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 AMP on bradykinin (BK)- and carbachol-induced accumulation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The BK-induced responses were mediated via a G protein which was not inhibited by CTX or pertussis toxin treatment. 2 BK-stimulated IPs accumulation and Ca2+ mobilization were potentiated by CTX (10 mu g ml(-1)) pretreatment which was time-dependent. Maximal increase of these responses occurred after 24 h treatment with CTX. The concentration-effect relationship of BK-induced responses were shifted to the left and BK was substantially more effective in CTX-treated cells than in the control cells. This enhancing effect of CTX did not occur with carbachol. 3 Short-term (< 4h) treatment with forskolin (10 mu M) or dibutyryl cyclic AMP (1 mM) failed to accentuate BK-induced responses, but long-term (> 4h) treatment of TSMCs with these agents mimicked the enhancing effect of CTX, suggesting that CTX-induced enhancement of BK responsiveness might be due to a rise in cyclic AMP. 4 Prolonged treatment of TSMCs with these agents was accompanied by an increase in cell surface [H-3]-BK binding sites, which was inhibited by concurrent incubation with cycloheximide, an inhibitor of protein biosynthesis. Cycloheximide also abolished the potentiating actions of CTX, forskolin, and dibutyryl cyclic AMP on BK-induced IPs formation and Ca2+ mobilization. 5 The locus of this enhancement 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, forskolin, or dibutyryl cyclic AMP treatment, supporting the contention that this stimulatory effect is located at the BK receptor level. 6 These results demonstrate that the augmentation of BK-induced IPs accumulation and Ca2+ mobilization produced by CTX, forskolin and dibutyryl cyclic AMP involves a cyclic AMP-dependent mechanism which is induced by a sustained increase in the level of intracellular cyclic AMP. CTX and forskolin may promote an increase of the synthesis of BK receptors, and thereby enhance BK-induced responses.