Effects of a water‐soluble forskolin derivative (NKH477) and a membrane‐permeable cyclic AMP analogue on noradrenaline‐induced Ca2+ mobilization in smooth muscle of rabbit mesenteric artery

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Abstract
1 Effects were studied of 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative and of dibutyryl-cyclic AMP, a membrane-permeable cyclic AMP analogue on noradrenaline (NA)-induced Ca2+ mobilization in smooth muscle strips of the rabbit mesenteric artery. The intracellular concentration of Ca2+ ([Ca2+]i), isometric force and cellular concentration of inositol 1,4,5-trisphosphate (InsP3) were measured. 2 NA (10 μm) produced a phasic, followed by a tonic increase in both [Ca2+]i and force in a solution containing 2.6 mm Ca2+. NKH477 (0.01–0.3 μm) attenuated the phasic and the tonic increases in both [Ca2+]i and force induced by 10 μm NA, in a concentration-dependent manner. 3 In Ca2+-free solution containing 2 mm EGTA with 5.9 mm K+, NA (10 μm) produced only phasic increases in [Ca2+]i and force. NKH477 (0.01 μm) and dibutyryl-cyclic AMP (0.1 mm) each greatly inhibited these increases. 4 NA (10 μm) led to the production of InsP3 in intact smooth muscle strips and InsP3 (10 μm) increased Ca2+ in Ca2+-free solution after a brief application of Ca2+ in β-escin-skinned smooth muscle strips. NKH477 (0.01 μm) or dibutyryl-cyclic AMP (0.1 mm) modified neither the NA-induced synthesis of InsP3 in intact muscle strips nor the InsP3-induced Ca2+ release in skinned strips. 5 In Ca2+-free solution, high K+ (40 and 128 mm) itself failed to increase [Ca2+]i but concentration-dependently enhanced the amplitude of the increase in [Ca2+]i induced by 10 μm NA with a parallel enhancement of the maximum rate of rise. The extent of the inhibition induced by NKH477 (0.01 μm) or dibutyryl-cyclic AMP (0.1 mm) on the NA-induced [Ca2+]i increase was inversely related to the maximum rate of rise of [Ca2+]i induced by NA in Ca2+-free solution containing various concentrations of K+. These results suggest that the increase in the rate of Ca2+ release induced by NA can conceal the inhibitory action on NA-induced Ca2+ mobilization of agents that increase cyclic AMP. 6 Repetitive application of 10 μm NA in Ca2+-free solution led to a disappearance of the NA-induced increase in [Ca2+]i, but NA could again increase [Ca2+]i in Ca2+-free solution after a brief application of Ca2+ with 40 mm K+ (‘Ca2+-loading’). The magnitude of this NA-induced increase in [Ca2+]i depended on the duration of the Ca2+-loading. With application of dibutyryl-cyclic AMP (0.1 mm) during the Ca2+-loading period, the loading duration required for the restoration of the maximum NA-response was shortened. 7 Cyclopiazonic acid (10 μm, an inhibitor of Ca2+-ATPase at intracellular storage sites) attenuated the inhibitory action of dibutyryl-cyclic AMP on the NA-induced increase in [Ca2+]i in Ca2+-free solution. When NA (10 μm) was applied twice for 30 s with a 10 min interval in Ca2+-free solution, the amplitude of response to the second application was about one third of the first response. With application of 0.1 mm dibutyryl-cyclic AMP during the first application of NA, the increase in [Ca2+]i induced by the first application of NA was inhibited, but the response induced by the second was enhanced. These results suggest that dibutyryl-cyclic AMP enhances Ca2+ uptake into the NA-sensitive storage sites. 8 We conclude that, in smooth muscle of the rabbit mesenteric artery, agents that increase cyclic AMP inhibit the NA-induced increase in [Ca2+]i through an activation of Ca2+ uptake into the cellular storage sites.