Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery

Abstract

It is controversial whether the endothelial cell release of nitric oxide (NO) or a different factor(s) accounts for endothelium-dependent hyperpolarization, because in many arteries endothelium-dependent relaxation and hyperpolarization resists inhibitors of NO synthase. The contribution of NO to acetylcholine-induced endothelium-dependent hyperpolarization and relaxation of the rabbit carotid artery was determined by measuring NO with electrochemical and chemiluminescence techniques. In the presence of phenylephrine to depolarize and contract the smooth muscle cells, acetylcholine caused concentration-dependent hyperpolarization and relaxation which were closely correlated to the release of NO. N(omega)-nitro-L-arginine methyl ester (30 microM) partially reduced the release of NO and caused a similar reduction in smooth muscle cell relaxation and hyperpolarization. To determine if the residual responses were mediated by another endothelium-derived mediator or NO released despite treatment with N(omega)-nitro-L-arginine methyl ester, N(omega)-nitro-L-arginine (300 microM) was added. The combined inhibitors further reduced, but did not eliminate, NO release, smooth muscle relaxation, and hyperpolarization. Hyperpolarization and relaxation to acetylcholine remained closely correlated with the release of NO in the presence of the inhibitors. In addition, the NO donor, SIN-1, caused hyperpolarization and relaxation which correlated with the concentrations of NO that it released. These studies indicate that (i) the release of NO by acetylcholine is only partially inhibited by these inhibitors of NO synthase when used even at high concentrations, and (ii) NO rather than another factor accounts fully for endothelium-dependent responses of the rabbit carotid artery.

Figure 1

Effect of l-NAME and l-NNA on ACh-induced release of NO and nitrite from rabbit carotid artery. (a) Tracings of typical amperograms obtained with porphyrinic microsensors showing release of NO (nM) caused by ACh (3 × 10⁻⁶ M) added at time zero under control conditions, or after treatment with l-NAME (3 × 10⁻⁵ M) or l-NNA (3 × 10⁻⁴ M, respectively). (b) Plot of peak concentration of NO released in response to each individual ACh concentration (−log M) shown on the left ordinate (n = 12), or cumulative amount of nitrite (pmol/cm²) following the entire concentration response to ACh (10⁻⁸–3 × 10⁻⁶ M) shown on the right ordinate (C, n = 11). NO release was determined in response to ACh under control conditions (○), or after treatment with l-NAME (3 × 10⁻⁵ M; •), l-NNA (3 × 10⁻⁴ M; □), or the combination of l-NAME and l-NNA (3 × 10⁻⁵ M and 3 × 10⁻⁴ M, respectively; ▴). Data are means ± SEM; error bars for NO release in most cases are not visible on the graph due to the size of the symbols.

Figure 2

Effect of l-NAME and l-NNA on ACh-induced relaxation and hyperpolarization. Tracings of recordings of isometric tension (mN) and membrane potential (mV) recorded simultaneously in (a) carotid artery rings under control conditions, (b) treated with l-NAME (3 × 10⁻⁵ M), or (c) treated with the combination of l-NAME (3 × 10⁻⁵ M) and l-NNA (3 × 10⁻⁴ M). The rings were depolarized and contracted with phenylephrine (PE) (1–3 × 10⁻⁶ M) and then exposed to cumulative increasing ACh concentrations (−log M) indicated by the arrows.

Figure 3

Effect of l-NAME and l-NNA on ACh-induced hyperpolarization, relaxation, and NO release. ACh-evoked relaxations (open symbols) and hyperpolarizations (filled symbols) recorded from arterial rings contracted and depolarized with phenylephrine under control conditions (circles), after treatment with l-NAME (3 × 10⁻⁵ M; squares), or after treatment with l-NAME (3 × 10⁻⁵ M) and l-NNA (3 × 10⁻⁴ M; triangles) are shown in a. (b) Close correlation between the release of NO measured with the porphyrinic microsensor from rabbit carotid arteries caused by ACh (3 × 10⁻⁶ M) under control conditions or after treatment with the same concentrations of l-NAME, or l-NAME combined with l-NNA and the maximal relaxation (filled circles) or hyperpolarization (open circles). (c) Close correlation between the maximal relaxation (filled circles) or hyperpolarization (open circles) to ACh (3 × 10⁻⁶ M) and the cumulative release of nitrite caused by the concentration response to ACh (10⁻⁸ to 3 × 10⁻⁶ M) under control conditions or after treatment with l-NAME alone or with the combination of l-NAME and l-NNA.

Figure 4

Relaxation, hyperpolarization, and NO release caused by SIN-1 in rings of carotid artery. (a) Relaxation and hyperpolarization to SIN-1 in arteries contracted to phenylephrine. The values shown are the maximal and stable relaxation attained 4 min after adding each concentration of SIN-1. (b) Correlation between the NO concentration measured with a porphyrinic sensor 4 min after adding SIN-1 to physiological buffer and the relaxation (○) and hyperpolarization (•) caused by each concentration.