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

Abstract

We showed previously that OX 1 orexin receptor stimulation produced a strong 3 H overflow response from [ 3 H]arachidonic acid (AA)-labeled cells. Here we addressed this issue with a novel set of tools and methods, to distinguish the enzyme pathways responsible for this response. CHO-K1 cells heterologously expressing human OX 1 receptors were used as a model system. By using selective pharmacological inhibitors, we showed that, in orexin-A-stimulated cells, the AA-derived radioactivity was released as two distinct components, i.e., free AA and the endocannabinoid 2-arachidonoyl glycerol (2-AG). Two orexin-activated enzymatic cascades are responsible for this response: cytosolic phospholipase A 2 (cPLA 2 ) and diacylglycerol lipase; the former cascade is responsible for part of the AA release, whereas the latter is responsible for all of the 2-AG release and part of the AA release. Essentially only diacylglycerol released by phospholipase C but not by phospholipase D was implicated as a substrate for 2-AG production, although both phospholipases were strongly activated. The 2-AG released acted as a potent paracrine messenger through cannabinoid CB 1 receptors in an artificial cell-cell communication assay that was developed. The cPLA 2 cascade, in contrast, was involved in the activation of orexin receptor-operated Ca 2ϩ influx. 2-AG was also released upon OX 1 receptor stimulation in recombinant HEK-293 and neuro-2a cells. The results directly show, for the first time, that orexin receptors are able to generate potent endocannabinoid signals in addition to arachidonic acid signals, which may explain the proposed orexincannabinoid interactions (e.g., in neurons).

Autocrine Endocannabinoid Signaling through CB1Receptors Potentiates OX1Orexin Receptor Signaling

“…We have recently shown that the endocannabinoid 2-AG is strongly produced and released in OX 1 receptor signaling, and that this 2-AG is able to act as a potent paracrine messenger via CB 1 receptors (Turunen et al, 2012). This, together with the studies in the CNS, suggests that 2-AG production is an important signal in orexin-mediated regulation of synaptic transmission.…”

“…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. Jarmo Laitinen and Juha Savinainen (University of Eastern Finland, Kuopio, Finland); the culture conditions for these have been described (Turunen et al, 2012). For Western blotting (WB) experiments, the cells were cultured on six-well plates (9.6-cm 2 well bottom area; Greiner Bio-One GmbH, Frickenhausen, Germany); for the luciferase assays, on either 24-or 96-well plates (Greiner Bio-One GmbH); and for the receptor binding experiments, on cell culture dishes (56-cm 2 bottom area) or 48-well plates (Greiner Bio-One GmbH).…”

“…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.…”

New horizons on the role of cannabinoid CB1 receptors in palatable food intake, obesity and related dysmetabolism

“…25 In an in vitro study, it was demonstrated that the coexpression of CB1 and OX1 receptors results in a higher potentiation of ERK signalling, possibly because of heterodimerization/oligomerization of these two receptors. 26 Recently, however, it was also shown that 2-AG produced and released through the activation of OX1 receptor acts as both a paracrine and autocrine messenger via CB1 receptors, 23,27 thus possibly explaining the above-mentioned findings in the rat dorsal raphe nucleus and periaqueductal gray, as well as the potentiation of ERK phoshorylation that follows OX1/CB1 receptor coexpression. These data suggest the involvement of 2-AG and CB1 receptors not only in orexin-A release but also in orexin-mediated regulation of synaptic transmission.…”

“…They have a pivotal role in the regulation of sleep/wakefulness, pain, reward and appetite, and a recent study demonstrates their interaction with the ECS. 23 Interestingly, in the rat dorsal raphe nucleus, orexin-Bmediated inhibition of glutamate release can be due to 2-AG release and subsequent activation of CB1 receptors on glutamatergic presynaptic terminals. 24 In the rat periaqueductal gray, orexin-A-induced analgesic responses rely on a similar 2-AG/CB1-mediated 'retrograde' inhibition of Y-aminobutyric acid (GABA) release.…”