POTENTIATION BY CHOLERA-TOXIN OF BRADYKININ-INDUCED INOSITOL PHOSPHATE PRODUCTION IN THE OSTEOBLAST-LIKE CELL-LINE MC3T3-E1

Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) iso enzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analys is with various anti-PLC antibodies. Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-bi nding protein. Inositol phosphate formation on stimulation by BK was n ot affected by pretreatment with pertussis toxin, whereas it was poten tiated by cholera toxin pretreatment. Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin f ailed to enhance the BK-mediated generation of inositol phosphates, bu t long-term preincubation with these agents partially mimicked the act ion of the cholera toxin. Cholera toxin also caused an increase in BK receptor number. Cycloheximide, a protein biosynthesis inhibitor, prev ented the potentiating actions of the cholera toxin and the cyclic AMP -elevating agents on BK-induced inositol phosphate production, and als o inhibited the increase in BK receptor number. The specific binding o f [H-3]BK to the whole MC3T3-E1 cells in the presence or absence of ch olera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg[Hyp3,Thi5.8,D-Phe7]BK, but not by the BI BK receptor agonist des -Arg9-BK. These data suggest that the activation of PI-PLC induced by cholera toxin in BK-stimulated MC3T3-E1 cells was caused by an enhance ment of the synthesis of BK receptor protein(s), at least part of whic h was mediated by a sustained increase in the intracellular level of c yclic AMP.