Regulation of OX₁orexin/hypocretin receptor‐coupling to phospholipase C by Ca²⁺influx

Abstract

Background and purpose: Orexin (OX) receptors induce Ca 2 þ elevations via both receptor-operated Ca 2 þ channels (ROCs) and the "conventional" phospholipase C (PLC)-Ca 2 þ release-store-operated Ca 2 þ channel (SOC) pathways. In this study we assessed the ability of these different Ca 2 þ influx pathways to amplify OX 1 receptor signalling to PLC in response to stimulation with the physiological ligand orexin-A. Experimental approach: PLC activity was assessed in CHO cells stably expressing human OX 1 receptors. Key results: Inhibition of total Ca 2 þ influx by reduction of the extracellular [Ca 2 þ ] to 1 mM effectively inhibited the receptor-stimulated PLC activity at low orexin-A concentrations (by 93% at 1 nM), and this effect was gradually reduced by higher orexin-A concentrations. A similar but weaker inhibitory effect (84% at 1 nM) was obtained on depolarization to B0 mV, which disrupts most of the driving force for Ca 2 þ entry. The inhibitor of the OX 1 receptor-activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca 2 þ ] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg 2 þ , and the SOC inhibitors, dextromethorphan, propoxy)-4-methoxyphenethyl]-1H-imidazole HCl) and 2-APB (2-aminoethoxydiphenyl borate), inhibited PLC activity at low concentrations of orexin-A, but were not as effective as TEA. Conclusions and implications: Both ROCs and SOCs markedly amplify the OX 1 receptor-induced PLC response, but ROCs are more central for this response. These data indicate the crucial role of ROCs in orexin receptor signalling.

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

“…2E), in agreement with Ca 2ϩ activation of cPLA 2 (reviewed in Ghosh et al, 2006). Although Ca 2ϩ level elevation should also stimulate DAGL (Bisogno et al, 2003), Ca 2ϩ level elevation (at least when triggered by thapsigargin or ionomycin) is a poor stimulant of PLD and an even poorer stimulant of PLC in CHO cells (Lund et al, 2000;Johansson et al, 2007;Jä ntti et al, 2012); therefore, in the absence of DAG production, there was no DAGL activity.…”

“…cPLA 2 but Not DAGL Activity Is Pivotal for OX 1 Receptor Activation of Receptor-Operated Ca 2؉ Influx. We showed previously that MAFP could strongly inhibit OX 1 receptor-operated Ca 2ϩ influx (Turunen et al, 2010a), which is central for orexin receptor-mediated Ca 2ϩ level increases because it also drives PLC activity at low orexin concentrations (Lund et al, 2000;Johansson et al, 2007). Because MAFP is a potent inhibitor of both the cPLA 2 and DAGL pathways, we tested selective inhibitors of these pathways, i.e., pyrrophenone and THL, in Ca 2ϩ imaging assays.…”

Implicación de los canales L de Ca2+ en el efecto de la orexina A sobre la entrada de Ca2+ extracelular en neuronas de la corteza prefrontal de rata

“…3B). Some orexins-mediated actions in the nervous system have also been associated with an increase in PLC activity (8,10,12,13,20,22,27,33,42 Ca 2+ releasing from intracellular store involved in such elevation of [Ca 2+ ] i in PFC neurons. In spite of pretreating these neurons with an endoplasmic reticulum Ca 2+ -ATPase inhibitor thapsigargin (2 μM) for at least 1 h, the increase in [Ca 2+ ] i evoked by 1 μM orexin A persisted during the experiment (Fig.…”

“…The contribution of PLC and PKC to the orexins-evoked [Ca 2+ ] i increase has been extensively investigated in other neurons and cells (1,8,10,13,14,20,22,27,33,42). In particular previous studies strongly support the idea that the primary response upon stimulation of the OX1R is the activation of a Ca 2+ influx pathway, which triggers the PLC signaling pathway (13,20). Similarly, orexin A evokes [Ca 2+ ] i signaling in the neuropeptide Y neurons from the arcuate nucleus via OX1R-PLC-PKC and IP 3 pathways (22).…”

Autocrine Endocannabinoid Signaling through CB1Receptors Potentiates OX1Orexin Receptor Signaling

“…Whether this is due to differences in complexing with CB 1 receptors or efficacy of 2-AG production remains unknown. However, it would be tempting to speculate that the coupling of OX 1 receptors to receptor-activated Ca 21 influx may produce stronger DAGL stimulation than that obtained with muscarinic receptors (Magga et al, 2006;Johansson et al, 2007).…”