Acetylcholine-induced relaxation in blood vessels from endothelial nitric oxide synthase knockout mice

Abstract

1. Isometric tension was recorded in isolated rings of aorta, carotid, coronary and mesenteric arteries taken from endothelial nitric oxide synthase knockout mice (eNOS(-/-) mice) and the corresponding wild-type strain (eNOS(+/+) mice). The membrane potential of smooth muscle cells was measured in coronary arteries with intracellular microelectrodes. 2. In the isolated aorta, carotid and coronary arteries from the eNOS(+/+) mice, acetylcholine induced an endothelium-dependent relaxation which was inhibited by N(omega)-L-nitro-arginine. In contrast, in the mesenteric arteries, the inhibition of the cholinergic relaxation required the combination of N(omega)-L-nitro-arginine and indomethacin. 3. The isolated aorta, carotid and coronary arteries from the eNOS(-/-) mice did not relax in response to acetylcholine. However, acetylcholine produced an indomethacin-sensitive relaxation in the mesenteric artery from eNOS(-/-) mice. 4. The resting membrane potential of smooth muscle cells from isolated coronary arteries was significantly less negative in the eNOS(-/-) mice (-64.8 +/- 1.8 mV, n = 20 and -58.4 +/- 1.9 mV, n = 17, for eNOS(+/+) and eNOS(-/-) mice, respectively). In both strains, acetylcholine, bradykinin and substance P did not induce endothelium-dependent hyperpolarizations whereas cromakalim consistently produced hyperpolarizations (- 7.9 +/- 1.1 mV, n = 8 and -13.8 +/- 2.6 mV, n = 4, for eNOS(+/+) and eNOS(-/-) mice, respectively). 5. These findings demonstrate that in the blood vessels studied: (1) in the eNOS(+/+) mice, the endothelium-dependent relaxations to acetylcholine involve either NO or the combination of NO plus a product of cyclo-oxygenase but not EDHF; (2) in the eNOS(-/-) mice, NO-dependent responses and EDHF-like responses were not observed. In the mesenteric arteries acetylcholine releases a cyclo-oxygenase derivative.

Figure 1

Concentration-relaxation curves to acetylcholine in aortas with endothelium from eNOS(+/+) (upper panel; n=13) and eNOS(−/−) mice (lower panel; n=11) in the absence and presence of indomethacin (5 μM) and/or Nω-nitro-L-arginine (L-NA, 100 μM). Data are shown as means±s.e.mean.

Figure 2

Concentration-relaxation curves to acetylcholine in carotid arteries with endothelium from eNOS(+/+) (upper panel; n=5–6) and eNOS(−/−) mice (lower panel; n=4–5) in the absence and presence of indomethacin (5 μM) and/or Nω-nitro-L-arginine (L-NA, 100 μM). Data are shown as means±s.e.mean.

Figure 3

Acetylcholine (10 μM)-induced relaxations in mesenteric arteries with endothelium from eNOS(+/+) (upper panel; n=8–10) and eNOS(−/−) mice (lower panel; n=8–9), in the absence and presence of indomethacin (5 μM) and/or Nω-nitro-L-arginine (L-NA, 100 μM). Data are shown as means±s.e.mean. Asterisks indicate a statistically significant difference with the corresponding control value.

Figure 4

Acetylcholine (10 μM)-induced relaxations in coronary arteries with endothelium from eNOS(+/+) (upper panel; n=6–8) and eNOS(−/−) mice (lower panel; n=6–8), in presence or not of indomethacin (5 μM) and/or Nω-nitro-L-arginine (L-NA, 100 μM). Data are shown as means±s.e.mean. Asterisks indicate a statistically significant difference with the corresponding control value.

Figure 5

Original traces showing the effects of acetylcholine (ACh, 10 μM) and cromakalim (10 μM) on the resting membrane potential of smooth muscle cell from isolated coronary artery with endothelium taken from eNOS(+/+) (upper panel) or eNOS (−/−) mice (lower panel) mice.