Larsson Kim P, Peltonen Hanna M, Bart Genevieve, Louhivuori Lauri M, Penttonen Annika, Antikainen Miia, Kukkonen Jyrki P, Akerman Karl E O
A. I. Virtanen Institute for Molecular Sciences, Department of Neurobiology, Laboratory of Cell Biology, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland.
J Biol Chem. 2005 Jan 21;280(3):1771-81. doi: 10.1074/jbc.M406073200. Epub 2004 Nov 10.
The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX(1) and OX(2) receptors. Our previous results have suggested that low (subnanomolar) concentrations of orexin-A activate Ca(2+) entry, whereas higher concentrations activate phospholipase C, Ca(2+) release, and capacitative Ca(2+) entry. As shown here, the Ca(2+) response to subnanomolar orexin-A concentrations was blocked by activation of protein kinase C by using different approaches (12-O-tetradecanoylphorbol acetate, dioctanoylglycerol, and diacylglycerol kinase inhibition) and protein phosphatase inhibition by calyculin A. The Ca(2+) response to subnanomolar orexin-A concentrations was also blocked by Mg(2+), dextromethorphan, and tetraethylammonium. These treatments neither affected the response to high concentrations of orexin-A nor the thapsigargin-stimulated capacitative entry. The capacitative entry was instead strongly suppressed by SKF96365. An inward membrane current activated by subnanomolar concentrations of orexin-A and the currents activated upon transient expression of trpc3 channels were also sensitive to Mg(2+), dextromethorphan, and tetraethylammonium. Responses to subnanomolar concentrations of orexin-A (Ca(2+) elevation, inward current, and membrane depolarization) were voltage-dependent with a loss of the response around -15 mV. By using reverse transcription-PCR, mRNA for the trpc1-4 channel isoforms were detected in the CHO-hOX1-C1 cells. The expression of truncated TRPC channel isoforms, in particular trpc1 and trpc3, reduced the response to subnanomolar concentrations of orexin-A but did not affect the response to higher concentrations of orexin-A. The results suggest that activation of the OX(1) receptor leads to opening of a Ca(2+)-permeable channel, involving trpc1 and -3, which is controlled by protein kinase C.
食欲肽是肽类递质/激素，可通过G蛋白偶联的OX(1)和OX(2)受体在多种类型的细胞中发挥刺激作用。我们之前的结果表明，低（亚纳摩尔）浓度的食欲肽A可激活Ca(2+)内流，而较高浓度则可激活磷脂酶C、Ca(2+)释放和容量性Ca(2+)内流。如下所示，通过不同方法（12-O-十四酰佛波醇-13-乙酸酯、二辛酰甘油和二酰甘油激酶抑制）激活蛋白激酶C以及用花萼海绵诱癌素A抑制蛋白磷酸酶，均可阻断对亚纳摩尔浓度食欲肽A的Ca(2+)反应。Mg(2+)、右美沙芬和四乙铵也可阻断对亚纳摩尔浓度食欲肽A的Ca(2+)反应。这些处理既不影响对高浓度食欲肽A的反应，也不影响毒胡萝卜素刺激的容量性内流。相反，SKF96365可强烈抑制容量性内流。亚纳摩尔浓度的食欲肽A激活的内向膜电流以及瞬时表达trpc3通道时激活的电流，对Mg(2+)、右美沙芬和四乙铵也敏感。对亚纳摩尔浓度食欲肽A的反应（Ca(2+)升高、内向电流和膜去极化）具有电压依赖性，在约-15 mV时反应消失。通过逆转录聚合酶链反应，在CHO-hOX1-C1细胞中检测到trpc1-4通道亚型的mRNA。截短的TRPC通道亚型，特别是trpc1和trpc3的表达，降低了对亚纳摩尔浓度食欲肽A的反应，但不影响对更高浓度食欲肽A的反应。结果表明，OX(1)受体的激活导致一个Ca(2+)可渗透通道的开放，该通道涉及trpc1和trpc3，受蛋白激酶C控制。
