BOMBESIN ACTIVATION OF PHOSPHOLIPASE-C BETA-1 IN RAT ACINAR PANCREATIC-CELLS INVOLVES THE PERTUSSIS-TOXIN-SENSITIVE G(ALPHA-I3) PROTEIN

Bombesin stimulation of inositol 1,4,5-trisphosphate (InsP(3)) formati on in rat sonicated pancreatic acinar cells was inhibited by an antibo dy directed against the pertussis toxin (PTX)-sensitive GTP-binding G( alpha i3) protein but not by an anti-G(alpha q-11) antibody. After sol ubilization and gel filtration, [I-125-Tyr(4)]bombesin binding sites w ere recovered in a peak of protein of 67 similar to 90 kDa with a maxi mal enrichment corresponding to a molecular mass of 83-kDa. Results ob tained from the non-hydrolysable GTP analog guanosine-5'-[gamma-thio]t riphosphate (GTP gamma S) binding, PTX-stimulated ADP-ribosylation and immunoblotting showed that the 83-kDa fraction contained the G(alpha 13) protein but not the G(alpha q-11) protein. Furthermore, GTP gamma S increased the bombesin binding dissociation constant (K-D) from 0.32 to 0.60 nM, while the anti-G(alpha i3) antibody decreased the maximal binding capacity (B-max) from 50 to 25 fmol/mg protein without affect ing the K-D. Mixing solubilized bombesin binding sites with a phosphol ipase C (PLC) preparation from rat pancreas reconstituted a bombesin-s timulated PLC activity which was markedly inhibited by the anti-G(alph a i3) antibody but unaffected by the anti-G(alpha q-11) antibody. In a ddition, this stimulation was inhibited by an anti-PLC beta 1 antibody . This result supports the involvement of the PLC beta 1 isoform in bo mbesin receptor activation.