THE EFFECT OF CYCLIC-AMP ELEVATING AGENTS ON BRADYKININ-INDUCED AND CARBACHOL-INDUCED SIGNAL-TRANSDUCTION IN CANINE CULTURED TRACHEAL SMOOTH-MUSCLE CELLS

1 The effects of cholera toxin (CTX), forskolin and dibutyryl cyclic A MP on bradykinin (BK)- and carbachol-induced accumulation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cul tured tracheal smooth muscle cells (TSMCs). The BK-induced responses w ere mediated via a G protein which was not inhibited by CTX or pertuss is toxin treatment. 2 BK-stimulated IPs accumulation and Ca2+ mobiliza tion were potentiated by CTX (10 mu g ml(-1)) pretreatment which was t ime-dependent. Maximal increase of these responses occurred after 24 h treatment with CTX. The concentration-effect relationship of BK-induc ed responses were shifted to the left and BK was substantially more ef fective in CTX-treated cells than in the control cells. This enhancing effect of CTX did not occur with carbachol. 3 Short-term (< 4h) treat ment with forskolin (10 mu M) or dibutyryl cyclic AMP (1 mM) failed to accentuate BK-induced responses, but long-term (> 4h) treatment of TS MCs with these agents mimicked the enhancing effect of CTX, suggesting that CTX-induced enhancement of BK responsiveness might be due to a r ise in cyclic AMP. 4 Prolonged treatment of TSMCs with these agents wa s accompanied by an increase in cell surface [H-3]-BK binding sites, w hich was inhibited by concurrent incubation with cycloheximide, an inh ibitor of protein biosynthesis. Cycloheximide also abolished the poten tiating actions of CTX, forskolin, and dibutyryl cyclic AMP on BK-indu ced IPs formation and Ca2+ mobilization. 5 The locus of this enhanceme nt was further investigated by examining the effects of CTX, forskolin and dibutyryl cyclic AMP on AlF4--induced IPs accumulation in canine TSMCs. AlF4--induced IPs accumulation was not affected by CTX, forskol in, or dibutyryl cyclic AMP treatment, supporting the contention that this stimulatory effect is located at the BK receptor level. 6 These r esults demonstrate that the augmentation of BK-induced IPs accumulatio n and Ca2+ mobilization produced by CTX, forskolin and dibutyryl cycli c AMP involves a cyclic AMP-dependent mechanism which is induced by a sustained increase in the level of intracellular cyclic AMP. CTX and f orskolin may promote an increase of the synthesis of BK receptors, and thereby enhance BK-induced responses.