蛋白激酶C α在内皮素-1刺激培养猫虹膜括约肌平滑肌细胞胞质磷脂酶A2和花生四烯酸释放中的作用

Shahid Husain, Ata A Abdel-Latif
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引用次数: 52

摘要

我们研究了蛋白激酶C (PKC)异构体在内皮素-1 (ET-1)诱导的猫虹膜括约肌平滑肌(CISM)细胞花生四烯酸(AA)释放中的作用和机制。ET-1以浓度(EC50=8 nM)和时间依赖性(t1/2=1.2 min)增加AA释放。胞质磷脂酶A2 (cPLA2)而非磷脂酶C (PLC)参与了受刺激细胞中AA的释放。结果表明,et -1诱导的AA释放可被PLA2抑制剂AACOCF3、quinacrine和manoalide抑制,而PLC抑制剂U-73122和二酰基甘油脂肪酶抑制剂RHC-80267抑制。PKC在et -1诱导的AA释放中的作用得到以下研究结果的支持:苯酚酯(PDBu)可使AA释放增加96%,PDBu长时间处理细胞可选择性下调PKCα并完全抑制et -1诱导的AA释放,staurosporine或PKC抑制剂ro31 -8220预处理细胞可阻断et -1诱导的AA释放。Gö-6976是一种特异性抑制PKCα和β的化合物,以浓度依赖的方式阻断et -1诱导的AA释放,IC50值为8 nM。胸腺毒素(0.1 μM)是PKCα、β和γ的特异性激活剂,诱导AA释放增加150%。ET-1处理细胞可引起PKCα从细胞质向颗粒部分的明显易位,而PKCβ则无明显易位。这些结果表明PKCα在et -1诱导的AA释放中起关键作用。免疫化学分析显示CISM细胞中存在cPLA2、p42mapk和p44mapk。所提供的数据与PKCα在et -1刺激的CISM细胞中cPLA2激活和AA释放中的作用一致,但与p42/p44丝裂原活化蛋白激酶(MAPK)的作用不一致,因为:(1) PKC抑制剂RO 31-8220抑制et -1诱导的AA释放、cPLA2磷酸化和cPLA2活性,但对p42/p44 MAPK激活无抑制作用;(2)酪氨酸激酶抑制剂染料木素抑制et -1刺激的MAPK活性,但对et -1刺激的细胞中AA释放无抑制作用。我们得出结论,在CISM细胞中,ET-1激活PKCα, PKCα激活cPLA2, cPLA2释放AA用于前列腺素合成。
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Role of protein kinase C α in endothelin-1 stimulation of cytosolic phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells

We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC50=8 nM) and time-dependent (t1/2=1.2 min) manner. Cytosolic phospholipase A2 (cPLA2), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF3, quinacrine and manoalide, PLA2 inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCα and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. Gö-6976, a compound that inhibits PKCα and β specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 μM), a specific activator of PKCα, β, and γ induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCα, but not PKCβ, from cytosol to the particulate fraction. These results suggest that PKCα plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA2, p42mapk and p44mapk in the CISM cells. The data presented are consistent with a role for PKCα, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA2 phosphorylation and cPLA2 activity, but had no inhibitory effect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhibitor, inhibited ET-1-stimulated MAPK activity but had no inhibitory effect on AA release in the ET-1-stimulated cells. We conclude that in CISM cells, ET-1 activates PKCα, which activates cPLA2, which liberates AA for prostaglandin synthesis.

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