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- Ammoun, Sylwia, et al.
(författare)
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Distinct Recognition of OX1 and OX2 Receptors by Orexin Peptides
- 2003
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Ingår i: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0022-3565 .- 1521-0103. ; 305:2, s. 507-514
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca(2+) responses (influx and release) in human OX(1) and OX(2) receptor- expressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca(2+), suggesting similar activation of Ca(2+) influx as we have previously shown for orexin-A and OX(1) receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX(1) and OX(2) receptors, but the response mediated by the OX(2) receptor was more resistant to truncation than the response mediated by the OX(1) receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A(14-33). Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX(2) receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca(2+) dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX(2) receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10- to 100-fold lower affinity and potency for the OX(1) receptor.
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| 2. |
- Ammoun, Sylwia, et al.
(författare)
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G-protein-coupled OX1 Orexin/hcrtr-1 Hypocretin Receptors Induce Caspase-dependent and -independent Cell Death through p38 Mitogen-/Stress-activated Protein Kinase
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281, s. 834-842
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the signaling of OX1 receptors to cell death using Chinese hamster ovary cells as a model system. OX1 receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca2+ elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominant-negative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.
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| 3. |
- Ammoun, Sylwia, 1968-
(författare)
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Orexin Receptors in Recombinant CHO Cells : Signaling to Short- and Long-Term Cell Responses
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Recently discovered neuropeptides orexins (orexin-A and -B) act as endogenous ligands for G-protein-coupled receptors called OX1 and OX2 receptors. Our previous studies have established model systems for investigation of the pharmacology and signaling of these receptors in recombinant CHO cells. OX1 receptor-expressing CHO cells were mainly utilized in this thesis.Orexin-A and -B activate both OX1 and OX2 receptors. However, orexin-B is less potent in activating OX1 receptors than orexin-A, whereas the peptides are equipotent on OX2 receptors. We have performed mutagenesis on orexin-A to investigate the basis for this selectivity. We show that OX2 receptor is generally less affected by the mutations and thus OX2 receptor appears to have less strict requirements for ligand binding, likely explaining the lack of difference in affinity/potency between orexin-A and orexin-B on OX2 receptor.The other studies focus on orexin receptor signaling. OX1 receptors are shown to regulate adenylyl cyclase both in positive and negative manner, activate different MAP-kinases (ERK1/2 and p38) and induce cell death after long-lasting stimulation. Adenylyl cyclase regulation occurs likely through three different G-protein families, Gi, Gs and Gq. For ERK1/2, several downstream pathways, such as Ras, Src, PI3-kinase and protein kinase C (PKC) are implicated. OX1 receptor-mediated activation of ERK is suggested to be cytoprotective whereas p38 MAP-kinase induces programmed cell death. Three particularly interesting findings were made. Firstly, novel PKC δ (delta) is suggested to regulate adenylyl cyclase, whereas conventional and atypical PKCs are involved in activation of ERK. Secondly, adenylyl cyclase and ERK activation is fully dependent on extracellular Ca2+. Further experiments suggest that the previously discovered receptor-operated Ca2+ influx is not affecting the downstream effectors of orexin receptors but that it instead enables orexin receptors to couple to several signal cascades. Thirdly, upon inhibition of caspases, classical mediators of programmed cell death, OX1 receptor-mediated cell death is not reversed, but instead the pathways to death are altered so de novo gene transcription is no longer required for cell death.
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| 4. |
- Ammoun, Sylwia, et al.
(författare)
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OX1 orexin receptors activate extracellular signal-regulated kinase in Chinese hamster ovary cells via multiple mechanisms : the role of Ca2+ influx in OX1 receptor signaling
- 2006
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 20:1, s. 80-99
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Tidskriftsartikel (refereegranskat)abstract
- Activation of OX1 orexin receptors heterologously expressedin Chinese hamster ovary (CHO) cells led to a rapid, strong,and long-lasting increase in ERK phosphorylation (activation).Dissection of the signal pathways to ERK using multiple inhibitorsand dominant-negative constructs indicated involvement of Ras,protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly,Ca2+ influx appeared central for the ERK response in CHO cells,and the same was indicated in recombinant neuro-2a cells andcultured rat striatal neurons. Detailed investigations in CHOcells showed that inhibition of the receptor- and store-operatedCa2+ influx pathways could fully attenuate the response, whereasinhibition of the store-operated Ca2+ influx pathway alone orthe Ca2+ release was ineffective. If the receptor-operated pathwaywas blocked, an exogenously activated store-operated pathwaycould take its place and restore the coupling of OX1 receptorsto ERK. Further experiments suggested that Ca2+ influx, as such,may not be required for ERK phosphorylation, but that Ca2+,elevated via influx, acts as a switch enabling OX1 receptorsto couple to cascades leading to ERK phosphorylation, cAMP elevation,and phospholipase C activation. In conclusion, the data suggestthat the primary coupling of orexin receptors to Ca2+ influxallows them to couple to other signal pathways; in the absenceof coupling to Ca2+ influx, orexin receptors can act as signalintegrators by taking advantage of other Ca2+ influx pathways.
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| 5. |
- Holmqvist, Tomas, et al.
(författare)
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OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:8, s. 6570-6579
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the mechanism of OX(1) 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 (EC(50) approximately 1 nm) inhibition and a strong (4-fold), low potency (EC(50) = approximately 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 (pEC(50) = approximately 500 nm). cAMP elevation was not caused by Ca(2+) elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, 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 (EC(50) of orexin-A = approximately 10 nm and EC(50) of orexin-B = approximately 100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s) antibodies. We conclude that OX(1) 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 activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX(1) receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca(2+) elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.
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