Wang X D, Kiang J G, Atwa M A, Smallridge R C
Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
J Investig Med. 1996 Dec;44(9):566-74.
FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of alpha-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2 (PLA2).
Norepinephrine (NE, 10 mumol/L) was used as an alpha-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo-1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits.
Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-alpha, -beta I, -beta II, -gamma, delta, -epsilon, -zeta, and -eta isoforms were identified. Norepinephrine induced translocation of PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon isoforms but not -zeta and -eta from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-alpha.
These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in alpha-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-alpha translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-alpha, to regulate AA generation is an intriguing question that remains to be clarified.
FRTL-5甲状腺细胞是一种广泛用于研究甲状腺功能的细胞系。α-1肾上腺素能受体的激活可刺激该细胞系中花生四烯酸（AA）的释放以及胞质Ca2+浓度的升高。已知胞质Ca2+和花生四烯酸是调节多种甲状腺功能的重要第二信使。这些信使的产生主要受两种不同类型的磷脂酶调节，即磷脂酶C（PLC）和磷脂酶A2（PLA2）。
使用去甲肾上腺素（NE，10 μmol/L）作为α-1肾上腺素能激活剂，采用荧光染料indo-1测定胞质游离Ca2+浓度（[Ca2+]i）。测量花生四烯酸释放以作为PLA2激活的指标，并使用商业试剂盒进行蛋白激酶C（PKC）活性测定和亚型鉴定。
去甲肾上腺素增加了[Ca2+]i和AA释放。通过去除细胞外Ca2+或使用Ca2+通道阻滞剂NiCl2或CoCl2来阻止NE诱导的胞质Ca2+内流，可完全抑制AA的生成。Ca2+螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）抑制NE诱导的[Ca2+]i升高，也显著抑制了NE诱导的AA释放。PKC抑制剂（H-7或星形孢菌素）抑制PKC活性或佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）或百里酰胺毒素（TX）长时间处理诱导的下调均显著阻断了NE诱导的AA释放，这表明PKC参与介导NE诱导的AA释放。蛋白激酶C活性测定表明，NE在5分钟内诱导了PKC的激活。为了进一步阐明PKC或Ca2+在调节AA释放中的作用，我们使用针对8种不同蛋白激酶C亚型的特异性抗体通过免疫印迹鉴定了PKC亚型。鉴定出了PKC-α、-βI、-βII、-γ、-δ、-ε、-ζ和-η亚型。去甲肾上腺素诱导PKC-α、-βI、-βII、-γ、-δ和-ε亚型从胞质向膜的转位，但不诱导-ζ和-η亚型的转位。细胞内Ca2+的螯合、Ca2+内流的阻止或百里酰胺毒素（TX）的长时间处理完全阻断了NE诱导的PKC-α转位。
这些结果与从AA实验获得的数据一起表明，PKC在α-1肾上腺素能受体介导的PLA2激活和随后的AA释放中起关键作用。细胞外Ca2+内流是PKC-α转位和AA释放的先决条件。Ca2+是直接作用于PLA2还是通过PKC-α来调节AA生成，这是一个有待阐明的有趣问题。
