α1-Adrenergic Receptor Stimulation of Mitogenesis in Human Vascular Smooth Muscle Cells: Role of Tyrosine Protein Kinases and Calcium in Activation of Mitogen-Activated Protein Kinase
α1-Adrenergic Receptor Stimulation of Mitogenesis in Human Vascular Smooth Muscle Cells: Role of Tyrosine Protein Kinases and Calcium in Activation of Mitogen-Activated Protein Kinase
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发表时间：
1999-07
影响因子：
3.5
通讯作者：
Zhuo-wei Hu;Xiao-you Shi;R. Lin;Jin Chen;B. Hoffman
中科院分区：
文献类型：
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作者：
Zhuo-wei Hu;Xiao-you Shi;R. Lin;Jin Chen;B. Hoffman
许多 G 蛋白偶联受体的信号传导途径与受体酪氨酸激酶的信号传导途径重叠。我们之前发现α1-肾上腺素能受体刺激人血管平滑肌细胞的DNA合成和细胞增殖；这些作用可被酪氨酸蛋白激酶 (TPK) 抑制剂金雀异黄素和丝裂原激活蛋白激酶 (MAPK) 拮抗剂 2-氨基嘌呤减弱。实验旨在确定 α1 受体的激活是否直接刺激人类血管平滑肌细胞中的 TPK 和 MAPK。去甲肾上腺素刺激多种蛋白质的时间和浓度依赖性酪氨酸磷酸化，包括 p52-、75-、85-、120-和 145-kDa 蛋白质。在自磷酸化测定和肽底物的作用下，抗磷酸酪氨酸抗体沉淀的蛋白质都证明了 TPK 活性增加。去甲肾上腺素的这些作用被α1受体拮抗剂完全阻断。一种膜渗透性 Ca2+ 螯合剂 [1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸四（乙酰氧基甲基）酯]，完全阻断去甲肾上腺素对酪氨酸蛋白磷酸化的刺激，表明细胞内 Ca2+ 在 α1 受体刺激酪氨酸蛋白磷酸化中起着关键作用。在酪氨酸磷酸化蛋白中，结果表明其中两个是 PLCγ1 和接头蛋白 Shc。此外，由于 p42/44ERK1/2 磷酸化增加，α1 受体刺激导致 MAPK 活性随时间增加。 TPK 抑制剂和细胞内 Ca2+ 螯合剂 [1,2-双(邻氨基苯氧基)乙烷-N,N,N',N'-四乙酸四(乙酰氧基甲基)酯]也减弱了 α1 受体介导的 MAPK 激活。这些结果表明酪氨酸蛋白和细胞内 Ca2+ 的磷酸化在 α1 受体刺激的 MAPK 信号通路中发挥着关键作用，可能有助于增加 DNA 合成和细胞增殖。
Signaling pathways of many G protein-coupled receptors overlap with those of receptor tyrosine kinases. We have found previously that α1-adrenergic receptors stimulate DNA synthesis and cell proliferation in human vascular smooth muscle cells; these effects were attenuated by the tyrosine protein kinase (TPK) inhibitor genistein and the mitogen-activated protein kinase (MAPK) antagonist 2-aminopurine. Experiments were designed to determine if activation of α1 receptors directly stimulated TPKs and MAPKs in human vascular smooth muscle cells. Norepinephrine stimulated time- and concentration-dependent tyrosine phosphorylation of multiple proteins, including p52-, 75-, 85-, 120-, and 145-kDa proteins. Increased TPK activity was demonstrated in proteins precipitated by an antiphosphotyrosine antibody, both in autophosphorylation assays and with a peptide substrate. These effects of norepinephrine were completely blocked by α1 receptor antagonists. A membrane-permeable Ca2+ chelator [1,2-bis( o -aminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid tetra(acetoxymethyl)ester], completely blocked norepinephrine stimulation of phosphorylation of tyrosine proteins, suggesting that intracellular Ca2+ plays a critical role in α1 receptor stimulation phosphorylation of tyrosine proteins. Of the tyrosine-phosphorylated proteins, the results suggest that two of them are PLCγ1 and adapter protein Shc. Also, α1 receptor stimulation caused a time-dependent increase in MAPK activity due to increased phosphorylation of p42/44ERK1/2. The α1 receptor-mediated activation of MAPK was also attenuated by TPK inhibitors and intracellular Ca2+ chelator [1,2-bis( o -aminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid tetra(acetoxymethyl)ester]. These results suggest that phosphorylation of tyrosine proteins and intracellular Ca2+plays a critical role in α1 receptor-stimulated MAPK signaling pathways, potentially contributing to increased DNA synthesis and cell proliferation.