Arachidonic acid release mediated by OX₁ orexin receptors

Abstract

Background and purpose:We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX1 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 A2 (PLA2) signalling system is also involved in orexin receptor signalling. Experimental approach: Recombinant Chinese hamster ovary-K1 cells, expressing human OX1 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. Key results: Orexins strongly stimulated [ 3 H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC50 = 8.90 and 8.38 respectively). The concentration-response curves appeared biphasic. The release was fully inhibited by the potent cPLA2 and iPLA2 inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA2 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 PLA2 activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA2 activation at low orexin concentrations. Conclusions and implications: Activation of OX1 orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA2 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.

Autocrine Endocannabinoid Signaling through CB1Receptors Potentiates OX1Orexin Receptor Signaling

“…Human orexin-A and -B were from NeoMPS (Strasbourg, France), N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M) was from RBI (Natick, MA), and Cell Culture. CHO-hOX 1 cells, expressing human OX 1 receptors, have been described previously, as have their culture conditions (see, e.g., Turunen et al, 2010Turunen et al, , 2012. CHO-hCB 1 cells, expressing human CB 1a receptor (Grimsey et al, 2010), were a kind gift from Dr. Michelle Glass (University of Auckland, Auckland, New Zealand) via Drs.…”

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

“…CHO-hOX 1 cells expressing ϳ500 fmol/mg of protein levels of high-affinity human OX 1 receptors (as determined from high-affinity 125 I-orexin-A binding (P. M. Turunen and J. P. Kukkonen, unpublished data) were described previously, as were their culture conditions (Lund et al, 2000;Turunen et al, 2010a). CHO-hCB 1 cells expressing human CB 1a receptors (Grimsey et al, 2010) were a kind gift from Dr. Michelle Glass (University of Auckland, Auckland, New Zealand) via Drs.…”

“…The full sensitivity of the response to the reputed phospholipase A 2 (PLA 2 ) inhibitor methyl arachidonyl fluorophosphonate (MAFP), in addition to partial sensitivity to inhibitors of other enzymes and intracellular signal pathways (Turunen et al, 2010a), suggests that this effect is mediated by PLA 2 enzymes. Although MAFP is very commonly used as a PLA 2 inhibitor, it is an analog of AA and is able to inhibit a number of serine hydrolases that hydrolyze ester-or amide-bound AA (De Petrocellis et al, 1997;Deutsch et al, 1997;Dinh et al, 2002;Savinainen et al, 2010).…”

“…We showed previously that OX 1 orexin receptor activation strongly stimulates 3 H overflow from [ 3 H]AA-labeled cells (Turunen et al, 2010a). The full sensitivity of the response to the reputed phospholipase A 2 (PLA 2 ) inhibitor methyl arachidonyl fluorophosphonate (MAFP), in addition to partial sensitivity to inhibitors of other enzymes and intracellular signal pathways (Turunen et al, 2010a), suggests that this effect is mediated by PLA 2 enzymes.…”

“…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).…”