OX₁ orexin/hypocretin receptor activation of phospholipase D

Abstract

BACKGROUND AND PURPOSEOrexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. EXPERIMENTAL APPROACHOrexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX1 orexin receptors using KEY RESULTSOrexin stimulation strongly increased PLD activity -even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration-response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCd. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gai/o nor Gbg seemed to play an important role in the orexin response. CONCLUSIONS AND IMPLICATIONSStimulation of OX1 receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCd but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCd in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCd. Abbreviations bARK1, b-adrenoceptor kinase 1; c-and nPKC, conventional and novel PKC, respectively; GF109203X (bisindolylmaleimide I, Gö6850), 2-(1-[3-dimethylaminopropyl]-1H-indol-3-yl)-3-(1H-indol-3-yl)-maleimide; GGTI-2133, N-([4-(imidazol-4-yl)methylamino]-2- [1-naphthyl]benzoyl)leucine trifluoroacetate salt; Gö6976, 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo(2,3-a)pyrrolo(3,4-c)carbazole-12-propanenitrile; HBM, HEPES-buffered medium; KAC1-1, a peptide cPKC activator; KIC1-1, a peptide cPKC inhibitor; KAD1-1, a peptide PKCd activator; KAE1-1, a peptide PKCe activator, KIE1-1, a peptide PKCe inhibitor; MAFP, methyl arachidonyl fluorophosphonate; PA, phosphatidic acid; pEC50, -logEC50; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP5K, type I

OX1 Orexin/Hypocretin Receptor Signaling through Arachidonic Acid and Endocannabinoid Release

“…1), but the enzymes responsible for this could not be resolved because of the low selectivity of the inhibitors available (Turunen et al, 2010a). We showed that OX 1 receptors also potently activate both phospholipase C (PLC) and phospholipase D (PLD) cascades (Johansson et al, 2008;Jä ntti et al, 2012), producing diacylglycerol (DAG) and phosphatidic acid (PA), respectively. Both of these compounds could act as substrates for AA release, through the DAG lipase (DAGL)-monoacylglycerol lipase (MAGL) and PA-PLA 2 cascades, respectively, and PA can also be hydrolyzed to DAG by PA phosphohydrolase (reviewed in Kukkonen, 2011).…”

“…The average retardation factor values for AA and 2-AG were 0.58 Ϯ 0.01 and 0.67 Ϯ 0.01, respectively. TLC data were quantitated both through imaging plate analysis and through scraping and scintillation counting; the two methods yielded comparable results (Jä ntti et al, 2012). For this reason, only the results from the imaging plate analysis are presented.…”

“…The molecular mechanisms of orexin receptor signaling are very diverse (reviewed in Kukkonen and Åkerman, 2005). Especially prominent seems to be coupling to the production of messengers through phospholipase action (Johansson et al, 2008;Turunen et al, 2010a;Jä ntti et al, 2012).…”

Autocrine Endocannabinoid Signaling through CB1Receptors Potentiates OX1Orexin Receptor Signaling

“…After thawing, cell debris was spun down and the PLC Activity. Total inositol phosphate release was measured essentially as described in Jäntti et al (2012). CHO-hCB 1 cells were transiently transfected with either OX 1 or M 1 cDNA and also the other vectors included in the Elk-1 luciferase assay in the same ratios to make the assays comparable.…”

Orexin receptors: a journey through their discovery to the development of suvorexant, the new sleeping pill

“…8 A recent study on Chinese hamster ovarian cell lines has revealed other signaling mechanisms like activation of phospholipase D and release of arachidonic acid upon activation of OX 1 orexin receptors. 9 Orexins being excitatory neuropeptides, stimulation of their receptors lead to depolarization by the following mechanisms; (i) activation of NSCC (non -selective cation channels), (ii) stimulation of NCX (sodium calcium exchanger) and (iii) inhibition of potassium channels which are active at rest. 10 Although their signaling mechanisms have been elucidated, their physiological significance still remains elusive.…”