hypocretin receptors

Biochimie xxx (2013) 1e8

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Review

Lipid signaling cascades of orexin/hypocretin receptors Jyrki P. Kukkonen* Biochemistry and Cell Biology, Department of Veterinary Biosciences, POB 66, FIN-00014, University of Helsinki, Finland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 March 2013 Accepted 18 June 2013 Available online xxx

Orexins e orexin-A and orexin-B e are neuropeptides with significant role in regulation of fundamental physiological processes such as sleep-wakefulness cycle. Orexins act via G-protein-coupled OX1 and OX2 receptors, which are found, in addition to the central nervous system, also in a number of peripheral organs. Orexin receptors show high degree of signaling promiscuity. One particularly prominent way of signaling for these receptors is via phospholipase cascades, including the phospholipase C, phospholipase D and phospholipase A2 cascades, and also diacylglycerol lipase and phosphoinositide-3-kinase pathways. Most analyses have been performed in recombinant cells; there are indications of some of these cascades in native cells while the significance of other cascades remains to be shown. In this review, I present these pathways, their activation mechanisms and their physiological significance. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Orexin Hypocretin Phospholipase Endocannabinoid Ion fluxes

1. Introduction Orexin/hypocretin receptors (OX1 and OX2 receptors) belong to the G-protein-coupled receptor (GPCR) superfamily. They play a key role in the regulation of many physiological processes, especially in the wakefulness/sleep pattern (reviewed in Refs. [1,2]). Orexin receptors show very diverse signaling originating from multiple G-protein species (and possible other signal transducers) and ranging from ion channel activation/inhibition to hormone release, cell differentiation and even cell death (reviewed in Refs. [2e4]). One striking feature of orexin receptor signaling is its coupling to generation of lipid messengers via phospholipases. This

Abbreviations: 2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; anandamide, N-arachidonoylethanolamine; CNS, central nervous system; cPLA2 and iPLA2, cytosolic (Ca2þ-dependent) and intracellular (Ca2þ-independent) PLA2, respectively; DAG, diacylglycerol; DAGL, DAG lipase; DOG, dioctanoylglycerol; ERK, extracellular signal-regulated kinase; GPCR, G-protein-coupled receptor; GPL, glycerophospholipid; IP3, inositol-1,4,5-trisphosphate; KB-R7943, a NCX inhibitor; lysoGPL, lyso(glycero)phospholipid; lysoPA, lysophosphatidic acid; MAFP, methyl arachidonyl fluorophosphonate; NAPE, N-acyl-phosphatidylethanolamine; NSCC, non-selective cation channel; OX1, orexin 1 receptor; OX2, orexin 2 receptor; PA, phosphatidic acid; PC, phosphatidylcholine; PC-PLC, PC-specific PLC; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide-3-kinase; PI, phosphatidylinositol; PIs, phosphatidylinositols (including differentially phosphorylated species PI, PIP, PIP2 and PIP3); PIP, phosphatidylinositolmonophosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5trisphosphate; PKB, PKC and PKD, protein kinase B, C and D, respectively; PLA1, PLA2, PLB, PLC and PLD, phospholipase A1, A2, B, C and D, respectively; pyrrophenone, a cPLA2a/z inhibitor; TRP (channel), transient receptor potential (channel); U73122, a PLC inhibitor. * Tel.: þ358 9 191 57024; fax: þ358 9 191 57033. E-mail address: jyrki.kukkonen@helsinki.fi.

is not surprising for GPCRs but the rich spectrum of phospholipase activation by orexin receptors may be less usual. Lipids and lipid-derived molecules represent a vast and very diverse group of bioactive compounds. These compounds can have different sources: some are absorbed from the diet, some are mere metabolites of these or the body’s own compounds, and some are actively produced for their physiological role in, e.g. signaling. The most classical cascades producing lipid derivatives for signaling purposes are instigated by reactions catalyzed by phospholipases, which themselves are targeted by particular signal pathways from, e.g. GPCRs. The knowledge of the diversity of these, as well as other, lipid pathways (reviewed in Ref. [5]) and the actions downstream of these, has increased tremendously. At the same time we need to admit that our understanding of the intricacies of the lipid signaling networks is still very limited. In this review, I present the known (and speculated) lipid signaling pathways of orexin receptors and their physiological significance. 2. Overview of orexin receptors signaling We have presented a general overview of orexin receptor signaling in some recent reviews [2e4]. All in all, the data available indicate that orexin receptor signaling is very multifaceted and complex. However, most of the analytic data originate from studies with recombinant cell lines, which makes conclusions of the significance of the findings somewhat unclear. Orexin receptors couple, at least, to the G-protein families of Gi/ o, Gq and Gs, and possibly also to b-arrestin, dynein light chain Tctex-types 1 and 3 and the protein phosphatase SHP-2 (reviewed

0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.06.015

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in Refs. [2,3]). Further downstream of the receptors, responses such as activation of phospholipases (below), modulation of second messenger levels (lipid-derived messengers, cAMP, Ca2þ), activation Ser/Thr and Tyr kinases, activation/potentiation of nonselective cation channels (NSCCs) and voltage-gated Ca2þ channels and inhibition and activation of Kþ channels have been seen. In the long run, effects seen involve activation of transcription factors and resultant plastic changes; even cell death is seen in some cell types (reviewed in Refs. [2e4]). For some responses signal cascades between the receptor and the response measured can be traced, but in many cases the pathways are not properly mapped. Remarkably, it seems that orexin receptors may preferentially utilize distinct signal cascades in each tissue/cell type. The molecular mechanisms behind this are not known, but an obvious guess would suggest that tissue-specific expression of signal transducers and distinct signal complex formation explains the behavior. 2.1. Orexin receptors and calcium Ever since their cloning, orexin receptor were known to strongly couple to Ca2þ elevation in a manner sensitive to the phospholipase C (PLC) inhibitor U73122 [6,7]. This led to the conclusions that the receptors would be of the Gq-coupled type with PLC-driven inositol-1,4,5-phosphate (IP3) generation and Ca2þ release. While the PLC coupling was soon verified by direct measurements [see, e.g. Section 3.3 and (Ref. [8])], also other properties of the Ca2þ elevations were discovered. We were able to show that the primary response to OX1 orexin receptor stimulation was indeed a Ca2þ influx, of the type “receptor-operated” as it did not require release from Ca2þ stores or IP3 [8,9]. In addition, orexin receptors couple to the more regular store-operated Ca2þ influx, which follows from the IP3-dependent Ca2þ store discharge [10]. Most of the investigations have been performed in recombinant Chinese hamster ovary-K1 (CHO) cells but neither the channel nor its activation

mechanism is fully known, although transient receptor potential (TRP) family NSCCs make good candidates. In the central nervous system (CNS) neurons, similar non-selective cation fluxes are seen. For details, see Section 3.4 and [2e4]. 3. Phospholipases Phospholipases by definition are enzymes that hydrolyze (membrane) glycerophospholipids (GPLs). Phospholipases fall in the principal classes of phospholipase A, B, C and D (PLA, PLB, PLC and PLD). Of the interest for this review are the recognized signaling phospholipases PLA2, PLC and PLD (Fig. 1A and B). 3.1. Orexin receptors and phospholipase A2 The name “PLA2” indicates that the enzymes hydrolyze the GPL sn2 ester bond. However, many of the enzymes belonging to this family may show less specificity for the sn2 bond over sn1, and they may thus act as PLA1, PLB or lyso-PLA enzymes (reviewed in Ref. [11]). When PLA2 enzymes hydrolyze the sn2 bond, the products are a free fatty acid and a sn2 lysophospholipid [lysoGPL]. The fatty acid in the sn2 position is often unsaturated (mono or poly); the most classical, though not really the sole product of PLA2 reaction, is arachidonic acid (AA). The family of PLA2 enzymes is the largest among phospholipases, and it is divided in several subfamilies (reviewed in Ref. [11]). While the enzymes within a subfamily are related to each other, the different subfamilies are not necessarily related to each other. The class IV and VI enzymes are usually associated with intracellular signaling. Class IV is known as cPLA2 (“c” for cytosolic and also Ca2þ-dependent). It harbors members IVA-F (a.k.a. cPLA2a, -b, -g, -d, -3 and -z) (reviewed in Refs. [11,12]). The most investigated form is IVA (cPLA2a). This enzyme is activated by Ca2þ and phosphorylation by protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and some other

Fig. 1. Phospholipase signaling and signal pathway interaction. (A) OX1 receptor stimulation-induced phospholipase pathways in CHO cells. (B) Some possible conversions of the lipid messengers. Please observe that the same messengers (e.g. DAG) from different pathways may not be equal (different fatty acid composition). (C) PIP2 and signaling. NCX and some Kþ channels require PIP2 for activity and some NSCCs are inhibited and some are stimulated by PIP2. PIP2 is required by PLD and it also stimulates cPLA2a. PLC hydrolyses PIP2 and thus reduces PIP2 levels while PLD signaling increases PIP2 synthesis. FFA, free fatty acid. Responses downstream of PIP2 metabolism are not shown (i.e. PLC and PI3K cascades).

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kinases, and it is also positively regulated by phosphatidylnositol4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIP2 and PIP3, respectively). Regulation of some other members of the family is significantly less well known (reviewed in Refs. [11,12]). cPLA2a is also known for its rather high selectivity for sn2 AA, making it a central enzyme for AA release for eicosanoid production. Class VI is also known as iPLA2, “i” referring to intracellular but later also to Ca2þ-independent. There are also six genes for class VI, VIA-F, with several splice variants of the gene for VIA (a.k.a. iPLA2/iPLA2b) (reviewed in Refs. [11,13,14]). The enzymes do not require Ca2þ for activity but may instead be inhibited by Ca2þ/ calmodulin (reviewed in Refs. [11,13,14]). Activation of class VI enzymes may be induced by e.g. ATP binding, proteolytic cleavage or oligomerization; whether PKC is involved, is unclear (reviewed in Refs. [11,13,14]). c- and iPLA2 species show variable specificity for different sn2 fatty acids and GPL headgroups. However, we should remember that phospholipase specificities are determined under test-tube conditions and not with the most of the physiological trigger stimuli. Investigations for different PLA2 enzymes are significantly hampered by multiple species and lack of inhibitors that would show selectivity for different families, let alone specific family members. Novel inhibitors for the class IV/cPLA2 (reviewed in Ref. [11]) are a much welcomed exception. PLA2 activity has been investigated for orexin receptors only in recombinant cells. Direct demonstration of PLA2 activity in orexin signaling comes from CHO cells expressing heterologous human OX1 receptors. In these cells, we observed release of 3H-radioactivity from cells labeled with 3H-AA upon stimulation with orexinA or orexin-B (the native orexin ligands) [15,16]. When the cells were labeled with 3H-oleic acid, the release was less potent. In a further study we utilized molecular species separation and novel pharmacological tools. We could show that the radioactivity released after orexin stimulation was partly composed of free AA and in part of 2-arachidonoylglycerol (2-AG), a pivotal endocannabinoid (see Section 3.5 for details) [17]. AA was released apparently via cPLA2 as the response was inhibited by pyrrophenone, an inhibitor of cPLA2a/z (Fig. 1A), and a similar response was triggered by the Ca2þ-elevating compound thapsigargin [17]. The cPLA2 cascade was, furthermore, responsible for supporting the receptor-operated Ca2þ influx (Fig. 2A) as this response could be inhibited by pyrrophenone and a structurally unrelated, lessselective inhibitor of serine hydrolases and proteases, methyl arachidonyl fluorophosphonate (MAFP) [15,17]. The involvement of cPLA2a and not cPLA2z is supported by the fact that the oleic acid label was released in solely DAGL-dependent manner in orexin signaling [17,18].

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In similarly recombinant human OX1-expressing human embryonic kidney (HEK-293) cells, orexin-A also induced release of 3 H-radioactivity from 3H-AA-labeled cells [19]. Stimulation with low concentration of orexin-A (1 nM) induced Ca2þ influxdependent Ca2þ oscillations, which could be blocked with MAFP. However, the effect of MAFP was reversible, which indicates that its effect is not on PLA2 or any other serine hydrolase. Thus it remains unclear whether the cascade is supported by PLA2 and AA. AA is able to induce Ca2þ oscillations in these cells and it is possible that MAFP, being and AA-analog, could inhibit these directly via the putative AA target protein. However, these are mere speculations until more selective inhibitors are applied on these cells. OX1 receptor signaling was also assessed in neuro-2a neuroblastoma cells and HEK-293 cells upon stable or transient OX1 expression, respectively [17]. Both AA and 2-AG were released at rather low potency; the source of AA was not determined. These are the only reports of (possible) PLA2 signaling of orexin receptors, and all of them originate from recombinant cells. It is difficult to predict their value with respect to orexin physiology. It is remarkable, though, that OX1 receptors in both CHO and HEK-293 cells are suggested to utilize AA/PLA2 in Ca2þ influx signaling. Orexin receptors signal to NSCCs in many native neurons and recombinant cells (see Section 3.4). For practical (and historical) reasons many investigations have been made in CHO cells. The channels type has not been clarified but TRP channels of the TRPC subfamily have been suggested to play a part (see Section 3.4). The signaling mechanisms have eluded researchers until recently when we managed to strongly attenuate the influx utilizing pyrrophenone and MAFP (above). Thus, we might expect that in these cells either AA (or another fatty acid released) or lysoGPL or a metabolite of these is required for this influx (Fig. 2A). Such metabolites could very well act on several TRP family members (reviewed in Ref. [5]). However, a factor complicating the conclusions is that Ca2þ influx, on the other hand, is required for orexin-mediated cPLA2 activation. How does the causality between Ca2þ influx and cPLA2 activation thus go? In the case of HEK cells it is even more difficult to draw firm conclusions due to the lack of similar pharmacological and molecular analysis. For any native tissues, it would be rather easy to apply pyrrophenone for cPLA2a/z inhibition, but this has so far not been tried, and neither has any analysis of AA (or other fatty acid) release been performed. 3.2. Orexin receptors and phospholipase D PLD enzymes constitute a much smaller and entirely intracellular group. The “general” isoforms are PLD1 and -2 (reviewed in

Fig. 2. (Putative) Targets for phospholipase-generated messengers in orexin receptor signaling. (A) cPLA2 signaling contributes to Ca2þ influx in OX1-expressing CHO cells. (B) OX1expressing CHO cells show robust activation of PLD via a nPKC, but the role of PLD signaling is unknown so far. It is also unknown how the nPKC is activated (Section 3.2). (C) PLC signaling has multiple roles in orexin receptor responses. DAG may directly regulate some NSCCs. DAG also activates PKC, which may activate NCX, NSCCs and voltage-gated Ca2þ channels (VGCC) and inhibit Kþ channels. PLC signaling may regulate ion channels and NCX also by reducing PIP2 (see Fig. 1). DAGL degrades DAG to 2-AG, which exits the cells and acts as a CB1 receptor agonist, and 2-AG degradation releases AA (not shown). IP3 acts as an IP3 receptor ligand releasing Ca2þ into the cytosol (not shown).

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Ref. [20]), while two additional enzyme species mitoPLD and Nacyl-phosphatidylethanolamineePLD (NAPEePLD) have very specific roles. The definition “D” indicates that the enzymes hydrolyze the phosphoester bond between the phosphate and the GPL headgroup. In the case of PLD1 and -2, the substrate is phosphatidylcholine (PC), and the products thus phosphatidic acid (PA) and choline. While the headgroup released (choline for PLD1 and -2) is usually not active in signaling, PA is. It is capable of binding a number of proteins (e.g. Raf-1, SOS) and it can also be processed to other bioactive molecules like diacylglycerol (DAG) and lysophosphatidic acid (lysoPA). Finally, PA is a fusogenic lipid due to its promotion of negative membrane curvature (reviewed in Ref. [20]). mitoPLD is involved in mitochondrial membrane fusion by hydrolyzing the inner membrane cardiolipin (1,3-bis[sn-30 phosphatidyl]-sn-glycerol) (reviewed in Ref. [21]). Cardiolipin is essentially a dimer of PAs joined by a shared glycerol, and the product of the hydrolysis is thus one PA and one phosphatidylglycerol. N-Arachidonoyl-phosphatidylethanolamine is a special inner plasma membrane GPL, which is hydrolyzed by NAPE-PLD to release N-arachidonoylethanolamine headgroup, which is a potent endocannabinoid/endovanilloid messenger, also known as anandamide [22]. PLD1 and PLD2 are associated with many signal cascades. Their regulation is far from completely understood, but they require PIP2 for membrane anchoring/activity [20]. PKC and Rho and Arf family small G-proteins have also been associated with their activation cascades. Investigations of the processes PLD enzymes participate in have suffered, similar to PLA2, of lack of proper tools. Recently, however, knock-outs of both isoforms have been produced [23,24], and also the novel inhibitors developed [25e27] will in future help to shed light on PLD-mediated processes. There is a single recent report about involvement of PLD in orexin receptor signaling, once again, from the human OX1expressing CHO cells. PLD is strongly and potently activated in these cells by orexin stimulation [28]. A strong response was also seen to the phorbol ester TPA, and a much weaker one to the specific peptide-activators of novel PKC (nPKC) isoforms PKCd and PKC3 but not to an activator of conventional PKCs (cPKCs). Responses to both orexin-A and TPA were potently inhibited by the inhibitor of cPKC and nPKC, GF108203X, while the potency of the inhibitor of cPKC and protein kinase D (PKD), Gö6976, was very weak. The responses were also not inhibited by dominant-negative PKD species. Further inhibitor studies suggested that PLD was activated via a nPKC, likely PKCd, upon stimulation of OX1 receptors (or with TPA) (Figs. 1A and 2B). The activation mechanism of nPKC would likely be DAG produced by PLC; however, inhibition of PLC with U73122 did not inhibit the orexin response, leaving the issue unresolved. In contrast, Rho family G-proteins were not involved in the orexin- or TPA-mediated PLD activation. Utilizing the novel PLD speciesspecific inhibitors [27], PLD1 was determined to be the active PLD species in the responses [28]. In the study, the significance of PLD signaling for orexin responses was not investigated, and thus far such has not been presented. It should be fairly easy, and very interesting, to apply the analysis of PLD signaling to native tissues. Both the somewhat subtype-selective PLD inhibitors [27] and the more potent non-selective inhibitor, FIPI (5-fluoro-2-indolyl deschlorohalopemide; Refs. [25,26]), are currently commercially available. 3.3. Orexin receptors and phospholipase C PLC, as we speak of it in the mammalian context, refers to an enzyme with hydrolytic activity toward the phosphatidylinositol phosphoester bond between the sn3-hydroxyl group and phosphate. The resultant products are DAG, on one side, and inositol

with a phosphate in position 1 of the inositol ring and possibly in other positions as well. The most classical substrate is PIP2 which gives IP3, but also phosphatidylinositols (PIs) with lower degree of phosphorylation, like PI and PIP, are substrates for PLC. PLC family is divided in subfamilies of b, g, d, 3, h and z, of which all but 3 and z contain several members and possible alternative spliced variants. PLC subfamilies and in some cases individual members have distinct regulatory mechanisms, which have been partly tracked down to their modular structure. The only common regulator is Ca2þ, which is required by all of them but it may also be the major regulatory pathway for d, h and z. PLCb is the classical GPCR target via the Gq family G-proteins as well as Gbg. However, essentially all the other subfamilies could also be targets of GPCR signaling: g via Tyr kinases or phosphoinositide-3-kinase (PI3K); d, h and z via Ca2þ; 3 via Rho (or Ras) family G-proteins; and h via Gbg (see also Ref. [5]). The lack of any subtype-selective PLC inhibitor makes distinction of PLC isoforms hard, and presence of multiple genes makes the use of RNA knock-down, likewise, uncertain. In fact there is not even any reliable and non-toxic subtype-non-selective inhibitor for PLC (see Refs. [2,3]); therewith the situation is somewhat similar to PLA2. Both orexin receptor subtypes have been shown to couple to PLC activation (Fig. 1A) in recombinant cells [2,3,29e31], while the receptor subtype involvement in PLC signaling seen in native testicular, hypothalamic and adrenal cells [32e35] is not clear. Some direct studies point out the ability of orexin receptors to couple to Gq [33,35]. However, without knockdown or other direct molecular evidence it is yet possible that orexin receptors also regulate PLC species other than PLCb. Indeed, we have seen indications of two different (unidentified) PLC species being activated in OX1 receptor signaling with specificities toward PI/PIP and PIP2, respectively [36]. Several of the signaling pathways implicated in PLC activation (see above) have also been identified for orexin receptors; for instance, the apparently potent coupling of orexin receptors to Ca2þ influx (Section 2.1) could easily represent such a trigger for PLC. Another classical indication of PLC activity is activation of PKC. cPKC and nPKC subfamily members are activated by DAG. However, plasma membrane DAG may also originate from metabolism of PA from PLD action, PC-specific PLC (PC-PLC) or reverse action of sphingomyelin synthase (reviewed in Ref. [5]). PKC involvement is easily assessed even in tissue samples where other analyses are hampered, like brain slices, utilizing mostly good pharmacological inhibitors and activators. In native CNS neurons, orexins often induce depolarization via post- and pre-synaptic mechanisms (Section 3.4). In some cases PKC has been associated with these processes [37e43] (Fig. 2C), but the target of PKC is often not clear. The physiological picture is unclear since in most cases the involvement of PKC has not been investigated. cPKC activation could also be promoted by AA and lysophosphatidylcholine (lysoPC), possible products of the PLA2 pathway [44e48]. Bacteria and protozoa express PC-PLC, but despite numerous propositions and even some possible demonstrations of such activity, no metazoan counterpart has been isolated so far. However, some studies report mammalian tissue PC-PLC activity. While some studies use molecular separation and pathway tracking by labeled substrates and thus build a more convincing case, most studies resort to the use of a reputed PC-PLC inhibitor, D609. Unfortunately, D609 is also an inhibitor of sphingomyelin synthase [49], which could account for the PC-PLC-like reaction observed. D609 is also a reactive compound [50], and its selective even against sphingomyelin synthase may be questioned. Some studies report that D609 inhibits orexin responses in the CNS (reviewed in Ref. [2]). While being an interesting finding as such, the dubious pharmacological profile of D609 precludes further conclusions at the moment.

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3.4. Lipid messengers in the regulation of ion fluxes by orexin receptors As indicated above, post-synaptic depolarization is an often seen response to orexin stimulation of native CNS neurons. Several mechanisms for the depolarization exist: a) inhibition of Kþ channels, b) activation of NSCCs, and c) activation of Naþ/Ca2þ exchanger (NCX). In addition, voltage-gated Ca2þ channels, with a possible role in regulating, e.g., NCX-dependent depolarization or presynaptic transmitter release, have been reported to be activated/ potentiated by orexin receptors in neurons (or other excitable cells) (Fig. 2C). Kþ channels are represented by a vast number of channel isoforms with multiple roles in regulation of (neuronal) firing. Several Kþ channel isoforms are sensitive to PKC phosphorylation, and there indeed are cases where PKC inhibitors have abolished orexinmediated inhibition of Kþ channels [40,43] (Fig. 2C). However, it should be pointed out that PKC inhibitors have been tested in only a fraction of cases where inhibition of Kþ channels has been observed (reviewed in Refs. [2e4]). Inward rectifier Kþ (Kir) channels may be one likely target for orexin regulation in the cases where depolarization is the response. Different Kir members may be inhibited by GPCR inputs either via PKC phosphorylation or hydrolysis of PIP2, which is elementarily required for the activity of these channels [51e53]. While inwardly rectifying Kþ channels are inhibited by orexin receptors in native tissues as well as upon recombinant expression [54e58], the mechanisms have not been investigated and the channel types are not always identified. Some Kþ channel types are also sensitive to AA (reviewed in Ref. [59]), suggesting that PLA2 (or DAG lipase) cascade might contribute to their regulation. The identity and activation mechanisms for NSCCs activated by orexin receptors are unclear. As presented above (Section 3.1), in recombinant CHO cells the PLA2 pathway is implicated. In the CNS, there is very little information. In one study, PKC has been associated with the activation [39] (Fig. 2C). The most obvious candidate for NSCCs would be TRP family channels. Regulation of TRP channels is diverse and only partially clarified (reviewed in Refs. [60,61]), but GPL-derived signals apparently represent one of the major positive or negative regulators (reviewed in Ref. [5]). The most usual regulators comprise free fatty acids or their derivatives (like eicosanoids), endovanilloids, DAG and lysoGPLs. Thus, the PLA2, PLC and potently even PLD pathways might be directly involved in the regulation (see also Section 3.1). PLC pathway has yet another interesting role in the regulation, as PIs, in particular PIP2, have been associated with positive or negative regulation of a variety of TRP channels (Fig. 1C). By its ability to stimulate PIP2 synthesis, even PLD may thus be associated with this regulation (Fig. 1C). Different TRPC subfamily channels are directly regulated by PIs, DAG, LPC and AA (reviewed in Ref. [5]). Yet another possibility of TRP channel regulation via DAG (or AA or LPC; see above) is via PKC. The regulation of different TRP family members by PKC has been only partially mapped. Among TRPC members, TRPC1 has been shown to be stimulated/potentiated [62e64]. Within other TRP subfamilies, TRPM1, -4, -6 and -7 [65e71] and TRPV1, -4, -5 and -7 [72e80] have been reported to be PKC-stimulated/potentiated. For some other isoforms, there are reports of PKC inhibition, while TRPP2 would be activated by protein kinase D1 (PKD1) [81]. The analysis, however, is not straight-forward. The stimulation by PKC as well as other signals may not be direct but manifested by sensitization to physiological stimuli, reduced sensitivity to inactivation, or modulation of trafficking (stimulated exocytosis or inhibited endocytosis). The tissue expression profile, including the heteromeric channel compositions (which are not easily determined), PKC isoforms and other interacting proteins, may determine whether the dominant response is inhibition or stimulation or none (see, e.g. Refs. [63,64,82,83]).

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Lipid signaling to NSCCs by orexin receptors has not been much investigated. Among recombinant systems, TRPC channels have been suggested to contribute to the signal in human OX1-expressing CHO, HEK-293 and IMR-32 cells [19,84]. In IMR-32 neuroblastoma cells, dominant-negative TRPC6, an inhibitor of TRPC3/6, is able to partially block the receptor-operated Ca2þ influx response to both orexin-A and a synthetic DAG, dioctanoylglycerol (DOG) [84]. Similar was seen with RNAi against TRPC3 [85] (see below). TRPC3, -6 -and -7 can be activated by DAG (reviewed in Ref. [5]). In CHO cells, orexin receptor-operated Ca2þ influx was partially inhibited by dominant-negative TRPC1 and TRPC3 channels, while their impact on the DOG response was not assessed [86]. Since these constructs were not expressed together it is impossible to know whether they are incorporated in the same or separate signal complexes; TRPC1 and -3 are known to be able to heteromerize [87]. These studies suggest that TRPC channels might be at least partially involved in the OX1 response via direct DAG or PI regulation or indirectly via PKC phosphorylation. In addition, we have recently shown that cPLA2 inhibition as well as non-selective serine hydrolase inhibition effectively blocks the receptor-operated Ca2þ influx response at low orexin concentrations (Section 3.1; (Refs. [15,17])), implicating either AA or a lysoGPL in the response. AA activates TRPC6 while TRPC5 is activated by lysoPC [5]. However, other TRP subfamily channels may be targeted as well (reviewed in Ref. [5]), and neither AA nor any lysoGPL are end products. Among native cells, NSCC activation by orexins has been reported in CNS neuron of many nuclei (reviewed in Refs. [2e4]). However, no molecular evidence is presented as to their identity, and the activation mechanisms of these channels in orexin signaling are essentially unknown. The only indication of lipid signaling is in a report of orexin activation of NSCCs in rat tractus solitarius neurons via a PKC-dependent mechanism [39]. The third mechanism suggested to depolarize neurons upon orexin receptor activation is NCX. NCX in the forward mode removes one Ca2þ from the cytosol for three Naþ entering, and it thus conducts an inward current. NCX is driven by the electrochemical driving forces of membrane potential and ion activities (wconcentrations) and can thus also act in a reverse mode carrying an outward current (reviewed in Ref. [88]). Interestingly, NCX activity has also been suggested to be stimulated by PIP2 and possibly also by PKC, and inhibited by polyunsaturated fatty acids (reviewed in Ref. [88]) (Fig. 2C). NCX has been implicated in orexin receptor signaling in CNS neurons [56,58,89e91]. NCX in forward mode may be difficult to distinguish from NSCCs but substitutions of extracellular Naþ or intracellular Ca2þ might be used to separate these systems. However, this is not without possible misinterpretations; some TRP channels (and other NSCCs) may require intracellular Ca2þ for activity (reviewed in Ref. [60]). Niþ and KB-R7943 have been utilized as inhibitors. KB-R7943 is suggested to have w10-fold higher (micromolar) affinity as an inhibitor for the reverse mode of NCX [92]. However, also the different NCX isoforms show affinity differences. KB-R7943 in the range of 10e100 mM inhibits the orexin-induced current [56,58,89e91]. However, KB-R7943 is not that selective; in fact, it blocks TRPC3, -6 and -7 with micromolar or submicromolar potency [93] while other TRP channels have not been assessed. Based on this and other unclarities, I therefore feel that the ability of orexin receptors to depolarize neurons via NCX is not clearly proven. There is, though, some evidence for NCX engagement in orexin signaling in recombinant systems. In recombinant IMR-32 neuroblastoma cells, the Ca2þ elevations induced by OX1 receptor stimulation are heterogeneous: in some cells, extracellular Naþ removal strongly inhibits Ca2þ elevation while in the others, no effect is seen [85]. The former type of responding is also attenuated by KB-R7943 (10 mM), dominantnegative TRPC6 and siRNA against TRPC3. The authors conclude

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that in this cell population, TRPC3-containing channels primarily mediate a Naþ influx which then triggers the Ca2þ influx via reversely acting NCX. It should be noted that KB-R7953 is likely to inhibit TRPC3 also directly, but Naþ replacement indeed suggests reverse action of NCX. Orexins also act via apparent presynaptic mechanisms (reviewed in Refs. [2e4]). These are seen in neuronal preparations as, for instance, increased or decreased excitability which is sensitive to block of action potentials (e.g. tetrodotoxin) and synaptic activity (e.g. high Mg2þ/low Ca2þ). While the sensitivity to tetrodotoxin may indicate similar excitatory actions on some presynaptic neuron as described above (e.g. inhibition of Kþ channels, stimulation of NCSS or NCX), sensitivity to synaptic block without sensitivity to tetrodotoxin may indicate an action of orexins on presynaptic terminals, i.e. regulation of transmitter release from presynaptic terminals. Similar mechanisms may also act here, but another mechanism would be modulation of voltage-gated Ca2þ channel activity. There are some indications of regulation of these channels via orexin receptors in neurons (as also in some other excitable cells), but it is in most cases not known, whether this is direct or via depolarization (reviewed in Refs. [3,4]). There also is little information about the signaling mechanisms. PKC, though, has been implicated in some cases (reviewed in Refs. [3,4]). PLC signaling produces IP3 as discussed under Section 3.3, which is likely to induce Ca2þ release from the endo-/sarcoplasmic reticulum. In one study, IP3-dependence of the orexin-induced Ca2þ release has been demonstrated by artificially lowering the IP3 levels [9]. 3.5. Endocannabinoid in orexin signaling Endocannabinoids are lipid-derived mediators utilized by the body for activation of cannabinoid receptors (CB1 and CB2; both GPCRs). The two most well known endocannabinoids are anandamide and 2-AG, both containing the AA moiety. Anandamide is produced from the unusual membrane GPL, Narachidonoyl-phosphatidylethanolamine, by the enzyme NAPEPLD, as described under Section 3.2. 2-AG is a “breakdown product” of AA-containing DAG by the action of the enzyme DAG lipase (DAGL). The regulation NAPE-PLD and DAGL are not well known but Ca2þ may be an important stimulant [22,94]. Endocannabinoid signaling is found throughout the body but especially important is its regulatory role for the CNS and the immune system (reviewed in Refs. [95,96]). We have recently seen that OX1 orexin receptor stimulation in recombinant CHO cells induces a robust production of 2-AG, which is able to act in the same paracrine manner as in the brain [17,97] (Fig. 2C). Two previous studies in the brain slices have produced indirect but strong evidence of the orexin signals being mediated by 2-AG. In both cases (dorsal raphé nucleus and ventrolateral periaqueductal gray matter), 2-AG is suggested to be released upon post-synaptic activation of the PLCeDAGL cascade by orexin, and to act on CB1 receptor on presynaptic terminals in the regulation of both sleep-wakefulness circuitry and antinociception [98,99], in agreement with the classical retrograde synaptic transmission of endocannabinoids (reviewed in Ref. [95]). The ability of orexin receptors to induce production 2-AG is not surprising taken their strong ability to stimulate PLC (Section 3.3). The pharmacological evidence from the brain yet suggests rather significant DAG production, which thus supports the idea that PLC activity in the brain is a significant cellular orexin response. 2-AG is degraded by hydrolysis to AA and glycerol, and it may thus constitute an important route for release of AA with a possible role in orexin responses in the brain.

3.6. Phosphoinositide-3-kinase PI3K cascade is one of the central regulators of cell survival, proliferation and differentiation. Class I PI3Ks phosphorylate PIP2 at 3-position of the inositol ring to PIP3, and the PIP3 formed acts as a scaffold for protein kinases, e.g. phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB) (reviewed in Ref. [100]). Class I PI3Ks have classically been thought to be regulated by growth factor receptors, but they are also widely targeted in GPCR signaling via Ras or Gbg (reviewed in Ref. [100]). There are only a few examples of orexin receptor regulation of PI3K. In InR1-G9 cells glucagonsecreting cell line of pancreas, PI3K activation by orexin inhibits proglucagon production [101]. In white adipocyte precursor 3T3-L1 cells, PI3K activation by orexin leads to enhancement of triglyceride synthesis [102]. The receptor subtypes mediating these effects and the signal cascades engaged in the activation of PI3K are unknown. We have obtained a partial inhibition of orexin-induced ERK phosphorylation in OX1-expressing CHO cells by inhibition of the PI3K pathway by pharmacological or molecular biological inhibitors [103]. Altogether it is difficult to predict the importance of PI3K in orexin receptor signaling based on the very few studies performed. 4. Conclusions Detailed studies in recombinant cell line, especially in OX1expressing CHO cells, show coupling to multiple phospholipaseinstigated signal pathways, and there is clear evidence for activation of the PI3K pathway, as well, in other cell lines. How these cascades are activated by orexin receptors, is much less wellknown. From the information available it is difficult to conclude anything concerning potential receptor subtype differences. In recombinant cells mainly OX1 receptors signaling has been investigated and in many native cells receptor subtype expression is not known or it is investigated utilizing inaccurate methods (see Refs. [1,2]). Another significant question is the physiological role of these cascades. There is plenty of evidence for the PLC cascade from the CNS neurons as well as other cell types. Currently this includes evidence based on PKC activation as well as DAG-dependent endocannabinoid production. PLA2 and PLD (and PI3K) cascades have not been assessed in native cells, but we may hope this to take place soon as novel specific inhibitors are now available. Molecular mechanisms of the NSCC activation, so prominent a response for orexin receptors both in the CNS neurons and in recombinant cells, are not known in detail, but lipid-mediated signaling constitutes a reasonable bet. Knowledge of possible disorders caused by dysfunction of the orexinergic system is extremely limited, partially because of the shortcomings of the tools available [1,2]. The known dysfunctions are very drastic, i.e. the incapacitating mutations in the OX2 receptor in narcoleptic dogs, the putative death of orexinergic neurons in human narcolepsy, and similarly manipulated rodent models. More subtle disorders are not known. The gross failure either in the receptor or in the peptide inevitably entirely blocks the signaling, and therefore does not tell us much about the importance of any specific signal cascade. Therefore, there is little we can tell about lipid (or any) signaling of orexin receptors in possible disorders. If the lipid cascades indeed are important for orexin receptor signaling, then the dietary intake of different fatty acids (e.g. n-3 vs. n-6 polyunsaturated fatty acids) may bear significance for orexin signaling. Thus far these are though mere speculations. Acknowledgments Professor Karl Åkerman (Institute of Biomedicine, University of Helsinki) is acknowledged for helpful discussions. This study was

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Please cite this article in press as: J.P. Kukkonen, Lipid signaling cascades of orexin/hypocretin receptors, Biochimie (2013), http://dx.doi.org/ 10.1016/j.biochi.2013.06.015