Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines

Abstract

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (β-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTPγS alone, the C-terminal octapeptide cholecystokinin (CCK-8), or gastrin. All compounds caused a significant 2- to 4.4-fold stimulation of PLC activity in the different cell lines, which was diminished by the non-hydrolyzable GDP analogue GDPβS. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCK A receptor antagonist L-364,718 ( K d = 0.24 nM) and tritiated CCK B receptor antagonist L-365,260 ( K d = 0.13 nM) were only present in Rin 1027-B2 cells. High-affinity binding sites for both ligands were not found in β-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCK A receptor-selective) and CR 2945 (CCK B receptor-selective), as well as microphysiometry experiments, resulted in the same receptor distribution. Reverse transcriptase–polymerase chain reaction confirmed the CCK receptor distribution pattern for Rin 1027-B2 cells, but in addition showed the existence of CCK B receptors in β-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein α-subunits demonstrated inhibition of CCK-stimulated PLC activity in β-TC3 cells by G q/11α antiserum (70%), in Rin 1027-B2 cells by G q/11α antiserum (70%) and G i-3α antiserum (23%), and in INR1-G9 cells by G q/11α antiserum (60%) and G oα antiserum (45%). We conclude that CCK receptor subtypes with different G-protein-coupling specificities to PLC are present in the different hormone-secreting cells of the endocrine pancreas.