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 GTPgammaS 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 GDPbetaS. 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 beta-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 beta-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein alpha-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)-3alpha 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.
Institute of Clinical Medicine, University of Tromso, Norway. ruthp@fagmed.uit.no
R H
RH
Paulssen
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N
Fraeyman
J
J
Florholmen
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002766:Cholecystokinin / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006728:Hormones / Q000378:metabolism; D006801:Humans; D051379:Mice; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
D006728:Hormones; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; D002766:Cholecystokinin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins