Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na(+)/K(+) ATPase on EDHF relaxations in the rat hepatic artery

Abstract

1. In the rat hepatic artery, the SK(Ca) inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 microM) and d-tubocurarine (100 microM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM). 2. The IK(Ca) inhibitors clotrimazole (3 microM) and 2-chlorophenyl-bisphenyl-methanol (3 microM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono-oxygenase inhibitor ketoconazole (10 microM) had no effect in the presence of apamin. 3. Ciclazindol (10 microM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated (86)Rb(+) influx via the Gardos channel (IK(Ca)) in human erythrocytes. 4. The Na(+)/K(+) ATPase inhibitor ouabain (500 microM) and the K(IR) blocker Ba(2+) (30 microM) neither alone nor in combination inhibited EDHF relaxations. Ba(2+) was also without effect in the presence of either apamin or charybdotoxin. 5. In contrast to EDHF, an increase in extracellular [K(+)] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K(+)-free physiological salt solution, re-admission of K(+) always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba(2+). 6. The present study provides pharmacological evidence for the involvement of SK(Ca) and IK(Ca) in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K(+) activating Na(+)/K(+) ATPase and K(IR) in this blood vessel.

Figure 1

Effects of SK_Ca inhibitors on EDHF relaxations induced by acetylcholine in the presence of N^ω-nitro-L-arginine (300 μM) and indomethacin (10 μM) in arteries contracted by phenylephrine. The SK_Ca blockers UCL 1684 (n=5), scyllatoxin (ScTx, n=6) and d-tubocurarine (d-TC, n=6) were each combined with charybdotoxin (ChTx) and the effect of these combinations on EDHF was compared to that of charybdotoxin alone (n=10). At this concentration (300 nM), charybdotoxin is without any effect on EDHF relaxations. Data are presented as means±s.e.mean.

Figure 2

Effect of IK_Ca inhibitors on EDHF relaxations induced by acetylcholine in the presence of N^ω-nitro-L-arginine (300 μM) and indomethacin (10 μM) in arteries contracted by phenylephrine. The IK_Ca blockers clotrimazole (CLT) and 2-chlorophenyl-bisphenyl-methanol (C23) were each combined with apamin and the effect of these combinations on EDHF was compared to that of apamin alone. At this concentration (300 nM), apamin is without any effect on EDHF relaxations. Data are presented as means±s.e.mean of six experiments.

Figure 3

Effects of K⁺ channel inhibitors on calcium ionophore stimulated ⁸⁶Rb⁺ influx in human erythrocytes. The K⁺ channel inhibitors iberiotoxin (IbTx), apamin (Apa), charybdotoxin (ChTx), ciclazindol (Cz), clotrimazole (CLT) and 2-chlorophenyl-bisphenyl-methanol (C23) and the cytochrome P450 mono-oxygenase inhibitor ketoconazole (KEC) were present 85 min before erythrocytes were stimulated for 5 min by A23187 and Ca²⁺. Data are expressed as percentage of ⁸⁶Rb⁺ influx in the absence of test drugs (control) and are presented as means±s.e.mean of six experiments (all from different individuals). Asterisks denote a statistically significant difference from control values (P<0.05).

Figure 4

Effects of ouabain and Ba²⁺ on EDHF relaxations induced by acetylcholine in the presence of N^ω-nitro-L-arginine (300 μM) and indomethacin (10 μM) in arteries contracted by phenylephrine. The effect of ouabain and Ba²⁺ either alone or combined was examined. Control denotes EDHF relaxations in the absence of these drugs. Data are presented as means±s.e.mean of 7–9 experiments.

Figure 5

Traces showing differences between K⁺ and EDHF relaxations in hepatic arteries contracted by phenylephrine (PhE) in the presence of N^ω-nitro-L-arginine (300 μM) and indomethacin (10 μM). Arterial segments were first exposed to acetylcholine (ACh) to demonstrate EDHF relaxations in a physiological salt solution with a normal K⁺ content (4.6 mM KCl). After a washout period of 30 min, the relaxant effect of increased extracellular K⁺ concentrations (from 4.6 mM to 9.6, 14.6 and 19.6 mM) was recorded to see whether K⁺ could mimic EDHF in these arterial segments. Dashed line indicates the basal tension level before addition of phenylephrine. Upper panel K⁺ failed to relax an arterial segment in which EDHF caused relaxation. Lower panel Although K⁺ could relax another arterial segment the relaxation was transient and partial in contrast to the EDHF relaxation.

Figure 6

Effects of ouabain and Ba²⁺ on relaxation evoked by K⁺ in the presence of N^ω-nitro-L-arginine (300 μM) and indomethacin (10 μM) in arteries contracted by phenylephrine. Preparations were incubated with either ouabain or Ba²⁺ or vehicle (control) for 30 min in K⁺-free physiological salt solution before re-admission of K⁺. Data are presented as means±s.e.mean of 6–9 experiments.