Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1

Abstract

Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) isoenzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analysis with various anti-PLC antibodies. Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-binding protein. Inositol phosphate formation on stimulation by BK was not affected by pretreatment with pertussis toxin, whereas it was potentiated by cholera toxin pretreatment. Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin failed to enhance the BK-mediated generation of inositol phosphates, but long-term preincubation with these agents partially mimicked the action of the cholera toxin. Cholera toxin also caused an increase in BK receptor number. Cycloheximide, a protein biosynthesis inhibitor, prevented the potentiating actions of the cholera toxin and the cyclic AMP-elevating agents on BK-induced inositol phosphate production, and also inhibited the increase in BK receptor number. The specific binding of [3H]BK to the whole MC3T3-E1 cells in the presence or absence of cholera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg[Hyp3,Thi5,8,D-Phe7]BK, but not by the B1 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 enhancement of the synthesis of BK receptor protein(s), at least part of which was mediated by a sustained increase in the intracellular level of cyclic AMP.

The effect of cyclic AMP elevating agents on bradykinin‐ and carbachol‐induced signal transduction in canine cultured tracheal smooth muscle cells

“…causes inhibition of PI turnover, which is partly due to a reduction in number of hormonal receptors (Gardner et al, 1989;Guillon et al, 1989;Socorro et al, 1990). It has also been shown that CTX potentiates the BK-stimulated PI turnover in human foreskin fibroblasts, the osteoblast-like cell line MC3T3-E1 and BALB/c/3T3 cells (Olashaw & Pledger, 1988;Etscheid & Villereal, 1989;Banno et al, 1993). This potentiating action was inhibited by cycloheximide.…”

“…This potentiating action was inhibited by cycloheximide. This response may therefore involve an increase in protein synthesis following elevation of the concentration of cyclic AMP, probably through BK receptors (Etscheid et al, 1991;Banno et al, 1993). Moreover, elevation of cyclic AMP concentration caused by CTX promotes transcription of mRNA encoding a protein that stimulates the release of arachidonic acid metabolites (Peterson et al, 1991).…”

Pharmacological characterization of bradykinin receptors in canine cultured tracheal smooth muscle cells

“…Cyclic AMP elevating agents have been shown to exert their effect by receptor up-regulation (Etscheid et al, 1991;Banno et al, 1993). We previously reported that pretreatment of TSMCs with cholera toxin, forskolin, and dibutyryl cyclic AMP enhanced bradykinin-induced inositol phosphates accumulation and a rise in [Ca2+]i through an increase in density of the bradykinin receptor (Yang et al, 1994c Moreover, preincubation with forskolin led to an increase in B. from 25.1 ± 0.3 to 218 ± 24 fmol mg'-protein without changing the KD value.…”

“…It has been shown that long-term cholera toxin treatment potentiates the bradykinin-stimulated phosphoinositide turnover in canine TSMCs, human foreskin fibroblasts, osteoblast-like cell line MC3T3-E1 and BALB/c/3T3 cells (Banno et al, 1993;Etscheid et al, 1991;Olashaw & Pledger, 1988;Yang et al, 1994c (Etscheid et al, 1991;Banno et al, 1993;Yang et al, 1994c). Although only B2 receptor density has been shown to increase in osteoblast-like MC3T3-E1 cells treated with cholera toxin (Banno et al, 1993) …”

“…Although only B2 receptor density has been shown to increase in osteoblast-like MC3T3-E1 cells treated with cholera toxin (Banno et al, 1993) …”