Effects of cAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

The effects of increases in intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) on carbachol-induced generation of inositol phosphates (IPs) and increases in intracellular Ca²⁺ ([Ca²⁺]_i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The cAMP elevating agents, cholera toxin (CTX) and forskolin, induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC₅₀) at concentrations of 7.6 ± 1.3 g/ml and 4.8 ± 0.9 M, respectively. Forskolin caused a concentration-dependent inhibition of carbachol-induced increase in [Ca²⁺]_i with half-maximal inhibition (-logEC₅₀) at 5.2 ± 0.7 M. Pretreatment of TSMCs with either CTX (10 μg/ml, 4 h), forskolin (10-100 μM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited carbachol-stimulated Ca²⁺ mobilization and IPs accumulation. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of carbachol without changing the EC₅₀ values. After treatment with forskolin for 24 h, carbachol-induced IPs accumulation and Ca²⁺ mobilization were close to those of control group. SQ-22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 μM], an inhibitor of adenylate cyclase, and HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, 50 μM], an inhibitor of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit carbachol-induced IPs accumulation. Moreover, the inactive analogue of forskolin, 1,9-dideoxy forskolin, did not inhibit these responses evoked by carbachol, suggesting that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The K_D and B_max values of the muscarinic receptor (mAChR) for [³H]-N-methyl scopolamine binding were not significantly changed by forskolin treatment for 30 min and 24 h, suggesting that the inhibitory effect of forskolin is distal to the mAChR. The locus of this inhibition was further investigated by examining the effect of forskolin treatment on AIF₄^--stimulated IPs accumulation in canine TSMCs. The AIF₄^--induced response was inhibited by forskolin, supporting the notion that G protein(s) are directly activated by AIF₄^- and uncoupled to phospholipase C by forskolin treatment. We conclude that cAMP elevating agents inhibit carbachol-stimulated generation of IPs and Ca²⁺ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca²⁺]_i are very early events in the activation of mAChRs, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.