Effect of forskolin on endothelin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

1 The effects of increase in intracellular adenosine 3′:5′-cyclic monophosphate (cAMP) on endothelin-1 (ET-1)-induced generation of inositol phosphates (IPs) and increase in intracellular Ca²⁺ ([Ca²⁺]_i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). 2 Pretreatment of TSMCs with either cholera toxin (CTX; 10 μg ml^??1, 4 h), forskolin (10 μm , 30 min), or dibutyryl cAMP (1 mm , 30 min) inhibited ET-1-stimulated Ca²⁺ mobilization (by 23 ± 5%, n = 8) and IPs accumulation (by 32 ± 6%, n = 4). While after treatment with forskolin for 24 h, the cells retained the ability to respond to ET-1-induced Ca²⁺ mobilization to the same extent as the control group. 3 Forskolin (1–100 μm ) inhibited the ET-1-induced increase in [Ca²⁺]_i, but the lower concentrations had little effect on this response. 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 ET-1 without changing the –logEC₅₀ values. 4 The water-soluble forskolin analogue L-858051, 7-deacetyl-7β-(γ-N-methylpiperazino)-butyryl forskolin, significantly inhibited ET-1-stimulated IPs accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive analogue of forskolin, had little effect on stimulated responses. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit ET-1-induced IPs accumulation. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. 5 The locus of this inhibition of forskolin treatment on AlF4??-stimulated IPs accumulation was investigated in canine TSMCs. The AlF4??-induced IPs accumulation was inhibited by forskolin, supporting that G protein(s) are directly activated by AlF4?? and uncoupled to phospholipase C by forskolin treatment. 6 We conclude that cAMP elevating agents inhibit ET-1-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 ET-1 receptors, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.