Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines
Rh. Paulssen et al., Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines, BIOCH PHARM, 60(6), 2000, pp. 865-875
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.