Effects of a water‐soluble forskolin derivative (NKH477) and a membrane‐permeable cyclic AMP analogue on noradrenaline‐induced Ca 2+ mobilization in smooth muscle of rabbit mesenteric artery

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