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-hydr olyzable GTP analogue GTP gamma S alone, the C-terminal octapeptide cholecy stokinin (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 dim inished by the non hydrolyzable GDP analogue GDP beta S. CCK receptor subty pes were characterized by radioligand binding experiments. High-affinity bi nding 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 onl y present in Rin 1027-B2 cells. High-affinity binding sites for both ligand s were not found in beta-TC3 or INR1-G9 cells. Competition binding experime nts with non-labeled CCK receptor antagonists CR 1505 (CCKA receptor-select ive) and CR 2945 (CCKB receptor-selective), as well as microphysiometry exp eriments, 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 recepto rs in beta-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different e-protein a subunits demonstrated inhibition of CCK-stim ulated PLC activity in beta-TC3 cells by G(q/11)alpha antiserum (70%), in R in 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-pr otein-coupling specificities to PLC are present in the different hormone-se creting cells of the endocrine pancreas. (C) 2000 Elsevier Science Inc.