OX1Orexin Receptors Activate Extracellular Signal-Regulated Kinase in Chinese Hamster Ovary Cells via Multiple Mechanisms: The Role of Ca2+Influx in OX1Receptor Signaling

Ammoun, Sylwia; Johansson, Lisa; Ekholm, Marie E.; Holmqvist, Tomas; Danis, Alexander S.; Korhonen, Laura; Sergeeva, Olga A.; Haas, Helmut L.; Åkerman, Karl E.O.; Kukkonen, Jyrki P.

doi:10.1210/me.2004-0389

Cited by 80 publications

(126 citation statements)

References 65 publications

Supporting

Mentioning

119

Contrasting

Unclassified

Order By: Relevance

“…While little is currently understood about the mechanisms of regulation by the OX1R in GT1-7 cells, our results on phosphorylation of ERK1 and ERK2 are compatible with a recent study that demonstrated activation of ERK1/2 by orexin A in HEK-293T cells and in Chinese hamster ovary cells transfected with OX1R [59, 60]. …”

Section: Discussionsupporting

confidence: 92%

Orexin A Induces GnRH Gene Expression and Secretion from GT1-7 Hypothalamic GnRH Neurons

et al. 2006

View full text Add to dashboard Cite

Orexin A, a recently discovered hypothalamic peptide, has been shown to have a stimulatory effect on release of gonadotropin-releasing hormone (GnRH) from rat hypothalamic explants in vitro. However, it is presently unclear whether in vivo this effect is mediated directly at the level of the GnRH neuron, or via multiple afferent neuronal connections. Therefore, in the present study, we investigated the direct action of orexin A on GnRH neurons using the immortalized GnRH-secreting GT1-7 hypothalamic cells. Orexin-1 receptor (OX1R) expression was detected in GT1-7 cells by RT-PCR and Western blot. Results showed that 0.1–1 nM orexin A, when administered in culture media for 4 h, can significantly stimulate GnRH mRNA expression in GT1-7 cells (p < 0.05). Administration of 1 µM OX1R antagonist, SB-334867, completely blocked the observed orexin A responses in these cells, indicating that orexin A stimulation of GnRH neurons is specifically through OX1R. Moreover, 0.1 nM orexin A stimulated GnRH release after 30–45 min. To examine possible signal transduction pathways involved in mediating these effects, a MEK inhibitor (UO-126), PKC inhibitor (calphostin C), and PKA inhibitor (H-89), were used, with each blocking orexin A-induced GnRH transcription and release from immortalized cells. Collectively, our results show that orexin A is capable of directly stimulating GnRH transcription and neuropeptide release from these immortalized hypothalamic neurons, and that the effects of orexin A appear to be mediated via the OX1R, coupled with activation of the PKC-, MAPK- and PKA-signaling pathways. It is suggested that the stimulatory effect of orexin A on GnRH transcription and release may also occur directly at the level of GnRH neurons in vivo.

show abstract

Section: Discussionsupporting

confidence: 92%

Orexin A Induces GnRH Gene Expression and Secretion from GT1-7 Hypothalamic GnRH Neurons

et al. 2006

View full text Add to dashboard Cite

show abstract

“…It has been well documented that intracellular protein kinase C (PKC) signaling pathway is involved in orexin receptorsmediated [Ca 2+ ] i mobilizing (1,14,22,23,33). We then determined whether orexin A regulates [Ca 2+ ] i of PFC neurons by modulation of PKC signaling pathway.…”

Section: Resultsmentioning

confidence: 99%

Implicación de los canales L de Ca2+ en el efecto de la orexina A sobre la entrada de Ca2+ extracelular en neuronas de la corteza prefrontal de rata

et al. 2009

View full text Add to dashboard Cite

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca2+ concentration ([Ca2+]i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca2+]i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1-1 microM) induced increase of [Ca2+]i in a dose-dependent manner. This elevation of [Ca2+]i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca2+ stores by the endoplasmic reticulum inhibitor thapsigargin (2 pM), [Ca2+]i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca2+-free condition, orexin A failed to induce any changes of Ca2+ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca2+]i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 microM) and D609 (10 microM), respectively. Selective blockade of L-type Ca2+ channels by nifedipine (5 microM) significantly suppressed the elevation of [Ca2+]i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca2+]i in neurons from deep layers of PFC, which depends on extracellular Ca2+ influx via L-type Ca2+ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.

show abstract

“…To investigate whether the orexin-A-induced cell death also required de novo synthesis of mRNA, we treated CHO cells with an inhibitor of gene transcription, actinomycin D. At the concentration of 10 nM, actinomycin D only weakly affected cell viability, but it fully reversed orexin-A-induced cell death (Fig. 2B) (10,17) and activation of ERK, PKC, and PI3K (4,7,14). Chelation of intracellular Ca 2ϩ with 1 M BAPTA-AM significantly improved cell viability in response to thapsigargin, as expected, but did not affect orexin-A-mediated cell death (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, orexin receptors have been suggested to regulate cell plasticity. ERK (extracellular signal-regulated kinase) is strongly activated in CHO cells heterologously expressing OX 1 receptors (4,6,7). In the hippocampus, orexin receptors regulate synaptic plasticity (8).…”

mentioning

confidence: 99%

G-protein-coupled OX1 Orexin/hcrtr-1 Hypocretin Receptors Induce Caspase-dependent and -independent Cell Death through p38 Mitogen-/Stress-activated Protein Kinase

Ammoun

Lindholm

Wootz

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

We have investigated the signaling of OX 1 receptors to cell death using Chinese hamster ovary cells as a model system. OX 1 receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca 2؉ 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 dominantnegative 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.There is a growing interest in the ability of G-protein-coupled receptors (GPCRs) 2 to affect synaptic plasticity and cell differentiation as well as cell growth, death, and survival (reviewed in Refs. 1-3). However, the mechanisms of GPCR signaling in these processes are poorly characterized. The pathways engaged seem to include classical GPCR signals like second messenger-dependent activation of protein kinase A and C but also (for GPCRs) more novel signal transducers like small G-proteins (of Ras and Rho families), PI3K (phosphatidylinositol 3-kinase) and nonreceptor tyrosine kinases (e.g. Src).In this study we have focused on the signaling of a novel GPCR, OX 1 orexin receptor. Little is known about the intracellular signaling of OX 1 receptor and its cognate receptor, OX 2 receptor. Orexin receptor-expressing neuronal, endocrine, and muscle cells often seem to be excited by orexins, an effect that may be related to activation of Ca 2ϩ or nonselective cation channels (reviewed in Refs. 4 and 5). Recently, orexin receptors have been suggested to regulate cell plasticity. ERK (extracellular signal-regulated kinase) is strongly activated in CHO cells heterologously expressing OX 1 receptors (4, 6, 7). In the hippocampus, orexin receptors regulate synaptic plasticity (8). Lo...

show abstract

OX₁Orexin Receptors Activate Extracellular Signal-Regulated Kinase in Chinese Hamster Ovary Cells via Multiple Mechanisms: The Role of Ca²⁺Influx in OX₁Receptor Signaling

Cited by 80 publications

References 65 publications

Orexin A Induces GnRH Gene Expression and Secretion from GT1-7 Hypothalamic GnRH Neurons

Orexin A Induces GnRH Gene Expression and Secretion from GT1-7 Hypothalamic GnRH Neurons

Implicación de los canales L de Ca2+ en el efecto de la orexina A sobre la entrada de Ca2+ extracelular en neuronas de la corteza prefrontal de rata

G-protein-coupled OX1 Orexin/hcrtr-1 Hypocretin Receptors Induce Caspase-dependent and -independent Cell Death through p38 Mitogen-/Stress-activated Protein Kinase

Contact Info

Product

Resources

About