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

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 microM histamine, NKH477 (1 nM-1 microM) caused a concentration-dependent decrease of isometric tension, resulting in a complete relaxation at 300 nM. The EC550 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 microM 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 microM 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 microM). 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 microM 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 microM 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.