Pancreatic acinar cell signalling and function exhibit an absolute requirement for activation of Gα.

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.