The Orexin OX1 Receptor Activates a Novel Ca2+ Influx Pathway Necessary for Coupling to Phospholipase C

Abstract

Ca2؉ elevations in Chinese hamster ovary cells stably expressing OX 1 receptors were measured using fluorescent Ca 2؉ indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca 2؉ elevations with an EC 50 around 1 nM. When the extracellular [Ca 2؉ ] was reduced to a submicromolar concentration, the EC 50 was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mM external Ca 2؉ was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.The recently described hypothalamic peptides called orexins (1) or hypocretins (2) mediate their effects through G proteincoupled receptors called OX 1 and OX 2 receptors (1). The peptides and their receptors are widespread in the hypothalamus, cortex, and brainstem (2-5). The orexin/hypocretin peptides are encoded by a single mRNA giving rise to a 33-residue orexin-A peptide containing disulfide bridges and a linear 28-residue orexin-B (1). Orexin-A has a 10 -100-fold higher affinity and potency for OX 1 receptor as compared with orexin-B, whereas no preference is displayed by the OX 2 receptor (1). The orexins cause robust increases in intracellular Ca 2ϩ both in neurons cultured from rat medial and lateral hypothalamus (6) and spinal cord (7), and when studied using recombinant receptors (1). This has led to the suggestion that the receptors are coupled to the G q family G proteins. Interestingly, the response in neurons is partially dependent on extracellular Ca 2ϩ , which may suggest that the receptors are connected to a Ca 2ϩ influx pathway in neurons (6). Several different pathways for receptor-stimulated Ca 2ϩ entry have been suggested based on functional studies with other G protein-coupled receptors. Suggested pathways include store-operated Ca 2ϩ channels, second messenger-operated channels, as well as Ca 2ϩ -activated Ca 2ϩ channels (reviewed in Refs. 8 and 9). The aim of this study was to examine in detail the Ca 2ϩ mobilizing actions of orexins on recombinant OX 1 receptors expressed in CHO 1 -K1 cells. The results reveal the presence of a novel amplification mechanism at the level of phospholipase C that is dependent on activation of Ca 2ϩ influx pathway upstream of phospholipase C. EXPERIMENTAL PROCEDURESCell Cultures-To prepare the CHO-hOX 1 -C1 cells used in this study CHO-K1 cells were transfected with a bicistronic vector containing the coding sequence of human OX 1 receptor as described previously for chemokine receptors (10). Neomycin resistant clones were then isolated by limited dilution. They were grown in nutrient mixture (Ham's F-12) medium (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 100 units/ml penicillin G (Sigma), 80 units/ml streptomycin (Sigma), 400 g/ml Geneticin (G418; Life Technologies, Inc.) and 10% (v/v) fetal calf serum (Life Te...

The Orexin OX1Receptor Regulates Ca2+Entry via Diacylglycerol-Activated Channels in Differentiated Neuroblastoma Cells

“…At low agonist concentrations, a measurable Ca 2ϩ release from intracellular stores was essentially absent. This is in agreement with previous studies van den Pol, 1999;Lund et al, 2000;Uramura et al, 2001;Holmqvist et al, 2002;Willie et al, 2003;Kohlmeier et al, 2004) and indicates Ca 2ϩ entry as a primary OX 1 R response. In neuronal cells, a Ca 2ϩ elevation in response to G-protein-coupled receptors could be the consequence of several different mechanisms, including activation of VGCC, reversal of electrogenic Na ϩ /Ca 2ϩ exchange, or phospholipase C-linked Ca 2ϩ store discharge with a consequent activation of storeoperated channels.…”

“…OX 1 R-activated Ca 2ϩ entry was also apparent in the presence of 10 M KB-R7943, a concentration at which this compound should selectively block the reverse mode of Na ϩ / Ca 2ϩ exchange (Iwamoto et al, 1996). Ca 2ϩ entry has been shown previously to be separated from release-induced Ca 2ϩ entry at low agonist concentrations in CHO cells (Lund et al, 2000;Larsson et al, 2005). High Mg 2ϩ concentrations or dextromethorphan inhibited the OX 1 R-mediated Ca 2ϩ entry in IMR-32 cells, whereas there was no or only a weak effect on thapsigargin-stimulated Ca 2ϩ entry.…”

“…[Ca 2ϩ ] i measurements combined with patch-clamp recordings have demonstrated that the OX 1 R activates a pathway for Ca 2ϩ entry, which closely follows an inward current and depolarization, in Chinese hamster ovary (CHO) cells (Lund et al, 2000;Larsson et al, 2005). This Ca 2ϩ entry pathway is well separated from store release at subnanomolar to low nanomolar concentrations of orexin-A.…”

Role of Calcium in Neurotensin-Evoked Enhancement in Firing in Mesencephalic Dopamine Neurons

“…This is in agreement with previous work showing that heparin or BAPTA blocks NTevoked cationic currents in acutely isolated DAergic neurons (Wu et al, 1995). The Ca 2ϩ dependency of this excitatory effect and of cationic channel activation could result from the partial Ca 2ϩ dependency of PLC (Billah et al, 1980;Lund et al, 2000). Calcium could also be required because of the involvement of Ca 2ϩ -activated nonselective cationic conductances.…”

Central functions of the orexinergic system

“…Fig. 1) [11,17] . on the other hand, besides the G q pathway, OX2Rs may also be independently coupled to other signal transduction pathways.…”