Potassium- and acetylcholine-induced vasorelaxation in mice lacking endothelial nitric oxide synthase

Abstract

1. The contribution of an endothelium-derived hyperpolarizing factor (EDHF) was investigated in saphenous and mesenteric arteries from endothelial nitric oxide synthase (eNOS) (-/-) and (+/+) mice. 2. Acetylcholine-induced endothelium-dependent relaxation of saphenous arteries of eNOS(-/-) was resistant to N(omega)-nitro-L-arginine (L-NNA) and indomethacin, as well as the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a) quinoxalin-1-one(ODQ). 3. Potassium (K(+)) induced a dose-dependent vasorelaxation which was endothelium-independent and unaffected by either L-NNA or indomethacin in both saphenous and mesenteric arteries from eNOS(-/-) or (+/+) mice. 4. Thirty microM barium (Ba(2+)) and 10 microM ouabain partially blocked potassium-induced, but had no effect on acetylcholine-induced vasorelaxation in saphenous arteries. 5. Acetylcholine-induced relaxation was blocked by a combination of charybdotoxin (ChTX) and apamin which had no effect on K(+)-induced relaxation, however, iberiotoxin (IbTX) was ineffective against either acetylcholine- or K(+)-induced relaxation. 6. Thirty microM Ba(2+) partially blocked both K(+)- and acetylcholine-induced relaxation of mesenteric arteries, and K(+), but not acetylcholine-induced relaxation was totally blocked by the combination of Ba(2+) and ouabain. 7. These data indicate that acetylcholine-induced relaxation cannot be mimicked by elevating extracellular K(+) in saphenous arteries from either eNOS(-/-) or (+/+) mice, but K(+) may contribute to EDHF-mediated relaxation of mesenteric arteries.

Figure 1

Concentration-response curves showing the effects of NOS (L-NNA, 100 μM) in combination with COX (indomethacin, 10 μM) inhibition, and NOS, COX and GC inhibition (ODQ, 10 μM) versus control (no inhibitors) on acetylcholine-induced vasorelaxation of phenylephrine (1 μM) precontracted mouse saphenous arteries. (n=10). (a) In vessels from eNOS +/+ mice both NOS and COX and the combination of NOS, COX, and GC inhibition significantly reduced acetylcholine-induced vasorelaxation (^*P<0.05). (b) In vessels from eNOS −/− mice neither NOS plus COX nor NOS, COX and GC inhibition had a significant effect on acetylcholine-mediated relaxation (P>0.05).

Figure 2

Concentration-response curves showing that increasing extracellular K⁺ by the addition of 2–15 mM KCl (for a final bath concentration of 6.8–19.8 mM K⁺) produces equivalent vasorelaxant responses in saphenous arteries from both eNOS +/+ and −/− mice (P>0.05) (n=7). Data are means±s.e.mean.

Figure 3

Relaxation responses to either 100 μM acetylcholine or the addition of 10 mM KCl in saphenous arteries in the absence or presence of a combination of NOS and COX inhibition. Acetylcholine, but not K⁺, induced relaxation in vessels from eNOS +/+ was significantly inhibited (^*P<0.05) in the presence of NOS and COX inhibition, whereas in vessels from eNOS −/− mice the relaxation responses to both acetylcholine and K⁺ were insensitive to the combination of NOS and COX inhibition (P>0.05, n=6).

Figure 4

Acetylcholine- and K⁺-induced vasorelaxation responses were compared in endothelium-intact and endothelium-denuded preparations of mouse saphenous arteries. The removal of the endothelium eliminated the acetylcholine-induced relaxation in saphenous arteries from both eNOS +/+ and −/− mice whereas the K⁺-induced relaxation was shown to be endothelium-independent (P>0.05, n=8). Data are means±s.e.mean.

Figure 5

The relaxation response induced by the addition of 10 M K⁺ to saphenous arteries from eNOS +/+ and eNOS −/− mice was partially inhibited in the presence of 30 micromolar Ba²⁺ or 10 μM ouabain (^*P<0.05, n=7). The combination of Ba²⁺ and ouabain, however, almost totally abolished the vasorelaxant response to K⁺ in saphenous arteries from both eNOS +/+ and eNOS −/− mice. (^*P<0.05, n=7). Data are means±s.e.mean.

Figure 6

Concentration-response curves showing the lack of effect of barium, or ouabain alone or the effect of a combination of barium and ouabain, on acetylcholine-induced vasorelaxation in saphenous arteries isolated from (a) eNOS +/+ and (b) eNOS −/− mice (n=7) obtained in the presence of a combination of NOS (L-NNA, 100 μM) and COX (indomethacin, 10 μM).

Figure 7

Concentration-response curves showing the effects of potassium channel blockade with ChTX (100 nM), apamin (1 μM), or the combination of ChTX and apamin, in the presence of NOS and COX inhibition (100 μM L-NNA and 10 μM indomethacin), on acetylcholine-induced relaxation in saphenous arteries from (a) eNOS +/+ and (b) eNOS −/− mice (n=5). In vessels from both eNOS +/+ and −/− animals a combination of apamin and ChTX almost completely abolished the acetylcholine-induced relaxation (^*P<0.05) whereas neither apamin nor ChTX alone had a significant inhibitory effect (P>0.05).

Figure 8

Concentration-response curves showing the effects of barium (30 μM) or ouabain (10 μM), in the presence of NOS and COX (100 μM L-NNA and 10 μM indomethacin), on acetylcholine-induced relaxation in mesenteric arteries isolated from (a) eNOS +/+ mice (n=7) and (b) eNOS −/− mice (n=7). Barium, but not ouabain, significantly inhibited the maximal relaxation response to acetylcholine in vessels from eNOS +/+ (^*P<0.05), but neither barium nor ouabain produced a significant inhibitory action on the relaxation response to acetylcholine in vessels from eNOS −/− (^*P<0.05). Data are mean±s.e.mean.

Figure 9

The relaxation responses mediated by the addition of 10 mM K⁺, in the presence of a combination of NOS (L-NNA, 100 μM) and COX (indomethacin, 10 μM) inhibition, to mesenteric arteries from eNOS +/+ and −/− mice were partially (^*P<0.05) inhibited by either barium (30 μM) or ouabain (10 μM), and completely inhibited by the combination of barium and ouabain (^*P<0.05, n=6).