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J. Biol. Chem., Vol. 280, Issue 8, 6570-6579, February 25, 2005

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OX₁ Orexin Receptors Couple to Adenylyl Cyclase Regulation via Multiple Mechanisms^*

Tomas Holmqvist, Lisa Johansson, Marie Östman, Sylwia Ammoun, Karl E. O. Åkerman, and Jyrki P. Kukkonen¶

From the Department of Neuroscience, Unit of Physiology, Uppsala University, BMC, SE-75123 Uppsala, Sweden and the A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Neulaniementie 2, FIN-70210 Kuopio, Finland

In this study, the mechanism of OX₁ orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC₅₀ 1nM) inhibition and a strong (4-fold), low potency (EC₅₀ = 300 nM) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G_i/o proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC₅₀ = 500 nM). cAMP elevation was not caused by Ca²⁺ elevation or by G. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKC, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G_s activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC₅₀ of orexin-A = 10 nM and EC₅₀ of orexin-B = 100 nM), and the response was fully dependent on PKC. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-G_s antibodies. We conclude that OX₁ receptors stimulate adenylyl cyclase via a low potency G_s coupling and a high potency phospholipase C PKC coupling. The former or some exogenous G_s activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX₁ receptors couple to G_s almost as efficiently as the orexin-A-activated receptors, in contrast to Ca²⁺ elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.

Received for publication, July 1, 2004 , and in revised form, November 4, 2004.

^* This work was supported by European Union Contract QLG3-CT-2002-00826, the Åke Wiberg Foundation, the Lars Hierta Foundation, the Göran Gustafsson Foundation, the Novo Nordisk Foundation, the Academy of Finland, and the Sigrid Jusélius Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Neuroscience, Division of Physiology, Uppsala University, BMC, P.O. Box 572, SE-75123 Uppsala, Sweden. Tel.: 46-18-471-4171; Fax: 46-18-50-6357; E-mail: jyrki.kukkonen{at}fysiologi.uu.se.

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