FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2033547042> ?p ?o ?g. }

• W2033547042 endingPage "6579" @default.
• W2033547042 startingPage "6570" @default.
• W2033547042 abstract "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 Gi/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 Gβγ. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCδ, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of Gs 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 PKCδ. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Gαs antibodies. We conclude that OX1 receptors stimulate adenylyl cyclase via a low potency Gs coupling and a high potency phospholipase C → PKC coupling. The former or some exogenous Gs activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX1 receptors couple to Gs 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. 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 Gi/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 Gβγ. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCδ, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of Gs 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 PKCδ. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Gαs antibodies. We conclude that OX1 receptors stimulate adenylyl cyclase via a low potency Gs coupling and a high potency phospholipase C → PKC coupling. The former or some exogenous Gs activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX1 receptors couple to Gs 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. The neuropeptides/hormones orexin-A and -B and the corresponding G-protein-coupled receptors OX1 and OX2 receptor were discovered in 1998 (1de Lecea L. Kilduff T.S. Peyron C. Gao X. Foye P.E. Danielson P.E. Fukuhara C. Battenberg E.L. Gautvik V.T. Bartlett F.S. Frankel W.N. van den Pol A.N. Bloom F.E. Gautvik K.M. Sutcliffe J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 322-327Crossref PubMed Scopus (3247) Google Scholar, 2Sakurai T. Amemiya A. Ishii M. Matsuzaki I. Chemelli R.M. Tanaka H. Williams S.C. Richardson J.A. Kozlowski G.P. Wilson S. Arch J.R. Buckingham R.E. Haynes A.C. Carr S.A. Annan R.S. McNulty D.E. Liu W.S. Terrett J.A. Elshourbagy N.A. Bergsma D.J. Yanagisawa M. Cell. 1998; 92: 573-585Abstract Full Text Full Text PDF PubMed Scopus (3144) Google Scholar). 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. 3Kukkonen J.P. Holmqvist T. Ammoun S. A ̊kerman K.E. Am. J. Physiol. 2002; 283: C1567-C1591Crossref PubMed Scopus (247) Google Scholar). 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 (2Sakurai T. Amemiya A. Ishii M. Matsuzaki I. Chemelli R.M. Tanaka H. Williams S.C. Richardson J.A. Kozlowski G.P. Wilson S. Arch J.R. Buckingham R.E. Haynes A.C. Carr S.A. Annan R.S. McNulty D.E. Liu W.S. Terrett J.A. Elshourbagy N.A. Bergsma D.J. Yanagisawa M. Cell. 1998; 92: 573-585Abstract Full Text Full Text PDF PubMed Scopus (3144) Google Scholar, 4Smart D. Jerman J.C. Brough S.J. Rushton S.L. Murdock P.R. Jewitt F. Elshourbagy N.A. Ellis C.E. Middlemiss D.N. Brown F. Br. J. Pharmacol. 1999; 128: 1-3Crossref PubMed Scopus (167) Google Scholar, 5Okumura T. Takeuchi S. Motomura W. Yamada H. Egashira Si S. Asahi S. Kanatani A. Ihara M. Kohgo Y. Biochem. Biophys. Res. Commun. 2001; 280: 976-981Crossref PubMed Scopus (63) Google Scholar, 6Holmqvist T. A ̊kerman K.E.O. Kukkonen J.P. FEBS Lett. 2002; 526: 11-14Crossref PubMed Scopus (61) Google Scholar, 7Ammoun S. Holmqvist T. Shariatmadari R. Oonk H.B. Detheux M. Parmentier M. Akerman K.E. Kukkonen J.P. J. Pharmacol. Exp. Ther. 2003; 305: 507-514Crossref PubMed Scopus (147) Google Scholar, 8Zhu Y. Miwa Y. Yamanaka A. Yada T. Shibahara M. Abe Y. Sakurai T. Goto K. J. Pharmacol. Sci. 2003; 92: 259-266Crossref PubMed Scopus (144) Google Scholar). 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 (9Kenakin T. Trends Pharmacol. Sci. 2003; 24: 346-354Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), and a similar process has even been suggested for orexin receptors (7Ammoun S. Holmqvist T. Shariatmadari R. Oonk H.B. Detheux M. Parmentier M. Akerman K.E. Kukkonen J.P. J. Pharmacol. Exp. Ther. 2003; 305: 507-514Crossref PubMed Scopus (147) Google Scholar). 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 Gi, Gq, and Gs proteins (10Randeva H.S. Karteris E. Grammatopoulos D. Hillhouse E.W. J. Clin. Endocrinol. Metab. 2001; 86: 4808-4813Crossref PubMed Scopus (121) Google Scholar), 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 Gi/o proteins are engaged in orexin signaling (8Zhu Y. Miwa Y. Yamanaka A. Yada T. Shibahara M. Abe Y. Sakurai T. Goto K. J. Pharmacol. Sci. 2003; 92: 259-266Crossref PubMed Scopus (144) Google Scholar, 11Bernard R. Lydic R. Baghdoyan H.A. Neuroreport. 2002; 13: 447-450Crossref PubMed Scopus (24) Google Scholar, 12Bernard R. Lydic R. Baghdoyan H.A. Eur. J. Neurosci. 2003; 18: 1775-1785Crossref PubMed Scopus (40) Google Scholar, 13Hoang Q.V. Bajic D. Yanagisawa M. Nakajima S. Nakajima Y. J. Neurophysiol. 2003; 90: 693-702Crossref PubMed Scopus (79) Google Scholar). On the other hand, most other responses seen in native cells and cell lines, e.g. Ca2+ elevation, phospholipase C (PLC) 1The 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. 1The 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. activation, and activation of cation channels, are unlikely to be mediated by G1/o proteins. Ca2+ elevation is a prominent response seen in all the cell lines where the receptors have been heterologously expressed (2Sakurai T. Amemiya A. Ishii M. Matsuzaki I. Chemelli R.M. Tanaka H. Williams S.C. Richardson J.A. Kozlowski G.P. Wilson S. Arch J.R. Buckingham R.E. Haynes A.C. Carr S.A. Annan R.S. McNulty D.E. Liu W.S. Terrett J.A. Elshourbagy N.A. Bergsma D.J. Yanagisawa M. Cell. 1998; 92: 573-585Abstract Full Text Full Text PDF PubMed Scopus (3144) Google Scholar, 6Holmqvist T. A ̊kerman K.E.O. Kukkonen J.P. FEBS Lett. 2002; 526: 11-14Crossref PubMed Scopus (61) Google Scholar, 8Zhu Y. Miwa Y. Yamanaka A. Yada T. Shibahara M. Abe Y. Sakurai T. Goto K. J. Pharmacol. Sci. 2003; 92: 259-266Crossref PubMed Scopus (144) Google Scholar, 14Lund P.E. Shariatmadari R. Uustare A. Detheux M. Parmentier M. Kukkonen J.P. A ̊kerman K.E.O. J. Biol. Chem. 2000; 275: 30806-30812Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and in all the native neurons where this has been investigated (15van den Pol A.N. Gao X.B. Obrietan K. Kilduff T.S. Belousov A.B. J. Neurosci. 1998; 18: 7962-7971Crossref PubMed Google Scholar, 16van den Pol A.N. J. Neurosci. 1999; 19: 3171-3182Crossref PubMed Google Scholar, 17Uramura K. Funahashi H. Muroya S. Shioda S. Takigawa M. Yada T. Neuroreport. 2001; 12: 1885-1889Crossref PubMed Scopus (117) Google Scholar, 18van den Pol A.N. Patrylo P.R. Ghosh P.K. Gao X.B. J. Comp. Neurol. 2001; 433: 349-363Crossref PubMed Scopus (84) Google Scholar, 19Kohlmeier K.A. Inoue T. Leonard C.S. J. Neurophysiol. 2004; 92: 221-235Crossref PubMed Scopus (82) Google Scholar). Two different mechanisms seem to be active: (i) Ca2+ influx via a non-voltage-gated pathway in recombinant cells (14Lund P.E. Shariatmadari R. Uustare A. Detheux M. Parmentier M. Kukkonen J.P. A ̊kerman K.E.O. J. Biol. Chem. 2000; 275: 30806-30812Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 20Kukkonen J.P. A ̊kerman K.E.O. Neuroreport. 2001; 12: 2017-2020Crossref PubMed Scopus (50) Google Scholar) and via voltage-gated Ca2+ channels in neurons and some endocrine cells (17Uramura K. Funahashi H. Muroya S. Shioda S. Takigawa M. Yada T. Neuroreport. 2001; 12: 1885-1889Crossref PubMed Scopus (117) Google Scholar, 18van den Pol A.N. Patrylo P.R. Ghosh P.K. Gao X.B. J. Comp. Neurol. 2001; 433: 349-363Crossref PubMed Scopus (84) Google Scholar, 19Kohlmeier K.A. Inoue T. Leonard C.S. J. Neurophysiol. 2004; 92: 221-235Crossref PubMed Scopus (82) Google Scholar, 21Xu R. Wang Q. Yan M. Hernandez M. Gong C. Boon W.C. Murata Y. Ueta Y. Chen C. Endocrinology. 2002; 143: 4609-4619Crossref PubMed Scopus (41) Google Scholar, 22Larsson K.P. A ̊kerman K.E.O. Magga J. Uotila S. Kukkonen J.P. Na ̈sman J. Herzig K.H. Biochem. Biophys. Res. Commun. 2003; 309: 209-216Crossref PubMed Scopus (26) Google Scholar) and (ii) PLC activation and inositol 1,4,5-trisphosphate-dependent Ca2+ release (4Smart D. Jerman J.C. Brough S.J. Rushton S.L. Murdock P.R. Jewitt F. Elshourbagy N.A. Ellis C.E. Middlemiss D.N. Brown F. Br. J. Pharmacol. 1999; 128: 1-3Crossref PubMed Scopus (167) Google Scholar, 7Ammoun S. Holmqvist T. Shariatmadari R. Oonk H.B. Detheux M. Parmentier M. Akerman K.E. Kukkonen J.P. J. Pharmacol. Exp. Ther. 2003; 305: 507-514Crossref PubMed Scopus (147) Google Scholar, 10Randeva H.S. Karteris E. Grammatopoulos D. Hillhouse E.W. J. Clin. Endocrinol. Metab. 2001; 86: 4808-4813Crossref PubMed Scopus (121) Google Scholar, 14Lund P.E. Shariatmadari R. Uustare A. Detheux M. Parmentier M. Kukkonen J.P. A ̊kerman K.E.O. J. Biol. Chem. 2000; 275: 30806-30812Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 23Holmqvist T. A ̊kerman K.E.O. Kukkonen J.P. Neurosci. Lett. 2001; 305: 177-180Crossref PubMed Scopus (40) Google Scholar, 24Mazzocchi G. Malendowicz L.K. Aragona F. Rebuffat P. Gottardo L. Nussdorfer G.G. J. Clin. Endocrinol. Metab. 2001; 86: 4818-4821Crossref PubMed Scopus (49) Google Scholar, 25Karteris E. Chen J. Randeva H.S. J. Clin. Endocrinol. Metab. 2004; 89: 1957-1962Crossref PubMed Scopus (86) Google Scholar). 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 (26Malendowicz L.K. Tortorella C. Nussdorfer G.G. J. Steroid. Biochem. Mol. Biol. 1999; 70: 185-188Crossref PubMed Scopus (122) Google Scholar, 27Mazzocchi G. Malendowicz L.K. Gottardo L. Aragona F. Nussdorfer G.G. J. Clin. Endocrinol. Metab. 2001; 86: 778-782Crossref PubMed Scopus (106) Google Scholar), probably, at least in man, via OX1 receptors (27Mazzocchi G. Malendowicz L.K. Gottardo L. Aragona F. Nussdorfer G.G. J. Clin. Endocrinol. Metab. 2001; 86: 778-782Crossref PubMed Scopus (106) Google Scholar). Although this effect appears to be very similar to the adrenocorticotropic hormone, which utilizes the Gs 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 (28de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1622) Google Scholar). 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. 29Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (742) Google Scholar and 30Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Crossref PubMed Scopus (560) Google Scholar). The most important (known) inputs include G-protein α-subunits (Gαi/o, Gαs), G-protein βγ-subunits (Gβγ), 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, Gβγ 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. Gαs or PKC) (31Tang W.J. Gilman A.G. Science. 1991; 254: 1500-1503Crossref PubMed Scopus (747) Google Scholar, 32Na ̈sman J. Kukkonen J.P. Holmqvist T. A ̊kerman K.E.O. J. Neurochem. 2002; 83: 1252-1261Crossref PubMed Scopus (13) Google Scholar). 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 (2Sakurai T. Amemiya A. Ishii M. Matsuzaki I. Chemelli R.M. Tanaka H. Williams S.C. Richardson J.A. Kozlowski G.P. Wilson S. Arch J.R. Buckingham R.E. Haynes A.C. Carr S.A. Annan R.S. McNulty D.E. Liu W.S. Terrett J.A. Elshourbagy N.A. Bergsma D.J. Yanagisawa M. Cell. 1998; 92: 573-585Abstract Full Text Full Text PDF PubMed Scopus (3144) Google Scholar, 4Smart D. Jerman J.C. Brough S.J. Rushton S.L. Murdock P.R. Jewitt F. Elshourbagy N.A. Ellis C.E. Middlemiss D.N. Brown F. Br. J. Pharmacol. 1999; 128: 1-3Crossref PubMed Scopus (167) Google Scholar, 14Lund P.E. Shariatmadari R. Uustare A. Detheux M. Parmentier M. Kukkonen J.P. A ̊kerman K.E.O. J. Biol. Chem. 2000; 275: 30806-30812Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 20Kukkonen J.P. A ̊kerman K.E.O. Neuroreport. 2001; 12: 2017-2020Crossref PubMed Scopus (50) Google Scholar, 33Kukkonen J.P. Lund P.E. A ̊kerman K.E.O. Cell Calcium. 2001; 30: 117-129Crossref PubMed Scopus (88) Google Scholar) and (ii) it does not appear to express Ca2+-sensitive adenylyl cyclase isoforms, minimizing interference from this strong signal of orexin receptors. Cell Culture—CHO-hOX1 cells, as described before (14Lund P.E. Shariatmadari R. Uustare A. Detheux M. Parmentier M. Kukkonen J.P. A ̊kerman K.E.O. J. Biol. Chem. 2000; 275: 30806-30812Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), 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. Chemicals—Cholera toxin (CTx), GF109203X (bisindolylmaleimide I, Gö6850 (or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide), Gö6976 (or 12-[2-cyanoethyl]-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]-carbazole), ionomycin, and the rabbit polyclonal anti-Gαs IgG were from Calbiochem (La Jolla, CA), and ATP, cAMP, forskolin, 3-isobutyl-1-methylxanthine, (–)-isoproterenol (isoprenaline), pertussis toxin, probenecid (or p-[dipropylsulfamoyl] benzoic acid), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were from Sigma. Human orexin-A and -B were from Neosystem (Strasbourg, France), and fura-2 acetoxymethyl ester was from Molecular Probes Inc. (Eugene, OR). Thapsigargin and UK14,304 (or 5-bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine) were from RBI (Natick, MA); digitonin was from Merck; and rottlerin, U-73122 (1-[6-([(17b)-3-methoxyestra-1,3,5 (10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione), and wortmannin were from Tocris Cookson Ltd (Bristol, UK). Membrane-permeable (carrier peptide-conjugated) selective PKCϵ inhibitor, KIE1–1 (34Gray M.O. Karliner J.S. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 30945-30951Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 35Chen L. Hahn H. Wu G. Chen C.H. Liron T. Schechtman D. Cavallaro G. Banci L. Guo Y. Bolli R. Dorn II, G.W. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11114-11119Crossref PubMed Scopus (478) Google Scholar), was from KAI Pharmaceuticals, Inc. (South San Francisco, CA). [3H]Adenine, [14C]cAMP, and [3H]inositol were from Amersham Biosciences, and [α-33P]ATP was from PerkinElmer Life Sciences. SB-334867 (or 1-[2-methylbenzoxazol-6-yl]-3-(1,5)naphthyridin-4-yl-urea hydrochloride) (36Porter R.A. Chan W.N. Coulton S. Johns A. Hadley M.S. Widdowson K. Jerman J.C. Brough S.J. Coldwell M. Smart D. Jewitt F. Jeffrey P. Austin N. Bioorg. Med. Chem. Lett. 2001; 11: 1907-1910Crossref PubMed Scopus (137) Google Scholar) was a generous gift from Dr. Neil Upton (Neurology CEDD, GlaxoSmithKline Pharmaceuticals, Harlow, UK). 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 μmp-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 β2-adrenoreceptor, human transducin (Gαt-rod), human Gβ1, and human Gγ2 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-βARK1-CT (C terminus of the human β-adrenergic receptor kinase 1 (βARK1)), Dr. J. Silvio Gutkind (NIDCR, National Institutes of Health, Bethesda, MD) for pcDNAIII T8-β ARK (a fusion of the extracellular and transmembrane part of CD8 and the C terminus of the human βARK1), and Dr. Johanna Ivaska (VTT Medical Biotechnology, Centre for Biotechnology, Turku, Finland) for pEGFP-C1-PKCϵwt (fusion of GFP and PKCϵ) (37Ivaska J. Whelan R.D. Watson R. Parker P.J. EMBO J. 2002; 21: 3608-3619Crossref PubMed Scopus (138) Google Scholar). 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 (β2-adrenoreceptor for Gs coupling and α2A-adrenoreceptor for Gβγ-dependent PLC activation (and Ca2+ release)), Gβγ sequestering peptides/proteins (βARK1 C terminus, CD8-βARK1 C terminus, Gαt), 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 (38Kukkonen J.P. Jansson C.C. A ̊kerman K.E.O. Br. J. Pharmacol. 2001; 132: 1477-1484Crossref PubMed Scopus (40) Google Scholar). 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).Fig. 6OX1 receptor activity stimulates adenylyl cyclase in CTx-treated cells via phosphatidylinositol-specific phospholipase C → PKCδ.A and B, PLC activity was assessed using measurement of total inositol phosphate accumulation (A) and GFP-PKCϵ 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 PKCδ inhibitor rottlerin (10 μm) and the PKCϵ inhibitor KIE1–1 (1 μm, 30 min). ctrl, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8The 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Measurement of Adenylyl Cyclase Activity in Permeabilized Cells— Unlabeled cells were detached, washed in TBM, suspended in 50 mm Tris-HCl, 10 mm MgCl2, and 1 mm EDTA (pH 7.6), and permeabilized with 10 μg/ml digitonin. After this they were diluted in 4 volumes of 25 mm Tris-HCl, 100 mm NaCl, 2.5 mm MgCl2, 25 mm cAMP, 25 μm GTP, 100 μm ATP, 0.8 mg/ml creatine phosphokinase, and 5 mm phosphocreatine (pH 7.6). When the effect of anti-Gαs IgG was assessed, permeabilized cells in a minimal volume were incubated with a 1:10 dilution of the antibody for 2 h on ice before diluting in the experimental buffer. The aliquots used as controls were similarly incubated on ice; however, incubation on ice itself only slightly reduced the responses. The reactions were carried out for 15 min at 30 °C in the presence of 0.24 μm [33P]ATP (∼100 000 cpm). The reactions were interrupted by the addition of perchloric acid. Dowex/alumina separation was carried out as above except that the [14C]cAMP tracer was not included. Ca2+Measurements—Ca2+ imaging was performed to evaluate the effectivity of the different Gβγ-sequestering peptides (βARK1 C terminus, CD8-βARK1 C terminus, Gαt). A clear Gβγ-mediated response, α2A-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, α2A-adrenoreceptor, and each Gβγ 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 °" @default.
• W2033547042 created "2016-06-24" @default.
• W2033547042 creator A5000419083 @default.
• W2033547042 creator A5024769409 @default.
• W2033547042 creator A5065899533 @default.
• W2033547042 creator A5074780249 @default.
• W2033547042 creator A5080765918 @default.
• W2033547042 creator A5091067872 @default.
• W2033547042 date "2005-02-01" @default.
• W2033547042 modified "2023-10-13" @default.
• W2033547042 title "OX1 Orexin Receptors Couple to Adenylyl Cyclase Regulation via Multiple Mechanisms" @default.
• W2033547042 cites W1557611662 @default.
• W2033547042 cites W1641731911 @default.
• W2033547042 cites W175842496 @default.
• W2033547042 cites W1810053083 @default.
• W2033547042 cites W1875087350 @default.
• W2033547042 cites W1885709862 @default.
• W2033547042 cites W1915837588 @default.
• W2033547042 cites W1947580298 @default.
• W2033547042 cites W1959892192 @default.
• W2033547042 cites W1967866139 @default.
• W2033547042 cites W1969583788 @default.
• W2033547042 cites W1975089308 @default.
• W2033547042 cites W1977998991 @default.
• W2033547042 cites W1980380395 @default.
• W2033547042 cites W1980734690 @default.
• W2033547042 cites W1984311579 @default.
• W2033547042 cites W1985579250 @default.
• W2033547042 cites W1987306101 @default.
• W2033547042 cites W1989051865 @default.
• W2033547042 cites W1992054477 @default.
• W2033547042 cites W1997463053 @default.
• W2033547042 cites W2009607168 @default.
• W2033547042 cites W2014826286 @default.
• W2033547042 cites W2017384587 @default.
• W2033547042 cites W2020790123 @default.
• W2033547042 cites W2023494473 @default.
• W2033547042 cites W2027721701 @default.
• W2033547042 cites W2027740042 @default.
• W2033547042 cites W2034825884 @default.
• W2033547042 cites W2035380810 @default.
• W2033547042 cites W2036325467 @default.
• W2033547042 cites W2036946165 @default.
• W2033547042 cites W2047308870 @default.
• W2033547042 cites W2051408504 @default.
• W2033547042 cites W2059836980 @default.
• W2033547042 cites W2061617773 @default.
• W2033547042 cites W2066205729 @default.
• W2033547042 cites W2068675667 @default.
• W2033547042 cites W2078447869 @default.
• W2033547042 cites W2082473607 @default.
• W2033547042 cites W2090803400 @default.
• W2033547042 cites W2093881154 @default.
• W2033547042 cites W2101674994 @default.
• W2033547042 cites W2104631706 @default.
• W2033547042 cites W2114591638 @default.
• W2033547042 cites W2115902692 @default.
• W2033547042 cites W2130028880 @default.
• W2033547042 cites W2144001627 @default.
• W2033547042 cites W2152211310 @default.
• W2033547042 cites W2154675993 @default.
• W2033547042 cites W2163162884 @default.
• W2033547042 cites W2164675383 @default.
• W2033547042 cites W2165583928 @default.
• W2033547042 cites W2170355588 @default.
• W2033547042 cites W2222548602 @default.
• W2033547042 doi "https://doi.org/10.1074/jbc.m407397200" @default.
• W2033547042 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15611118" @default.
• W2033547042 hasPublicationYear "2005" @default.
• W2033547042 type Work @default.
• W2033547042 sameAs 2033547042 @default.
• W2033547042 citedByCount "123" @default.
• W2033547042 countsByYear W20335470422012 @default.
• W2033547042 countsByYear W20335470422013 @default.
• W2033547042 countsByYear W20335470422014 @default.
• W2033547042 countsByYear W20335470422015 @default.
• W2033547042 countsByYear W20335470422016 @default.
• W2033547042 countsByYear W20335470422017 @default.
• W2033547042 countsByYear W20335470422018 @default.
• W2033547042 countsByYear W20335470422020 @default.
• W2033547042 countsByYear W20335470422021 @default.
• W2033547042 countsByYear W20335470422022 @default.
• W2033547042 countsByYear W20335470422023 @default.
• W2033547042 crossrefType "journal-article" @default.
• W2033547042 hasAuthorship W2033547042A5000419083 @default.
• W2033547042 hasAuthorship W2033547042A5024769409 @default.
• W2033547042 hasAuthorship W2033547042A5065899533 @default.
• W2033547042 hasAuthorship W2033547042A5074780249 @default.
• W2033547042 hasAuthorship W2033547042A5080765918 @default.
• W2033547042 hasAuthorship W2033547042A5091067872 @default.
• W2033547042 hasBestOaLocation W20335470421 @default.
• W2033547042 hasConcept C118303440 @default.
• W2033547042 hasConcept C126322002 @default.
• W2033547042 hasConcept C134018914 @default.
• W2033547042 hasConcept C169760540 @default.
• W2033547042 hasConcept C170493617 @default.
• W2033547042 hasConcept C185592680 @default.
• W2033547042 hasConcept C2751546 @default.

• next