Relaxant effects of NKH477, a new water-soluble forskolin derivative, on guinea-pig tracheal smooth muscle: the role of Ca²⁺-activated K⁺ channels

Abstract

1. Mechanisms underlying the bronchorelaxant action of NKH477, a newly developed water-soluble forskolin derivative, were investigated in guinea-pig isolated tracheal smooth muscle.

2. In muscles precontracted with 3 μM histamine, NKH477 (1 nM–1 μM) caused a concentration-dependent decrease of isometric tension, resulting in a complete relaxation at 300 nM. The EC₅₀ for the relaxation was 32.6±4.3 nM (n=6).

3. In the presence of 30 or 90 nM iberiotoxin (IbTX), a selective blocker of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel, the relaxing action of NKH477 on the histamine-induced contraction was inhibited, giving rise to a parallel shift of the concentration-response curves; the EC₅₀ of NKH477 was increased to 131.4±20.4 nM at 30 nM IbTX (n=4), and 125.3±12.2 nM at 90 nM IbTX (n=4).

4. Pretreatment of muscles with 30 mM tetraethylammonium (TEA) caused a similar rightward shift of the concentration-response curve to NKH477 with an increase of the EC₅₀ to 139.8±18.4 nM (n=5). In contrast, the relaxing action of NKH477 was unaffected by 10 μM glibenclamide, an ATP-sensitive K⁺ channel blocker, or by 100 nM apamin, a blocker of small conductance Ca²⁺-activated K⁺ channels.

5. In muscles pretreated with 1 μM nifedipine, a blocker of the voltage-dependent Ca²⁺ channel (VDC), 30–90 nM IbTX did not affect the relaxant effects of NKH477 on the histamine-induced contraction.

6. In muscles precontracted by a K⁺-rich (40 mM) solution, NKH477 caused only minimal relaxation (19.8±1.7%, n=4) even at the highest concentration (1 μM).

7. In experiments to measure the ratio of fura-2 fluorescence signals (R_340/380) as an index of the intracellular Ca²⁺ concentration ([Ca²⁺]_i), the application of 100 nM NKH477 or 200 nM isoprenaline to the preparation precontracted by 3 μM histamine resulted in a decrease in [Ca²⁺]_i in association with a decrease in tension. The reduction of [Ca²⁺]_i and tension by NKH477 was 47.0±5.6% and 62.8±7.0%, respectively (n=5), and that with isoprenaline 60.6±7.4% and 67.4±6.4%, respectively (n=5). These effects of NKH477 and isoprenaline on [Ca²⁺]_i and tension were inhibited by 30 nM IbTX. The inhibitory action of IbTX was abolished in the presence of 1 μM nifedipine.

8. These results suggest that the bronchorelaxant action of NKH477 may result, at least in part, from activation of BK_Ca channels, which may cause a hyperpolarization of smooth muscle cell membranes and a secondary decrease in Ca²⁺ influx through VDCs, leading to a decrease in [Ca²⁺]_i.

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