Endothelium-derived hyperpolarizing factor and potassium use different mechanisms to induce relaxation of human subcutaneous resistance arteries

Abstract

This investigation examined the hypothesis that release of K(+) accounts for EDHF activity by comparing relaxant responses produced by ACh and KCl in human subcutaneous resistance arteries. Resistance arteries (internal diameter 244+/-12 microm, n=48) from human subcutaneous fat biopsies were suspended in a wire myograph. Cumulative concentration-response curves were obtained for ACh (10(-9) - 3x10(-5) M) and KCl (2.5 - 25 mM) following contraction with noradrenaline (NA; 0.1 - 3 microM). ACh (E(max) 99.07+/-9.61%; -LogIC(50) 7.03+/-0.22; n=9) and KCl (E(max) 74.14+/-5.61%; -LogIC(50) 2.12+/-0.07; n=10)-induced relaxations were attenuated (P<0.0001) by removal of the endothelium (E(max) 8.21+/-5.39% and 11.56+/-8.49%, respectively; n=6 - 7). Indomethacin (10 microM) did not alter ACh-induced relaxation whereas L-NOARG (100 microM) reduced this response (E(max) 61.7+/-3.4%, P<0.0001; n=6). The combination of ChTx (50 nM) and apamin (30 nM) attenuated the L-NOARG-insensitive component of ACh-induced relaxation (E(max): 15.2+/-10.5%, P<0.002, n=6) although these arteries retained the ability to relax in response to 100 microM SIN-1 (E(max) 127.6+/-13.0%, n=3). Exposure to BaCl(2) (30 microM) and Ouabain (1 mM) did not attenuate the L-NOARG resistant component of ACh-mediated relaxation (E(max), 76.09+/-8.92, P=0.16; n=5). KCl-mediated relaxation was unaffected by L-NOARG+indomethacin (E(max); 68.1+/-5.6%, P=0.33; n=5) or the combination of L-NOARG/indomethacin/ChTx/apamin (E(max); 86.61+/-14.02%, P=0.35; n=6). In contrast, the combination of L-NOARG, indomethacin, ouabain and BaCl(2) abolished this response (E(max), 5.67+/-2.59%, P<0.0001, n=6). The characteristics of KCl-mediated relaxation differed from those of the nitric oxide/prostaglandin-independent component of the response to ACh, and were endothelium-dependent, indicating that K(+) does not act as an EDHF in human subcutaneous resistance arteries.

Figure 1

Cumulative concentration-response curves to ACh (10⁻⁹ – 3×10⁻⁵ M) before and after incubation with the following combination of inhbitors (a) the cyclooxygenase inhibitor indomethacin (10 μM for 45 min), (b) the NO synthase inhibitor L-NOARG (100 μM for 45 min) or (c) L-NOARG (100 μM for 45 min) plus the K⁺ channel blockers ChTx (50 nM for 10 min) and apamin (30 nM for 10 min). Results are shown as mean±s.e.mean, for (n) arteries.

Figure 2

Comparison of ACh- and KCl-mediated relaxation. (a) Representative traces showing (i) relaxation responses of an intact artery to acetylcholine and KCl and (ii) the effect of removal of the endothelium on these responses. (b) Cumulative concentration-response curves for (i) acetylcholine and (ii) KCl obtained in arteries with and without an intact endothelium or in the presence of L-NOARG (100 μM) and indomethacin (10 μM) alone or combined with either ChTx (50 nM) and apamin (30 nM) or BaCl₂ (30 μM) plus ouabain (1 mM). Results are shown as mean±s.e.mean, for (n) arteries.