An evaluation of potassium ions as endothelium-derived hyperpolarizing factor in porcine coronary arteries

Abstract

In the rat hepatic artery, the endothelium-derived hyperpolarizing factor (EDHF) was identified as potassium. Potassium hyperpolarizes the smooth muscles by gating inward rectified potassium channels and by activating the sodium-potassium adenosine triphosphatase (Na(+)-K(+)ATPase). Our goal was to examine whether potassium could explain the EDHF in porcine coronary arteries. On coronary strips, the inhibition of calcium-dependent potassium channels with 100 nM apamin plus 100 microM charibdotoxin inhibited the endothelium-dependent relaxations, produced by 10 nM substance P and 300 nM bradykinin and resistant to nitro-L-arginine and indomethacin. The scavenging of potassium with 2 mM Kryptofix 2.2.2 abolished the endothelium-dependent relaxations produced by the kinins and resistant to nitro-L-arginine and indomethacin. Forty microM 18alpha glycyrrethinic acid or 50 microM palmitoleic acid, both uncoupling agents, did not inhibit these kinin relaxations. Therefore, EDHF does not result from an electrotonic spreading of an endothelial hyperpolarization. Barium (0.3 nM) did not inhibit the kinin relaxations resistant to nitro-L-arginine and indomethacin. Therefore, EDHF does not result from the activation of inward rectified potassium channels. Five hundred nM ouabain abolished the endothelium-dependent relaxations resistant to nitro-L-arginine and indomethacin without inhibiting the endothelium-derived NO relaxation. The perifusion of a medium supplemented with potassium depolarized and contracted a coronary strip; however, the short application of potassium hyperpolarized the smooth muscles. These results are compatible with the concept that, in porcine coronary artery, the EDHF is potassium released by the endothelial cells and that this ion hyperpolarizes and relaxes the smooth muscles by activating the Na(+)-K(+)ATPase.

Figure 1

Concentration-response curve to (A) ouabain and (B) KCl. In (B), strips were tonically contracted by 0.1 μM U46619 P in the presence of 100 μM nitro-L-arginine (L-NA) and 10 μM indomethacin.

Figure 2

Original recording of the smooth muscle cell membrane potential on a strip with (A+B) or without (C) intact endothelium. Effect of a fast injection of KCl in the perfused tissue bath close to the perifusion inlet (A), and close to the outlet, which caused a shorter, localized application of the ion (B and C).

Figure 3

Original recordings of mechanical activity of strips tonically contracted with 0.3 μM U46619 P in the presence of 100 μM nitro-L-arginine (L-NA) and 10 μM indomethacin. Effect of 300 nM bradykinin (BK) and 10 nM substance P (SP) (IC₅₀-concentrations) and 4 μM nitroglycerin (NG). The experiments were done in a medium without potassium in the panels C and D. In the panels B and D the experiments were done in the presence of the potassium chelator 2 mM kryptofix 2.2.2.

Figure 4

Concentration-response curves of bradykinin- and substance P-induced EDHF relaxations in the presence of 100 μM nitro-L-arginine and 10 μM indomethacin. The relaxations caused by the peptides are expressed in % of the tonic contraction produced throughout the experiment by 0.3 μM U 46619. Effect of 2 μM BaCl2, an inhibitor of inward rectified potassium channels (A and D); of 40 μM 18α glycyrrethinic acid (B and E) and of 50 μM palmitoleic acid, two inhibitors of cell to cell communication through gap junctions (C and F).

Figure 5

Concentration-response curves of bradykinin- and substance P-induced EDHF relaxations in the presence of 10 μM indomethacin and 100 μM nitro-L-arginine. The relaxations caused by the peptides are expressed in % of the tonic contraction produced throughout the experiment by 0.3 μM U 46619. Effect of 500 nM ouabain. The effect of ouabain is also shown in experiments done without indomethacin and nitro-L-arginine to show the role of nitric oxide.