Department of Basic Veterinary Sciences, Biochemistry, University of Helsinki, Helsinki, Finland.
Br J Pharmacol. 2010 Jan;159(1):212-21. doi: 10.1111/j.1476-5381.2009.00535.x. Epub 2009 Dec 4.
We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX(1) orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A(2) (PLA(2)) signalling system is also involved in orexin receptor signalling.
Recombinant Chinese hamster ovary-K1 cells, expressing human OX(1) receptors, were used as a model system. Arachidonic acid (AA) release was measured from (3)H-AA-labelled cells. Ca(2+) signalling was assessed using single-cell imaging.
Orexins strongly stimulated [(3)H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC(50) = 8.90 and 8.38 respectively). The concentration-response curves appeared biphasic. The release was fully inhibited by the potent cPLA(2) and iPLA(2) inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA(2) inhibitors, R- and S-bromoenol lactone, caused only a partial inhibition. The response was also fully dependent on Ca(2+) influx, and the inhibitor studies suggested involvement of the receptor-operated influx pathway. The receptor-operated pathway, on the other hand, was partially dependent on PLA(2) activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA(2) activation at low orexin concentrations.
Activation of OX(1) orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA(2) species, and this response may be important for the receptor-operated Ca(2+) influx. The response coincided with other high-potency lipid messenger responses, and may interact with these signals.
我们之前已经表明，磷脂酶 D 和 C 产生的脂质介质在 OX(1)食欲素受体信号中具有高效能，并且可能介导了一些对食欲素的生理反应。在这项研究中，我们研究了普遍存在的磷脂酶 A(2)（PLA(2)）信号系统是否也参与了食欲素受体信号。
使用表达人 OX(1)受体的重组中国仓鼠卵巢-K1 细胞作为模型系统。从（3）H-AA 标记的细胞中测量花生四烯酸（AA）释放。使用单细胞成像评估 Ca(2+)信号。
食欲素强烈刺激[(3)H]-AA 释放（最大 4.4 倍）。食欲素-A 比食欲素-B 略为有效（pEC(50)分别为 8.90 和 8.38）。浓度-反应曲线呈双相性。强效 cPLA(2)和 iPLA(2)抑制剂甲基花生四烯酰氟磷酸酯完全抑制释放，而 iPLA(2)抑制剂 R-和 S-溴烯醇内酯仅引起部分抑制。该反应也完全依赖于 Ca(2+)内流，抑制剂研究表明涉及受体操作的内流途径。另一方面，受体操作途径部分依赖于 PLA(2)活性。在低食欲素浓度下，细胞外信号调节激酶而不是蛋白激酶 C 参与了 PLA(2)的激活。
激活 OX(1)食欲素受体诱导了强烈的、高效能的 AA 释放，可能通过多种 PLA(2)物种，并且该反应可能对受体操作的 Ca(2+)内流很重要。该反应与其他高效能脂质信使反应同时发生，并且可能与这些信号相互作用。
