Takakura Ko, Taniguchi Takanobu, Muramatsu Ikunobu, Takeuchi Kenji, Fukuda Satoru
Department of Anesthesiology and Reanimatology, Fukui Medical University, Matsuoka, Fukui, Japan.
Crit Care Med. 2002 Apr;30(4):894-9. doi: 10.1097/00003246-200204000-00030.
It is well known that nitric oxide synthase is induced by endotoxin or inflammatory cytokines, and consequently large amounts of nitric oxide cause vascular hyporeactivity to vasoconstrictor agents and myocardial dysfunction, hence hypotension. However, there is considerable controversy as to whether these pathologic cardiovascular features are mediated directly by nitric oxide or also through the formation of secondary reaction products such as peroxynitrite (ONOO-1). Our objective was to investigate inhibitory effects of ONOO-1 on alpha1-adrenoceptors.
Prospective, controlled, in vitro, laboratory study.
Laboratory of a health sciences university.
Chinese hamster ovary cells that expressed the human recombinant alpha1a-, alpha1b-, or alpha1d-adrenoceptors, rat aorta strips.
Binding experiments of [3H]prazosin were done in the Chinese hamster ovary cell membranes pretreated with 100 microM to 3 mM ONOO-1. Displacement experiments with noradrenaline or 3-nitro-l-tyrosine also were conducted. Mobilization of intracellular Ca2+ evoked by 1 nM to 10 microM noradrenaline was monitored in a fluorescence spectrophotometer with dual excitation at 340 nm/380 nm and emission at 500 nm in fura-2/AM-loaded Chinese hamster ovary cells. Contractile force produced by noradrenaline was monitored in rat aorta strips that have alpha1a- and alpha1d-adrenoceptors, pretreated with 1 mM ONOO-1. Either 0.3 N NaOH or the decomposed ONOO-1 was used as the control.
The specific binding of [3H]prazosin to alpha1a- and alpha1d-adrenoceptor was inhibited by ONOO-1 in a concentration-dependent manner. We found that 3 mM ONOO-1 decreased maximum binding sites by 40% to 50% in alpha1a- and alpha1d-adrenoceptors. Binding affinities for prazosin and noradrenaline were not affected by 1 mM ONOO-1 in all subtypes. We found that 3-nitro-l-tyrosine did not affect the prazosin binding to three adrenoceptor subtypes. Noradrenaline increased intracellular Ca2+ concentration ([Ca2+]i) concentration-dependently, which was inhibited by ONOO-1 in alpha1a- and alpha1d-adrenoceptors. ONOO-1 had no effect on alpha1b-adrenoceptor. Contractile force produced by noradrenaline decreased significantly in aorta strips pretreated with ONOO-1.
ONOO-1 reduces the binding capacity of alpha1a- and alpha1d- but not alpha1b-adrenoceptors without changing the affinities. Treatment with ONOO-1 attenuates noradrenaline-stimulated increase in [Ca2+]i in alpha1a- and alpha1d-adrenoceptors but not in alpha1b-adrenoceptor. ONOO-1 also weakens noradrenaline-induced contractions in rat aorta that has alpha1a- and alpha1d-adrenoceptors. Cardiovascular hyporeactivity to catecholamines in septic shock may be caused in part by the inactivation of alpha-adrenoceptors by ONOO-1.
众所周知，一氧化氮合酶可由内毒素或炎性细胞因子诱导产生，大量一氧化氮会导致血管对血管收缩剂反应性降低以及心肌功能障碍，进而引起低血压。然而，关于这些病理性心血管特征是直接由一氧化氮介导，还是也通过诸如过氧亚硝酸盐（ONOO⁻）等二级反应产物的形成介导，存在相当大的争议。我们的目的是研究ONOO⁻对α1-肾上腺素能受体的抑制作用。
前瞻性、对照、体外实验室研究。
一所健康科学大学的实验室。
表达人重组α1a-、α1b-或α1d-肾上腺素能受体的中国仓鼠卵巢细胞、大鼠主动脉条。
在经100微摩尔/升至3毫摩尔/升ONOO⁻预处理的中国仓鼠卵巢细胞膜中进行[³H]哌唑嗪结合实验。还进行了去甲肾上腺素或3-硝基-L-酪氨酸的置换实验。在配备双激发波长（340纳米/380纳米）和发射波长（500纳米）的荧光分光光度计中，监测在负载fura-2/AM的中国仓鼠卵巢细胞中，1纳摩尔/升至10微摩尔/升去甲肾上腺素引起细胞内Ca²⁺动员情况。在经1毫摩尔/升ONOO⁻预处理、具有α1a-和α1d-肾上腺素能受体的大鼠主动脉条中，监测去甲肾上腺素产生的收缩力。使用0.3 N氢氧化钠或分解后的ONOO⁻作为对照。
ONOO⁻以浓度依赖性方式抑制[³H]哌唑嗪与α1a-和α1d-肾上腺素能受体的特异性结合。我们发现，3毫摩尔/升ONOO⁻使α1a-和α1d-肾上腺素能受体的最大结合位点减少40%至50%。在所有亚型中，1毫摩尔/升ONOO⁻对哌唑嗪和去甲肾上腺素的结合亲和力均无影响。我们发现，3-硝基-L-酪氨酸不影响哌唑嗪与三种肾上腺素能受体亚型的结合。去甲肾上腺素可浓度依赖性增加细胞内Ca²⁺浓度（[Ca²⁺]i），这在α1a-和α1d-肾上腺素能受体中受到ONOO⁻抑制。ONOO⁻对α1b-肾上腺素能受体无影响。在经ONOO⁻预处理的主动脉条中，去甲肾上腺素产生的收缩力显著降低。
ONOO⁻降低α1a-和α1d-肾上腺素能受体的结合能力，但不影响α1b-肾上腺素能受体，且不改变亲和力。用ONOO⁻处理可减弱去甲肾上腺素刺激引起的α1a-和α1d-肾上腺素能受体中[Ca²⁺]i增加，但不影响α1b-肾上腺素能受体。ONOO⁻还减弱了去甲肾上腺素在具有α
