诱导的胰腺外分泌严格需要G激活，导致IP₃生成、DAG产生以及对生理功能至关重要的下游信号传导。要点：进食后CCK和ACh刺激后细胞质钙离子和PKC活性增加是胰腺腺泡细胞液体分泌和胞吐激活的关键事件。与ACh不同，有人提出在生理浓度下，CCK刺激会导致烟酰胺腺嘌呤二核苷酸磷酸的产生，而不激活经典的G途径，以及肌醇1,4,5 - 三磷酸（IP）和二酰基甘油（DAG）的产生。在确定YM - 254890是一种高度选择性的G抑制剂后，我们表明YM - 254890完全抑制了体外和体内对M3R和CCK1R刺激的钙离子信号。YM - 254890完全消除了钙离子激活的氯离子电流激活，这对于M3R和CCK1R激活刺激的液体分泌以及淀粉酶分泌至关重要。我们得出结论，ACh和CCK刺激导致Gq/11激活，IP和DAG增加，这一事件对外分泌功能至关重要。
