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

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (beta-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTP gamma S 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 GDP beta S. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCKB receptor antagonist L-364,718 (K-d = 0.24 nM) and tritiated CCKB 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 beta-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCKA receptor-selective) and CR 2945 (CCKB 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 CCKB receptors in beta-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different e-protein a subunits demonstrated inhibition of CCK-stimulated PLC activity in beta-TC3 cells by G(q/11)alpha antiserum (70%), in Rin 1027-B2 cells by G(q/11)alpha antiserum (70%) and G(i)-3 alpha antiserum (23%), and in INR1-G9 cells by G(q/11)alpha antiserum (60%) and G(o)alpha 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. (C) 2000 Elsevier Science Inc.