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J. Biol. Chem., Vol. 280, Issue 8, 6570-6579, February 25, 2005

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OX₁ Orexin Receptors Couple to Adenylyl Cyclase Regulation via Multiple Mechanisms^*

Tomas Holmqvist, Lisa Johansson, Marie Östman, Sylwia Ammoun, Karl E. O. Åkerman, and Jyrki P. Kukkonen¶

From the Department of Neuroscience, Unit of Physiology, Uppsala University, BMC, SE-75123 Uppsala, Sweden and the A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Neulaniementie 2, FIN-70210 Kuopio, Finland

Received for publication, July 1, 2004 , and in revised form, November 4, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, the mechanism of OX₁ orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC₅₀ 1nM) inhibition and a strong (4-fold), low potency (EC₅₀ = 300 nM) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G_i/o proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC₅₀ = 500 nM). cAMP elevation was not caused by Ca²⁺ elevation or by G. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKC, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G_s activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC₅₀ of orexin-A = 10 nM and EC₅₀ of orexin-B = 100 nM), and the response was fully dependent on PKC. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-G_s antibodies. We conclude that OX₁ receptors stimulate adenylyl cyclase via a low potency G_s coupling and a high potency phospholipase C PKC coupling. The former or some exogenous G_s activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX₁ receptors couple to G_s almost as efficiently as the orexin-A-activated receptors, in contrast to Ca²⁺ elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The neuropeptides/hormones orexin-A and -B and the corresponding G-protein-coupled receptors OX₁ and OX₂ receptor were discovered in 1998 (1, 2). Orexin-A (33 amino acids) and orexin-B (28 amino acids) share the property of being able to activate both orexin receptors. Orexins are signal substances both in the central nervous system and in the periphery. In the central nervous system, all of the orexinergic neurons have their origin in the lateral hypothalamus from where they project widely to regulate especially wakefulness and paradoxical sleep, appetite and food intake, and endocrine and autonomic processes. At most of the projection sites both OX₁ and OX₂ receptors are expressed. The orexins most often act in an excitatory manner both via putative pre-, post-, and extrasynaptic mechanisms. In the periphery, orexins and orexin receptors have been have been found in the gastrointestinal tract and in the endocrine organs. The prominent periferal effects seen so far include regulation of gastrointestinal motility and hormone production and release, especially in the adrenal gland (reviewed in Ref. 3).

Based on measurements of binding affinity and the ability to elevate intracellular Ca²⁺ and liberate inositol phosphates in heterologous expression systems, the OX₁ receptor shows a 10-fold preference for orexin-A over orexin-B in contrast to the OX₂ receptor, which shows no preference (2, 4–8). This postulated selectivity profile is often used to determine the involved orexin receptor subtype in native cells and in vivo. However, some doubt has been cast on the validity of this practice. It is well known that different G-protein pathways can be differentially activated by different receptor agonists via agonist-selective receptor conformations (9), and a similar process has even been suggested for orexin receptors (7). Therefore, functional selectivity of receptor agonists may not be valid for all systems and responses.

The cellular signals triggered upon orexin receptor activation are relatively unclear. OX₂ receptors have been shown to be able to activate G_i, G_q, and G_s proteins (10), but the efficacy of the interaction and the role of these in the orexin receptor signaling is unknown. Some other studies, by the use of pertussis toxin or other techniques, suggest that G_i/o proteins are engaged in orexin signaling (8, 11–13). On the other hand, most other responses seen in native cells and cell lines, e.g. Ca²⁺ elevation, phospholipase C (PLC)¹ activation, and activation of cation channels, are unlikely to be mediated by G_1/o proteins. Ca²⁺ elevation is a prominent response seen in all the cell lines where the receptors have been heterologously expressed (2, 6, 8, 14) and in all the native neurons where this has been investigated (15–19). Two different mechanisms seem to be active: (i) Ca²⁺ influx via a non-voltage-gated pathway in recombinant cells (14, 20) and via voltage-gated Ca²⁺ channels in neurons and some endocrine cells (17–19, 21, 22) and (ii) PLC activation and inositol 1,4,5-trisphosphate-dependent Ca²⁺ release (4, 7, 10, 14, 23–25). Both orexin receptor subtypes seem to share these pathways. Ca²⁺ elevation could well explain some of the excitatory properties of orexin. Activation of adenylyl cyclase has also been shown to be important for orexin receptor signaling, although this pathway has seldom been investigated. In rat and human adrenal cortex, orexins strongly elevate cAMP, leading to activation of protein kinase A and increased synthesis and release of glucocorticoids (26, 27), probably, at least in man, via OX₁ receptors (27). Although this effect appears to be very similar to the adrenocorticotropic hormone, which utilizes the G_s pathway, the mechanisms of orexin signaling to adenylyl cyclase has not been investigated.

cAMP is a ubiquitous second messenger involved in a vast array of physiological processes such as regulation of glycogen metabolism, regulation of hormone synthesis, modulation of ion channels, and regulation of gene transcription. Most of these effects are mediated through binding and activation of protein kinase A, but novel targets of cAMP, such as Epacs, guanine nucleotide exchange factors of the small G-protein Rap, have been identified recently (28). Adenylyl cyclases (AC) are the enzymes responsible for cAMP production. Membrane-bound adenylyl cyclases with nine known mammalian isoforms are subject to many positive and negative regulatory inputs from especially G-protein-coupled receptors but also from other pathways (reviewed in Refs. 29 and 30). The most important (known) inputs include G-protein -subunits (G_i/o, G_s), G-protein -subunits (G), Ca²⁺/calmodulin, and protein kinase C (PKC). Three features are characteristic: (i) each isoform responds to a different subset of these factors; (ii) for one isoform a particular factor can be inhibitory, whereas it can be stimulatory for another isoform; and (iii) the factors can show strong cooperativity or even conditionality for stimulation of adenylyl cyclase. Thus, for instance, G can inhibit AC1, whereas it stimulates AC2, AC4, and AC7. However, it cannot stimulate AC2 (and probably neither AC4 nor AC7) unless the AC is simultaneously stimulated by other factors (e.g. G_s or PKC) (31, 32). The consequence of this isoform-specific signal integration is that the cAMP response will differ from cell to cell according to the expression profile of adenylyl cyclase isoforms as well as other proteins participating in the adenylyl cyclase regulation.

Recognizing the important role of cAMP in cellular processes and its putative importance of orexin receptor signaling, we wanted to investigate the intrinsic ability of OX₁ orexin receptors to regulate adenylyl cyclase activity in an isolated system, where manipulations are possible. For this purpose we chose a Chinese hamster ovary (CHO) cell line recombinantly expressing OX₁ receptors. This cell line has two advantages: (i) orexin receptor signaling in it has been relatively well characterized in previous studies by us and others (2, 4, 14, 20, 33) and (ii) it does not appear to express Ca²⁺-sensitive adenylyl cyclase isoforms, minimizing interference from this strong signal of orexin receptors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—CHO-hOX₁ cells, as described before (14), were cultured in nutrient mixture (Ham's F-12) medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), 100 units/ml penicillin (Sigma), 80 units/ml streptomycin (Sigma), and 400 µg/ml geneticin (G418; Invitrogen) in an air-ventilated humidified incubator in 260-ml plastic culture flasks (75-cm² bottom area; Greiner Bio-One GmbH, Frickenhausen, Germany). For microfluorometry and Ca²⁺ measurements, the cells were grown on uncoated circular glass coverslips (diameter, 25 mm; Menzel-Gläser, Braunschweig, Germany) and for other experiments on circular plastic culture dishes (inner diameter, 52 or 82 mm; Greiner). When the effect of pertussis toxin pretreatment was investigated, the cells were treated with 100 ng/ml pertussis toxin for 24–48 h. For cholera toxin, different concentrations (10, 100, and 1000 ng/ml) for 18 h were initially tested (see Fig. 4); subsequent experiments were then performed using 10 ng/ml for 18 h.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Comparison of the effect of different concentrations of CTx on the basal and orexin-stimulated cAMP production. The cells were pretreated with CTx for 18 h. ctrl, control.

Media—TES-buffered medium (TBM) consisted of 137 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1.2 mM MgCl₂, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, 10 mM glucose, and 20 mM TES adjusted to pH 7.4 with NaOH. Lysis buffer was composed of 50 mM HEPES and 150 mM NaCl (pH.7.5) supplemented with 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1.5 mM MgCl₂, 1 mM EDTA, 10 mM Na⁺-pyrophosphate, 1 mM Na⁺-orthovanadate, 10 mM Na⁺-fluoride, 250 µM p-nitrophenol phosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Laemmli sample buffer was composed of 50 mM Tris-HCl (pH 6.8) supplemented with 1 mM dithiothreitol, 2% SDS (w/v), 10% glycerol (v/v), and 0.1% bromphenol blue (w/v).

Expression Vectors—pcDNA3.1 plasmids harboring human ₂-adrenoreceptor, human transducin (G_t-rod), human G₁, and human G₂ were from the Guthrie cDNA Resource Center (www.cdna.org). The plasmid for expression of enhanced green fluorescent proteins (GFP), pEGFP-C1, was from Clontech (Palo Alto, CA). We gratefully acknowledge Dr. Robert J. Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC) for pRK5-ARK1-CT (C terminus of the human -adrenergic receptor kinase 1 (ARK1)), Dr. J. Silvio Gutkind (NIDCR, National Institutes of Health, Bethesda, MD) for pcDNAIII T8- ARK (a fusion of the extracellular and transmembrane part of CD8 and the C terminus of the human ARK1), and Dr. Johanna Ivaska (VTT Medical Biotechnology, Centre for Biotechnology, Turku, Finland) for pEGFP-C1-PKCwt (fusion of GFP and PKC) (37).

Transfection—CHO-OX₁ cells were grown on plastic culture dishes or on glass coverslips to 40–50% confluence. The dishes were washed with PBS, and the cells were transfected in OPTI-MEM (Invitrogen) using Lipofectamine (Invitrogen). After 5 h this medium was replaced with fresh Ham's F-12 medium with all of the usual supplements (see "Cell Culture"). Transfection efficiency was 40–70% as determined using expression of green fluorescent protein and function of transfected proteins (e.g. receptors, phosphodiestarases; not shown). Transfection of the cells was performed to introduce control receptors (₂-adrenoreceptor for G_s coupling and _2A-adrenoreceptor for G-dependent PLC activation (and Ca²⁺ release)), G sequestering peptides/proteins (ARK1 C terminus, CD8-ARK1 C terminus, G_t), and GFP-PKC constructs. The total amount of DNA was kept the same in all of the transfections using empty plasmids.

Measurement of cAMP Production in Intact Cells—The cellular ATP was prelabeled with 5 µCi/ml [³H]adenine for 2 h in culture medium, after which the cells were detached using PBS + 0.02% (w/v) EDTA, washed, and incubated at 37 °C in TBM containing 500 µM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, for 10 min (38). Thereafter the cells were stimulated for 10 min at 37 °C, after which the reactions were interrupted by centrifugation, removal of the supernatant, addition of perchloric acid, and freezing. [³H]ATP + [³H]ADP and [³H]cAMP fractions of the cell extracts were isolated by sequential Dowex/alumina chromatography. Radioactivity was determined using scintillation counting. The conversion of [³H]ATP to [³H]cAMP was calculated as a percentage of the total eluted [³H]ATP + [³H]ADP and normalized to the recovery of [¹⁴C]cAMP tracer. The effects of inhibitors (e.g. PKC inhibitors) used in measurements were tested both under basal and stimulated conditions (e.g. TPA, orexin-A), and any slight effects on the basal level were compensated when calculating (and showing) the inhibitory potency (e.g. Figs. 5, 6, and 8).

View larger version (29K): [in this window] [in a new window]   FIG. 5. cAMP elevation in response to PKC and OX1 receptor stimulation in intact CHO cells pretreated with 10 ng/ml CTx for 18 h. A, the cells were stimulated with 2 µM TPA, 1 µM thapsigargin (thaps) or the both together. The first comparison (for TPA and thapsigargin) is to the resting level (ctrl), and the second (for TPA+thapsigargin) is to the TPA response. B and C, inhibition of the TPA-stimulated (white bars) and TPA+thapsigargin-stimulated (black bars) responses by the PKC inhibitors Gö6976 (B) and GF109203X (C). The cells were pretreated with the PKC inhibitors for 30 min. ctrl corresponds to the noninhibited TPA or TPA+thapsigargin response. The comparison for each set of bars is to the corresponding control (ctrl). D, inhibition of the orexin-stimulated responses by the PKC inhibitors Gö6976 and GF109203X. The first ctrl corresponds to the resting CTx-stimulated level, and the second ctrl corresponds to orexin-A response in the similarly treated cells. The comparison is with the control orexin-A response. For all of the graphs, please note that the level obtained with CTx alone is 1 (indicated with dashed lines in B–D).

View larger version (31K): [in this window] [in a new window]   FIG. 6. OX1 receptor activity stimulates adenylyl cyclase in CTx-treated cells via phosphatidylinositol-specific phospholipase C PKC. A and B, PLC activity was assessed using measurement of total inositol phosphate accumulation (A) and GFP-PKC translocation (B), which is essentially dependent on diacylglycerol generation. The concentration of orexin-A in B is 100 nM. C, inhibition of orexin-induced cAMP generation by the phosphatidylinositol-specific phospholipase C inhibitor U-73122 (10 µM, 30 min of preincubation), phosphatidylcholine-specific phospholipase C inhibitor D609 (10 µM, 30 min) and the phosphoinositide 3-kinase inhibitor wortmannin (100 nM, 30 min). D, expression of novel PKC isoforms in CHO cells as indicated by Western blots with PKC subtype-specific antibodies. TPA stands for 24 h treatment with 2 µM TPA. E, inhibition of orexin-induced cAMP generation by the PKC inhibitor rottlerin (10 µM) and the PKC inhibitor KIE1–1 (1 µM, 30 min). ctrl, control.

View larger version (19K): [in this window] [in a new window]   FIG. 8. The sensitivity of the OX1 receptor response to PKC and PLC inhibition in intact CHO cells not pretreated with CTx. The cells were pretreated with the inhibitors for 30 min. The first comparison is with the basal response, and the second is with the control orexin-A response. ctrl, control.

Ca²⁺ Measurements—Ca²⁺ imaging was performed to evaluate the effectivity of the different G-sequestering peptides (ARK1 C terminus, CD8-ARK1 C terminus, G_t). A clear G-mediated response, _2A-adrenoreceptor-induced Ca²⁺ elevation, was selected as the test. This was verified using pertussis toxin, which fully inhibited this signal but not the endogenous P2Y-purinoceptor response. The cells were transfected with GFP, _2A-adrenoreceptor, and each G scavenger or empty vector (DNA ratio 1:2:7) as described above. 48 h later, the cells on coverslips were loaded with 4 µM fura-2 for 20 min at 37 °C in TBM + 0.5 mM probenecid (an inhibitor of the anion pump, which otherwise effectively extrudes fura-2), rinsed once, and used immediately. TILLvisION version 4.01 imaging system (TILL Photonics GmbH, Gräfelding, Germany) was used for the measurements (39). The cells were excited by alternating 340- and 380-nm light with the use of a monochromator, and the emission light was collected through a dichroic mirror and a 475-nm barrier filter with a CCD camera. The coverslips were constantly perfused at a rate of 1 ml/min in a 160-µl perfusion chamber at 37 °C. The additions into the chamber were made by perfusion. The data were analyzed using the TILL software and Microsoft Excel.

Inositol Phosphate Measurements—The cells were prelabeled with 3 mCi/ml myo-[³H]inositol for 20 h (14), after which they were harvested as for cAMP measurements in intact cells. The cells were preincubated at 37 °C in TBM containing 10 mM LiCl for 10 min, after which they were stimulated for 20 min. The reactions were terminated by spinning, replacement of TBM with ice-cold perchloric acid, and freezing. The supernatants were neutralized with KOH + KHCO₃, and the total inositol phosphate fraction was isolated with Dowex anion exchange chromatography. Radioactivity was determined using scintillation counting.

Translocation of GFP Fusion Proteins—CHO cells were transfected with the plasmid coding for GFP-PKC (pEGFP-C1-PKCwt) as described above. The experiments were performed 24 later. The experiments were performed essentially as Ca²⁺ measurements, except that 490-nm light was used for excitation, and the emission was collected through 505-nm dichroic mirror and 520-nm barrier filter. 100x/1.3 oil immersion objective was used for measurements (39).

SDS-PAGE and Immunoblotting—Cells on plastic culture dishes were washed once with ice-cold PBS and lysed then with lysis buffer (100 µl/85-mm dish). The protein concentration was quantified using DC protein assay (Bio-Rad) and adjusted accordingly. Volumes corresponding to 20 µg of protein were diluted in Laemmli buffer, boiled, separated by SDS-PAGE (10% acrylamide), and transferred to methanol-soaked polyvinylidene difluoride membranes (Amersham Biosciences). The membranes were blocked with 0.1% (v/v) Tween 20 and 5% (w/v) milk in PBS (TPBS-milk) for 1–2 h in room temperature before probing with antisera. Primary antibody incubation was done in TPBS at +4 °C overnight, and secondary antibody incubation was done in TPBS-milk at room temperature for 1 h after two 20-min washes with TPBS.

Specific protein detection was performed using rabbit polyclonal anti-PKC (SC-213), - (SC-214), - (SC-215), and - (SC-210) antibodies (1:500; St. Cruz Biotechnology, Santa Cruz, CA). Primary antibodies were detected using horseradish peroxidase-conjugated donkey antirabbit antibody F(ab')2 fragments (1:2000; Amersham Biosciences). Peroxidase enzymatic activity was visualized using the Enhanced Chemiluminescence assay (Amersham Biosciences). After this, the membranes were washed for 2 h with TPBS-milk, and the actin in each lane was probed for using rabbit polyclonal anti-actin antibody (A2066; 1:1000; Sigma) and the same secondary antibody as above. Same detection system was used as above. The densities of the exposed films were quantified using a "homemade" system described in Ref. 40. The system was calibrated using films of known densities (Wratten ND 0.1, 0.3, 0.5, 0.7, and 1) as described in Ref. 40, and the absorbances of the lanes were always kept in the range of 0.1–1. The controls, which the data were to be compared with, were always run on the same gel.

Data Analysis—Student's two-tailed t test was used in all pairwise comparisons and analysis of variance, followed by Tukey's post-hoc test, for multiple comparisons. In most cases, there was some variation in the basal and stimulated cAMP levels in different batches of cells, and the data were therefore normalized to the basal/control level to allow reliable comparison (see e.g. Fig. 2). The significances are as follows: not significant (ns), p > 0.05; p < 0.05 (*); p < 0.01 (**); and p < 0.001 (***). Any second comparison is marked with ns and . Significances are indicated only for the data where the results are not self-evident. In the figures, the mean ± S.E. is given. Each experiment was performed at least three times. SigmaPlot 4.1 (Jandel Scientific, Corte Madera, CA) was used for nonlinear curve fitting.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Regulation of adenylyl cyclase activity by different intracellular messengers in intact CHO cells. The effect of elevated Ca2+ (1 µM thapsigargin (thaps), 1 µM ionomycin (io)), PKC (2 µM TPA), G12 (G), Gs (10 µM prostaglandin E1, 10 ng/ml CTx), and forskolin (10 µM) were evaluated. The other compounds were added to the cells at the start of the experiments, but G1 and G2 were expressed from plasmids introduced 48 h earlier, and the CTx pretreatment time was 18 h. In A and B, the effects of the treatments are evaluated on the basal level, and in C, the effects were evaluated in cells pretreated with CTx. There are no error bars in the bars indicating basal/control levels because the data from different batches of cells were normalized to the resting cAMP levels in the absence (basal; A and B) and presence (ctrl; C) of CTx. The comparisons are with the basal/control levels.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (18K): [in this window] [in a new window]   FIG. 1. OX1 orexin receptor activation stimulates cAMP production in intact CHO cells. The cells treated with 10 µM SB-334867 were preincubated with it for 10 min before the stimulation with orexin-A. The first comparison is with the basal level and the second to the control response to 1 µM orexin-A.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Concentration response curves for orexin-A and -B with respect to cAMP elevation (filled symbols) in intact CHO cells compared with similar data with respect to Ca2+ elevation (empty symbols). The data for Ca2+ elevation has been previously published in Ref. 7 (published with permission from ASPET).

As previously shown in Fig. 2, activation of PKC with TPA stimulates cAMP production in the presence of activated G_s (CTx). This is further stimulated by Ca²⁺ elevation using thapsigargin (Fig. 5A), although thapsigargin by itself does not stimulate adenylyl cyclase in the presence or absence of G_s-stimulation (Figs. 2, A and C, and 5A). This suggests that at least the Ca²⁺-sensitive, conventional PKC isoforms might be involved in this response. The sensitivity of the TPA-mediated stimulation and combined TPA and thapsigargin-mediated stimulation was therefore tested for sensitivity to two different PKC inhibitors, Gö6976 and GF109203X. These two PKC inhibitors show some specificity for the three PKC families. Gö6976 essentially only inhibits conventional PKC isoforms (43). TPA-stimulated cAMP elevation was not inhibited by Gö6976 at concentrations up to 10 µM, but the Ca²⁺ elevation-dependent part of the response was inhibited in a concentration-dependent manner (Fig. 5B, +TPA+thapsigargin). GF109203X inhibits both conventional and novel PKC isoforms and at higher concentrations even the atypical PKC (43). In agreement with this, GF109203X inhibited both the TPA- and TPA+thapsigargin-stimulated adenylyl cyclase activity with equal potency (Fig. 5C). Thus, for some reason, TPA does basally only stimulate the novel, i.e. diacylglycerol-stimulated but not Ca²⁺-stimulated, PKC isoforms.

OX₁ receptors are known to strongly and with high potency activate PLC and elevate Ca²⁺ in CHO cells (14), and therefore PKC activation would be a likely way for them to activate adenylyl cyclase in the presence of activated G_s. The effectivity of the PKC inhibitors was thus evaluated with respect to the orexin-A-induced cAMP elevation in the presence of G_s-stimulation. GF109203X fully and concentration-dependently inhibited the cAMP elevation caused by stimulation of orexin receptors in the CTx-treated cells (Fig. 5D). In contrast, Gö6976 was completely ineffective at the concentrations up to 10 µM (Fig. 5D). Thus, in the presence of activated G_s, adenylyl cyclase stimulation via OX₁ receptors appears to fully rely on some novel PKC isoform(s). The fact that no conventional PKC isoforms appear to be involved is somewhat surprising considering that OX₁ receptor stimulation strongly elevates intracellular Ca²⁺ level.

We further wanted to investigate the activation pathway for PKC. In agreement with our previous study (14), OX₁ receptors strongly activated PLC in CHO cells, as assessed using biochemical measurement of total inositol phosphate generation (Fig. 6A) and measurement of translocation of GFP-labeled PKC or - (only GFP-PKC shown in Fig. 6B), which should be a measure of diacylglycerol generation. Release of diacylglycerol by PLC (likely the isoform) could thus make a plausible signal pathway for OX₁ receptor signaling to novel PKC. In agreement with this, U-73122, inhibitor of the phosphatidylinositol-specific phospholipase C, concentration-dependently inhibited orexin-A-induced cAMP accumulation (IC₅₀ = 3 µM; Fig. 6C). The inhibition occurred at similar potency as the inhibition of PLC (IC₅₀ = 1.5 µM) and appears specific because U-73122 did not inhibit TPA-mediated adenylyl cyclase activation (not shown). Orexin receptors have also been suggested to signal via phosphatidylcholine-specific phospholipase C. This suggestion is based on the inhibitory effect of D609 (see e.g. Ref. 17), a putative inhibitor of this enzyme. However, we did not observe any inhibition with D609 (10 µM, 30 min; Fig. 6C). Phosphorylation of PKC by 3-phosphoinositide-dependent kinase 1 (PDK1) seems to be essential for the ability of PKC to be activated. 3-Phosphoinositide-dependent kinase 1 is activated upon phosphoinositide 3-kinase-dependent production of phosphatidylinositol-3,4,5-trisphosphate, and some of our data in CHO cells suggest that phosphoinositide 3-kinase is activated upon OX₁ receptor stimulation.² We therefore treated the cells with the phosphoinositide 3-kinase inhibitor wortmannin (100 nM, 30 min) prior to orexin stimulation. This did not affect the orexin-induced cAMP elevation. Altogether, the data with the inhibitors and direct measurements of inositol phosphate and diacylglycerol liberation suggest that orexin receptor signaling to novel PKC relies on activation of the classical phosphatidylinositol-specific phospholipase C.

We wanted to more specifically identify the novel PKC isoform involved in the OX₁ receptor stimulation of adenylyl cyclase. The novel class of PKC contains PKC, PKC, PKC, and PKC. CHO cells have previously been suggested to express both PKC and PKC (44). In agreement with this, we could detect the presence of PKC and - but not PKC or PKC in Western blot (Fig. 6D). As expected, TPA pretreatment (2 µM, 24 h) led to a partial down-regulation of both PKC and PKC (Fig. 6D). We applied specific inhibitors of each expressed isoform, rottlerin for PKC (45) and KIE1–1 for PKC (34, 35), and measured cAMP generation in the CTx-treated cells. Rottlerin (30 min of preincubation) produced a concentration-dependent (IC₅₀ =3.5 µM) inhibition of orexin-A-induced cAMP accumulation (Fig. 6E). In contrast, KIE1–1 (1 µM, 30 min of preincubation) had no effect on orexin-A-mediated cAMP elevation (Fig. 6E), although this concentration fully inhibited orexin-A-induced translocation of GFP-PKC from the cytosol to the plasma membrane (data not shown). Thus, the data suggests that only PKC is involved in the orexin response to adenylyl cyclase.

PLC is activated by OX₁ receptors at relatively high potency (EC₅₀ = 1.5–15 nM, depending on the method used; Ref. 14; see also Fig. 6A), which suggests that PKC activation should be of similar potency. This was indeed true; when the potency of orexin-A to stimulate adenylyl cyclase in CTx-treated cells was investigated, it was shown to be very similar to PLC activation (pEC₅₀ = 8.06 ± 0.14 (EC₅₀ = 8.78 nM); number of independent measurements = 8) (Fig. 7A, circles). Thus, orexin-A was 35-fold more potent in CTx-treated cells than in control cells. Orexin-B was also more potent in the CTx-treated than in nontreated cells (pEC₅₀ = 6.99 ± 0.15 (EC₅₀ = 103 nM); number of independent measurements = 3) (Fig. 7A, triangles), although the difference was only 5-fold. This leads to more than 10-fold higher potency of orexin-A than orexin-B in CTx-treated cells. Thus, in CTx-treated cells, both the absolute and relative potencies of both orexin-A and -B were very similar to those observed for instance for Ca²⁺ elevations (Fig. 3, open symbols).

View larger version (16K): [in this window] [in a new window]   FIG. 7. Concentration response relationship for orexin-A and -B in the presence of activated Gs (CTx or 2-adrenoreceptor stimulus) in intact CHO cells. A, the cells were pretreated with 10 ng/ml CTx for 18 h. B and C, the cells were transfected with the 2-adrenoreceptor harboring vector 48 h in advance. isoprot refers to the 2-adrenoreceptor agonist isoproterenol. ctrl, control.

In the cells where adenylyl cyclase is maximally stimulated with G_s, OX₁ receptor signaling to adenylyl cyclase is thus entirely a result of activation of PKC. However, we have in Fig. 2A shown that the PKC activator TPA only very weakly stimulates adenylyl cyclase under basal conditions, i.e. in the absence of activated G_s. Thus, it appears unlikely that OX₁ receptor signaling to adenylyl cyclase under basal conditions would fully rely on PKC. In agreement with this logic, the PKC inhibitor GF109203X, but not Gö6976, reduced the orexin-A-stimulated cAMP elevation maximally by about 60% under basal conditions (Fig. 8). Both the PKC inhibitor rottlerin and the PLC inhibitor U-73122 inhibited orexin-A-induced cAMP elevation in the same degree as GF109203X (Fig. 8).

Based on the low activity of TPA (Fig. 2A) and from the incomplete inhibitory effect of GF109203X on orexin-A signal (above) in non-CTx-treated cells, it appears likely that OX₁ receptor stimulation also generates some other signal that enhances the ability of activated PKC to activate adenylyl cyclase. Because we have shown that the adenylyl cyclase isoform(s) expressed in CHO cells are not Ca²⁺-sensitive (Fig. 2), two possible signals are left, G and G_s. An effect mediated by G appears unlikely because overexpression of G₁₂-subunits was found to have no effect on cAMP production in the absence of simultaneous G_s stimulation (Fig. 2A) However, we further wanted to verify this by expressing G scavengers. The use of G scavengers may be tricky; low expression levels of the scavengers produce no inhibition, whereas higher levels can be toxic to the cells (own observation). This latter was also found to be the case in this study (data not shown). To find an expression level high enough to be fully active but still not toxic, several scavengers were tested for their ability to inhibit the signal in a paradigm generally accepted to rely on G signaling, _2A-adrenoreceptor-induced Ca²⁺ elevations (via G-mediated PLC activation). The cells were thus transiently transfected with the different constructs as described under "Ca²⁺ Measurements." The only G scavenger that had an inhibitory effect on this signal in our hands was transducin, which inhibited the UK14,304 (a selective ₂-adrenoreceptor agonist)-induced Ca²⁺ signal by about 50% upon overexpression, which is the effectivity often seen in other studies with G scavengers (46, 47). Having established the effectiveness of this scavenger, we examined its effects on cAMP signaling in our cells. No effect on orexin-induced cAMP elevation in cells under basal conditions or pretreated with CTx could be seen (Fig. 9); we thus conclude that G-subunits are unlikely to mediate orexin-induced cAMP elevation.

View larger version (20K): [in this window] [in a new window]   FIG. 9. The effect of transducin (Gt) expression on the resting and orexin-A-elevated cAMP level in control (–CTX) and CTx-pretreated (10 ng/ml, 18 h; +CTX) intact CHO cells. All of the control data (white bars) were normalized to 100%, and the transducin data were normalized to its corresponding control, with which it also is compared.

View larger version (14K): [in this window] [in a new window]   FIG. 10. Adenylyl cyclase activity in permeabilized CHO cells. A, stimulation of adenylyl cyclase activity by different compounds. The first comparison is with the basal activity and the second to the to 1 µM isoproterenol alone. B, the effect of the anti-Gs IgG on the stimulated adenylyl cyclase activity. The antibody data (black bars) are normalized to the corresponding controls in the absence of the antibody (white bars), with which they also are compared. C, concentration response curve for orexin-A. ctrl, control.

Inhibitory Coupling of OX₁ Receptors to Adenylyl Cyclase—A thorough analysis of OX₁ receptor regulation of adenylyl cyclase showed that in addition to the stimulatory components characterized above, it also incorporated an inhibitory component. This was seen as an inhibition of the basal cAMP production at low orexin concentrations (by 21 ± 5%, number of independent measurements = 5) in non-CTx-treated cells (Fig. 11, filled circles). This component was abolished when the cells were preincubated with pertussis toxin (Fig. 11, open circles). The inhibition of cAMP generation is most likely due to the action of G_i (or G_o; see Ref. 32), because G-subunits did not appear to have any effect on orexin receptor regulation of adenylyl cyclase (Figs. 2 and 9). The inhibitory component was not seen in the CTx-treated cells, most likely because the potency of the inhibitory and stimulatory components overlapped (data not shown)

View larger version (16K): [in this window] [in a new window]   FIG. 11. The effect of pertussis toxin on the cAMP production stimulated by orexin-A in intact CHO cells. Because there was no large difference in the basal or maximum stimulated levels in control (filled circles) or pertussis toxin-pretreated cells (empty circles), the data were normalized to same basal and maximum levels to better visualize the effect of pertussis toxin (PTx).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As indicated in the Introduction, different adenylyl cyclase subtypes are distinguishable based on their specific regulatory inputs. The specific adenylyl cyclase expression profile of CHO cells is not known, but, as with all the cell types, they can be expected to express several isoforms. The basal testing suggests that Ca²⁺-stimulated isoforms AC1, AC3, and AC8 are not expressed in high amounts. In contrast, some PKC-stimulated isoforms, AC2, AC5, and AC7, are likely to be present. In agreement with previous studies in native cells and upon overexpression (32, 49, 51–53), the effect of PKC on adenylyl cyclase was clearly enhanced by simultaneous G_s activation. There possibly also was a small response to overexpression of G, suggesting that some isoform stimulated by G, AC2, AC4, or AC7, could be present, which is reasonable considering that AC2 and AC7 are also stimulated by PKC. It may be difficult to make conclusions of the effects of exogenous G because there might be compensatory mechanisms activated by the long term G elevation (coexpression), and the overexpression of G₁₂ might distort the signaling of G-proteins. Yet what seems clear is that G are not important for the OX₁ receptor stimulation of adenylyl cyclase. Instead, the results most strongly suggest that adenylyl cyclase stimulation via OX₁ receptors occurs via G_s and protein kinase C, apparently the novel -isoform, in a manner where G_s is essentially permissive for the PKC effect. For G_s to be permissive for the PKC effect, it should act on the same adenylyl cyclase molecule as PKC. Therefore, it appears that the different stimulatory signals from the OX₁ receptor in most part target a single adenylyl cyclase isoform or a few isoforms with apparently similar regulatory properties.

The data suggest that PKC coupling of OX₁ receptors occurs via activation of PLC. In the light of the previous direct (10) and indirect data (14, 54), it appears thus likely that this is a result of "the classical" G_q PLC pathway. This is in agreement with our recent data showing high affinity interaction between OX₁ receptors and G_q/11³. Although this pathway would suggest rather nonselective signaling to different PKC via diacylglycerol and Ca²⁺, we could see that only novel PKC, in particular PKC, and not conventional PKC were involved in the OX₁ receptor stimulation of adenylyl cyclase activity. In a similar manner, the basal response to the diacylglycerol mimetic TPA in the presence of activated G_s seemed to be mediated by novel PKC, but the response could be further stimulated by Ca²⁺ elevations (thapsigargin) via a conventional PKC-dependent manner. The latter condition should apparently simulate OX₁ receptor stimulation, which leads to diacylglycerol and inositol 1,4,5-trisphosphate release via PLC and Ca²⁺ elevation via receptor- and store-operated Ca²⁺ influx and inositol 1,4,5-trisphosphate-dependent Ca²⁺ release (14, 20). However, no involvement of conventional PKC could be seen in the orexin response, although the Ca²⁺ elevation is even larger than that induced by thapsigargin. The reason for this can only be speculated. One possible explanation would be that orexin receptors, PLC, and particularly PKC (and adenylyl cyclase) somehow congregate with each other. In contrast, TPA causes a general (nonlocalized) PKC stimulation.

In this study we performed cAMP measurements both in intact cells and in permeabilized cells. Intact cells are a method of choice, but permeabilization allowed us to manipulate the intracellular side of the cells. We decided to use permeabilized cells instead of membrane preparations because this appeared to be more gentle for the cells, seen as better responses. The use of permeabilized cells confirmed the hypothesis of a direct activation of G_s by OX₁ receptors and also the fact that this component determines the potency of the cAMP elevation in the absence of other G_s stimulus. However, we also saw that the PKC dependence of the OX₁ receptor-mediated regulation of adenylyl cyclase vanished upon permeabilization of the cells. In some studies, PKC-dependent regulation of adenylyl cyclase activity is retained in membrane preparations. However, the stimulatory responses to adenylyl cyclase are in general strongly reduced by cell permeabilization/membrane preparation (48–50). This holds true for PKC-stimulated adenylyl cyclase activity; AC2, heterologously expressed in DDT₁-MF2 cells, is stimulated by 172% in intact cell, whereas the stimulation is only 29% in the membranes (49). If PKC activity is reduced upon cell permeabilization, the rather modest ability of PKC to stimulate adenylyl cyclase activity in our CHO cells is probably reduced significantly enough to be undetectable.

In the present study we observed an interesting difference in the ability of orexins to elevate cAMP in the absence and in the presence of activated G_s (CTx or ₂-adrenoreceptor activation). Both orexins were markedly more potent in the presence of activated G_s, and in permeabilized cells, where no PKC effect on adenylyl cyclase was seen, the potency of orexin-A was low. This led to the hypothesis, supported by other data, that the coupling of OX₁ receptors to phospholipase C is of high efficacy and to G_s is of low efficacy. In the presence of activated G_s, a typical 10-fold higher potency of orexin-A than orexin-B (see Introduction) was observed. This is logical in the light of the fact that this response is fully dependent on the activation of PKC, which occurs via PLC. The much lower potency observed in the absence of activated G_s is in the light of the present study fully determined by the low efficacy coupling of OX₁ receptors to G_s, because the high efficacy coupling to PKC cannot alone stimulate adenylyl cyclase. Most interestingly, there was only a 2-fold difference in the potency of orexin-A and -B peptides in the absence of activated G_s. Thus, one important conclusion of this study is that OX₁ receptors do not have to strongly prefer orexin-A over orexin-B, as commonly thought (see Introduction), and the use of orexin-A and -B in pharmacological distinction of orexin receptor subtypes should be discouraged.

In one previous study, OX₁ and OX₂ receptor coupling to adenylyl cyclase regulation has been investigated in recombinant BIM cells (8). In this study, surprisingly, none of the receptors stimulated cAMP production, and only OX₂ receptor was able to inhibit forskolin-stimulated adenylyl cyclase activity in a pertussis toxin-sensitive manner. This is in sharp contrast to the findings of the present study. As OX₁ receptors couple to G_s they should be able to elevate cAMP in all the cells types, because all the membrane bound adenylyl cyclase isoforms respond to G_s. Also, we can clearly see a coupling of OX₁ receptors to pertussis toxin-sensitive inhibition of adenylyl cyclase. It is possible that this difference between our results and the results of Zhu et al. (8) is caused by the lack of expression of some particular G_i/o isoform in BIM cells. However, it is rather clear that the results of Zhu et al. (8) do not represent any general difference between orexin receptor subtypes.

In conclusion, we have in this study shown that OX₁ orexin receptors couple to at least three different signal pathways to regulate adenylyl cyclase activity: 1) G_i/o to inhibit cAMP generation, 2) G_s to stimulate cAMP generation, and 3) PKC (via PLC and, putatively, G_q) to stimulate cAMP generation. This is the first demonstration of the functional significance of the previously suggested G_s coupling (for the OX₂ receptor; Ref. 10). This coupling may be able to explain the orexin-stimulated cAMP accumulation and glucocorticoid synthesis in the adrenal cortex, which is unlikely to occur via Ca²⁺- or PKC-stimulated adenylyl cyclases (27, 55, 56). The general of role of adenylyl cyclase in orexin receptor signaling is speculative, because almost no studies have addressed this question. However, because orexin receptors can connect to G_s, they should have a principle ability to produce some adenylyl cyclase activation in all cell types, something that might be further regulated via the effect of Ca²⁺ and PKC on specific adenylyl cyclase isoforms. Therefore, it appears likely that cAMP is a signal of importance for orexin receptors. On the pharmacological side, the results of the present study also demonstrate that the OX₁ receptor does not have to display any absolute selectivity between orexin-A and -B.

	FOOTNOTES

 
^* This work was supported by European Union Contract QLG3-CT-2002-00826, the Åke Wiberg Foundation, the Lars Hierta Foundation, the Göran Gustafsson Foundation, the Novo Nordisk Foundation, the Academy of Finland, and the Sigrid Jusélius Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Neuroscience, Division of Physiology, Uppsala University, BMC, P.O. Box 572, SE-75123 Uppsala, Sweden. Tel.: 46-18-471-4171; Fax: 46-18-50-6357; E-mail: jyrki.kukkonen{at}fysiologi.uu.se.

¹ The abbreviations used are: PLC, phospholipase C; AC, adenylyl cyclase; CTx, cholera toxin; GF109203X, bisindolylmaleimide I (or Gö6850), 2-(1-[3-dimethylaminopropyl]-1H-indol-3-yl)-3-(1H-indol-3-yl)-maleimide; PKC, protein kinase C; TBM, TES-buffered medium; TES, 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino) ethane sulfonic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate; U-73122, 1-(6-[([17b]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione; CHO, Chinese hamster ovary; GFP, green fluorescent protein; PBS, phosphate-buffered saline; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo-(3,4-c)-carbazole.

² S. Ammoun, L. Johansson, T. Holmqvist, A. S. Danis, L. Korhonen, K. E. O. Åkerman, and J. P. Kukkonen, manuscript in preparation.

³ J. Magga, G. Bart, C. Oker-Blom, K. E. O. Åkerman, and J. Näsman, manuscript in preparation.

	ACKNOWLEDGMENTS

 
All of the scientists who have provided us with plasmids and compounds (see "Chemicals" and "Expression Vectors" under "Experimental Procedures") are gratefully acknowledged. We also gratefully acknowledge Professor Jari Koistinaho (A. I. Virtanen Institute, Kuopio, Finland) for useful comments and Ludwig Petersson for technical assistance.

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