Li S, Huang F L, Feng Q, Liu J, Fan S X, McKenna T M
Resuscitative Medicine Program, Naval Medical Research Institute, Bethesda, Maryland, USA.
J Cell Physiol. 1998 Aug;176(2):402-11. doi: 10.1002/(SICI)1097-4652(199808)176:2<402::AID-JCP19>3.0.CO;2-4.
Our previous studies showed that lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis in cardiovascular tissues is attenuated by protein kinase C (PKC) inhibitors. In the current study, we identify a specific PKC isotype involved in the LPS signal transduction pathway that leads to NO formation in rat vascular smooth muscle cells (VSMC). VSMC were transfected with a mammalian expression vector containing a full length PKCalpha cDNA insert, and a stable transfectant overexpressing PKCalpha was obtained as evidenced by increased expression of PKCalpha mRNA and protein. In response to 100 ng/ml LPS stimulation, the PKCalpha transfectants showed a 1.8-fold increase in PKC activity at 30 min and a twofold increase in NO production over 24 hr compared with cells transfected with control plasmids. The LPS-stimulated increase in NO synthesis in PKCalpha transfectants was blocked by the specific PKCalpha inhibitor Gö 6976. After 6 hr LPS treatment, PKCalpha-transfected and control cells showed equivalent increases in mRNA and protein for the inducible NO synthase. NO synthase activity of the cell extracts assayed in the presence of excess substrate and cofactors was not significantly different between PKCalpha-transfected and control cells after LPS stimulation. However, mRNA levels for GTP cyclohydrolase I, a key enzyme in (6R)-tetrahydro-L-biopterin synthesis, and cationic amino acid transporter-2, involved in L-arginine transport, was enhanced in cells overexpressing PKCalpha compared with control cells. These results suggest that PKCalpha plays an important role in LPS-induced NO formation and that a significant portion of this effect may be by means of enhanced substrate availability to the inducible nitric oxide synthase enzyme.
我们之前的研究表明，蛋白激酶C（PKC）抑制剂可减弱脂多糖（LPS）诱导的心血管组织中一氧化氮（NO）的合成。在本研究中，我们确定了参与LPS信号转导途径的一种特定PKC亚型，该途径导致大鼠血管平滑肌细胞（VSMC）中NO的形成。用含有全长PKCα cDNA插入片段的哺乳动物表达载体转染VSMC，并获得了稳定转染且过表达PKCα的细胞株，PKCα mRNA和蛋白表达的增加证明了这一点。与用对照质粒转染的细胞相比，在100 ng/ml LPS刺激下，PKCα转染细胞在30分钟时PKC活性增加了1.8倍，在24小时内NO生成增加了两倍。PKCα转染细胞中LPS刺激的NO合成增加被特异性PKCα抑制剂Gö 6976阻断。LPS处理6小时后，PKCα转染细胞和对照细胞中诱导型NO合酶的mRNA和蛋白增加量相当。在LPS刺激后，在过量底物和辅因子存在下测定的细胞提取物的NO合酶活性在PKCα转染细胞和对照细胞之间没有显著差异。然而，与对照细胞相比，过表达PKCα的细胞中（6R）-四氢-L-生物蝶呤合成中的关键酶GTP环水解酶I以及参与L-精氨酸转运的阳离子氨基酸转运体-2的mRNA水平有所提高。这些结果表明，PKCα在LPS诱导的NO形成中起重要作用，并且这种作用的很大一部分可能是通过增强诱导型一氧化氮合酶的底物可用性来实现的。
