Mechanisms underlying β2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells
Mechanisms underlying β2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells
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DOI：
10.1113/jphysiol.2006.115998
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发表时间：
2006-10-15
影响因子：
5.5
通讯作者：
Ferro, Albert
中科院分区：
文献类型：
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作者：
Queen, Lindsay R.;Ji, Yong;Ferro, Albert
内皮细胞β(2)-肾上腺素能受体(β(2)AR)刺激可增加一氧化氮(NO)的生成，但其潜在的细胞机制尚不清楚。我们研究了L-精氨酸转运和一氧化氮合酶3(NOS3)磷酸化在β(2)AR介导的人脐静脉内皮细胞合成NO中的作用。为此，我们检测了经β-AR激动剂或环状AMP升高剂处理的人脐静脉内皮细胞对L-精氨酸的摄取、一氧化氮合酶活性(从L-精氨酸到L-瓜氨酸的转化)、膜电位(使用[H-3]四苯基膦)以及一氧化氮合酶-3的丝氨酸磷酸化(用Western blotting和质谱法)。刺激β(2)AR可增加L-精氨酸转运，Forsklin或二丁酰环AMP均可使cAMP升高，这种增加可被N-乙基马来酰亚胺抑制。用L赖氨酸阻断L精氨酸摄取抑制一氧化氮合酶的活性，反之，用N-omega-硝基-L-精氨酸甲酯阻断一氧化氮合酶抑制L-精氨酸的转运。刺激β(2)AR还引起膜超极化，这种超极化可被L-NAME抑制，提示L-精氨酸摄取增加是对NO介导的超极化的反应。β(2)AR的激活也增加了NOS活性和NOS-3在丝氨酸-1177上的磷酸化，这种增加可被蛋白激酶A(PKA)、磷脂酰肌醇3-激酶(PI3K)或Akt抑制而减弱，并可被PKA和Akt共同抑制而取消。这些结果表明，β(2)AR介导的HUVEC中一氧化氮合酶-3的活化是通过PKA和PI3K/AKT系统通过丝氨酸-1177上的一氧化氮合酶-3的磷酸化来实现的，并由NO介导的膜超极化导致的L-精氨酸摄取增加所维持。
Endothelial beta(2)-adrenoceptor (beta(2)AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of L-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in beta(2)AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed L-arginine uptake, NOS activity (from L-arginine to L-citrulline conversion), membrane potential (using [H-3]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with beta AR agonists or cyclic AMP-elevating agents. beta(2)AR stimulation increased L-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade of L-arginine uptake by L-lysine inhibited NOS activity and, conversely, blockade of NOS using N-omega-nitro-L-arginine methyl ester (L-NAME) inhibited L-arginine transport. beta(2)AR stimulation also caused a membrane hyperpolarization inhibitable by L-NAME, suggesting that the increase in L-arginine uptake occurred in response to NO-mediated hyperpolarization. beta(2)AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that beta(2)AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in L-arginine uptake resulting from NO-mediated membrane hyperpolarization.