Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human corticotropin-releasing factor (hCRF) receptors CRF1 and CRF2(a) couple to the Gs protein. It has been postulated that CRF receptors may also signal through phospholipase C (PLC). To test this hypothesis, binding and signaling properties were determined for both receptor subtypes stably expressed in human embryonic kidney 293 (HEK293) and human SK-N-MC neuroblastoma cells. CRF receptors were highly expressed and strongly coupled to Gs in HEK293 and SK-N-MC cells. However, when the calcium mobilization pathway was investigated, marked differences were observed. In SK-N-MC cells, neither CRF receptor stimulated calcium mobilization in the fluorometric imaging plate reader (FLIPR) assay, whereas activation of orexin type 1 and 2 receptors stably expressed in SK-N-MC cells revealed robust calcium responses. In contrast, intracellular calcium was strongly mobilized by agonist stimulation of hCRF1 and hCRF2(a) receptors in HEK293 cells. In HEK293 cells, potency rank orders for calcium and cAMP responses were identical for both receptors, despite a rightward shift of the dose-response curves. Complete inhibition of calcium signaling of both hCRF1 and hCRF2(a) receptors was observed in the presence of the PLC inhibitor U-73,122 whereas ryanodine, an inhibitor of calcium release channels and the protein kinase A inhibitor Rp-cAMPS were ineffective. Finally, CRF agonists produced a small but significant stimulation of inositol 1,4,5-triphosphate (IP3) accumulation in hCRF1-and hCRF2(a)-transfected HEK293 cells. These data clearly show that phospholipase C-mediated signaling of CRF receptors is dependent upon the cellular background and that in HEK293 cells human CRF receptors robustly respond in the FLIPR format.
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PMID:Cell-type specific calcium signaling by corticotropin-releasing factor type 1 (CRF1) and 2a (CRF2(a)) receptors: phospholipase C-mediated responses in human embryonic kidney 293 but not SK-N-MC neuroblastoma cells. 1545 Sep 49

We have investigated the direct excitatory effects of hypocretin-1 on acutely isolated prefrontal cortical pyramidal neurons and explored the signaling mechanisms of these actions. Puff application of hypocretin-1 caused an excitation in the recorded neurons. These effects of hypocretin-1 were abolished by a phospholipase C inhibitor D609, demonstrating that phospholipase C mediates the actions of hypocretin-1. A specific protein kinase C inhibitor, bisindolylmaleimide II, blocked the excitatory actions of hypocretin-1, suggesting that protein kinase C plays a key role. Finally, protein kinase A inhibitor applied intracellularly did not affect the responses. These results indicate that hypocretin-1 excites prefrontal neurons by activation of phospholipase C and protein kinase C pathways, but not protein kinase A.
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PMID:Signaling pathways of hypocretin-1 actions on pyramidal neurons in the rat prefrontal cortex. 1611 Feb 84

Orexins A and B are hypothalamic peptides, that act via two subtypes of receptors, named OX1-R and OX2-R. Rat and human adrenal cortexes are provided with both OX1-R and OX2-R, and we have previously shown that orexin-A, but not orexin-B, enhances glucocorticoid secretion from dispersed adrenocortical cells. Since OX1-Rs preferentially bind orexin-A and OX2-Rs are non-selective for both orexins, the hypothesis has been advanced that the secretagogue effect of orexin-A is exclusively mediated by the OX1-R. Here, we aimed to verify this contention and to gain insight into the signaling mechanism(s) underlying the secretagogue effect of orexins using primary cultures of rat and human adrenocortical cells. Reverse transcription-polymerase chain reaction showed that cultured cells, as freshly dispersed cells, expressed both OX1-R and OX2-R mRNAs. Orexin-A, but not orexin-B, concentration-dependently increased corticosterone and cortisol secretion from cultured rat and human adrenocortical cells, respectively. The blockade of OX1-Rs by selective antibodies abrogated the secretagogue effect of orexin-A, while the immuno-blockade of OX2-Rs was ineffective. The glucocorticoid response of cultured cells to orexin-A was annulled by the adenylate cyclase and protein kinase (PK) A inhibitors SQ-22536 and H-89, and unaffected by the phospholipase C and PKC inhibitors U-73122 and calphostin-C. Orexin-A, but not orexin-B, enhanced cyclic-AMP production from cultured cells, and did not alter inositol-3-phosphate release. Collectively, our present results allow us to conclude that orexins stimulate glucocorticoid secretion from rat and human adrenocortical cells, exclusively acting through OX1-Rs coupled to the adenylate cyclase/PKA-dependent signaling cascade.
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PMID:Orexins stimulate glucocorticoid secretion from cultured rat and human adrenocortical cells, exclusively acting via the OX1 receptor. 1615 81

Hypocretinergic (orexinergic) neurons in the hypothalamus project to the nucleus pontis oralis, a nucleus which plays a crucial role in the generation of active (rapid eye movement) sleep. We recently reported that the microinjection of hypocretin into the nucleus pontis oralis of chronically-instrumented, unanesthetized cats induces a behavioral state that is comparable to naturally-occurring active sleep. The present study examined the intracellular signaling pathways underlying the active sleep-inducing effects of hypocretin. Accordingly, hypocretin-1, a protein kinase C inhibitor and a protein kinase A inhibitor were injected into the nucleus pontis oralis in selected combinations in order to determine their effects on sleep and waking states of chronically instrumented, unanesthetized cats. Microinjections of hypocretin-1 into the nucleus pontis oralis elicited active sleep with a short latency. However, a pre-injection of bisindolylmaleimide-I, a protein kinase C-specific inhibitor, completely blocked the active sleep-inducing effects of hypocretin-1. The combined injection of bisindolylmaleimide-I and hypocretin-1 significantly increased the latency to active sleep induced by hypocretin-1; it also abolished the increase in the time spent in active sleep induced by hypocretin-1. On the other hand, the injection of 2'5'-dideoxyadenosine, an adenylyl cyclase inhibitor, did not block the occurrence of active sleep by hypocretin-1. We conclude that the active sleep-inducing effect of hypocretin in the nucleus pontis oralis is mediated by intracellular signaling pathways that act via G-protein stimulation of protein kinase C.
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PMID:Neuronal mechanisms of active (rapid eye movement) sleep induced by microinjections of hypocretin into the nucleus pontis oralis of the cat. 1653 74

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 microM 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.
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PMID:Orexin A induces GnRH gene expression and secretion from GT1-7 hypothalamic GnRH neurons. 1719 2

The effect of group I metabotropic glutamate receptor (mGluR1 and mGluR5) activation on identified melanin-concentrating hormone (MCH) neurons was studied using patch-clamp recording in hypothalamic slices from green fluorescent protein-expressing transgenic mice. S-3,5-dihydroxyphenylglycine (DHPG), a selective group I mGluR agonist, depolarized MCH cells and increased spike frequency. The mGluR-mediated depolarization was not blocked with tetrodotoxin but was significantly reduced by replacement of extracellular Na+ with Tris, by Ni2+ or the Na+/Ca2+ exchanger blocker KB-R7943, or with BAPTA in the pipette, consistent with a mechanism based on activation of the Na+/Ca2+ exchanger. DHPG also decreased potassium currents. DHPG-induced depolarization was reduced by either mGluR1 or mGluR5 antagonists, suggesting involvement of both receptor subtypes. DHPG-induced depolarization desensitized; blockade of mGluR1 prevented the desensitization. Group I mGluR activation enhanced NMDA-evoked currents; this enhancement was remarkably long lasting and could be blocked by protein kinase A or C blockers. DHPG potentiated electrically evoked NMDA receptor-mediated postsynaptic currents, and mGluR5 antagonists blocked this action. Group I mGluRs increased spontaneous EPSCs in MCH neurons, possibly by stimulation of nearby mGluR-expressing hypocretin neurons. We found no tonic activation of mGluRs. However, electrical stimulation produced a slow inward current, which could be blocked by group I mGluR antagonists, suggesting high, but not low, levels of synaptically released glutamate activated mGluRs. Together, group I mGluRs increase MCH neuron activity by multiple presynaptic and postsynaptic mechanisms, suggesting mGluRs may therefore play a role in hypothalamic signaling relating to MCH neuron modulation of food intake and energy metabolism.
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PMID:Rapid direct excitation and long-lasting enhancement of NMDA response by group I metabotropic glutamate receptor activation of hypothalamic melanin-concentrating hormone neurons. 1795 99

Orexins mediate a variety of physiological processes, including feeding behavior, the circadian pathway, and cortisol secretion. Steroidogenesis is regulated by a variety of neuropeptides, and one of the key rate-limiting steps is cholesterol transport across the mitochondrial membrane by the steroidogenic acute regulatory protein (StAR). StAR expression can be regulated through several different signaling pathways. Despite the clear link between orexins and steroid production, the actions of the orexin family of hormones on steroid biosynthesis are not fully understood. We present data showing that 100 nm of both orexins A and B for 4 or 24 h significantly up-regulates StAR, in H295R pluripotent adrenocortical cells. We present the dose-dependent and time-dependent characteristics of StAR up-regulation at the protein level, showing significant increases after 4 h at a relatively low agonist concentration (1 nm). We have provided a key analysis of the precise G protein-coupled signaling pathways required for the up-regulation of StAR in response to orexins A and B. This has involved dominant-negative G protein analysis, and the direct inhibition of the protein kinase A, protein kinase C, ERK1/2, and p38 pathways. This shows a fundamental role for multiple G protein-coupled and MAPK-mediated signaling pathways leading to StAR expression. Antagonist analysis also showed that orexin effects on StAR were primarily, but not exclusively, acting through the orexin receptor type 1. This is the first study linking orexin action on StAR expression and comprehensively describes the signaling pathways involved in regulating the complexity of hormone biosynthesis.
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PMID:Orexins stimulate steroidogenic acute regulatory protein expression through multiple signaling pathways in human adrenal H295R cells. 1845 Sep 61

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, OX1R and OX2R, which activate multiple G-proteins. In many tissues, orexins activate extracellular signal-regulated kinase (ERK(1/2)) and p38 mitogen-activated protein kinase (MAPK); however, the mechanism by which OX2R alone mediates MAPK activation is not understood. This study describes the intracellular signalling pathways involved in OX2R-mediated ERK(1/2) and p38 MAPK activation. In HEK-293 cells stably over-expressing recombinant human OX2R, orexin-A/B resulted in a rapid, dose and time dependent increase in activation of ERK(1/2) and p38 MAPK, with maximal activation at 10 min for ERK(1/2) and 30 min for p38 MAPK. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK(1/2) and p38 MAPK activation through multiple G-proteins and different intracellular signalling pathways. ERK(1/2) activation involves Gq/phospholipase C (PLC)/protein kinase C (PKC), Gs/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and Gi cascades; however, the Gq/PLC/PKC pathway, as well as PKA is not required for OX2R-mediated p38 MAPK activation. Interestingly, orexin-B-induced ERK(1/2) activation is predominantly mediated through the Gq/PLC/PKC pathway. In conclusion, this is the first comprehensive signalling study of the human OX2R recombinant receptor, showing ERK(1/2) and p38 MAPK activation are regulated by differential signalling pathways in HEK-293 cells, and that the ERK(1/2) activation is severely affected by naturally occurring mutants associated with narcolepsy. Moreover, it is evident that the human OX2R has ligand specific effects, with orexin-B being more potent in this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.
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PMID:The signalling profile of recombinant human orexin-2 receptor. 1859 70

Burst firing mediated by a low-threshold spike (LTS) is the hallmark of many thalamic neurons. However, postburst afterhyperpolarizations (AHPs) are relatively uncommon in thalamus. We now report data from patch-clamp recordings in rat brain slice preparations that reveal an LTS-induced slow AHP (sAHP) in thalamic paraventricular (PVT) and other midline neurons, but not in ventrobasal or reticular thalamic neurons. The LTS-induced sAHP lasts 8.9 +/- 0.4 s and has a novel pharmacology, with resistance to tetrodotoxin and cadmium and reduction by Ni(2+) or nominally zero extracellular calcium concentration, which also attenuate both the LTS and sAHP. The sAHP is inhibited by 10 mM intracellular EGTA or by equimolar replacement of extracellular Ca(2+) with Sr(2+), consistent with select activation of LVA T-type Ca(2+) channels and subsequent Ca(2+) influx. In control media, the sAHP reverses near E(K(+)), shifting to -78 mV in 10.1 mM [K(+)](o) and is reduced by Ba(2+) or tetraethylammonium. Although these data are consistent with opening of Ca(2+)-activated K(+) channels, this sAHP lacks sensitivity to specific Ca(2+)-activated K(+) channel blockers apamin, iberiotoxin, charybdotoxin, and UCL-2077. The LTS-induced sAHP is suppressed by a beta-adrenoceptor agonist isoproterenol, a serotonin 5-HT(7) receptor agonist 5-CT, a neuropeptide orexin-A, and by stimulation of the cAMP/protein kinase A pathway with 8-Br-cAMP and forskolin. The data suggest that PVT and certain midline thalamic neurons possess an LTS-induced sAHP that is pharmacologically distinct and may be important for information transfer in thalamic-limbic circuitry during states of attentiveness and motivation.
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PMID:Properties of a T-type Ca2+channel-activated slow afterhyperpolarization in thalamic paraventricular nucleus and other thalamic midline neurons. 1932 37

Hypocretins/orexins act through two receptor subtypes: OX(1) and OX(2). Outside the brain, orexin receptors are expressed in adrenal glands, where orexins stimulate the release of glucocorticoids. To further address the regulation of steroidogenesis, we analyzed the effect of orexins on the expression of steroidogenic enzymes in human adrenocortical National Cancer Institute (NCI) H295R cells by qPCR. In NCI H295R cells, OX(2) receptors were highly expressed, as they were in human adrenal glands. After treatment of NCI H295R cells with orexin A for 12-24 h, the cortisol synthesis rate was significantly increased, whereas 30 min of treatment showed no effect. While CYP11B1 and CYP11B2 mRNA levels were increased already at earlier time points, the expression of HSD3B2 and CYP21 mRNA was significantly up-regulated after treatment with orexin A for 12 h. Likewise, orexin B increased CYP21 and HSD3B2 mRNA levels showing, however, a lower potency compared with orexin A. The mRNA levels of CYP11A and CYP17 were unaffected by orexin A. OX(2) receptor mRNA levels were down-regulated after 12 and 24 h of orexin A treatment. Orexin A increased intracellular Ca(2+) but not cAMP concentrations in NCI H295R cells. Furthermore, inhibition of PKC and MAPK kinase/ERK kinase (MEK1/2) prevented the increase of HSD3B2 expression by orexin A. Accordingly, orexin A treatment of NCI H295R cells markedly enhanced ERK1/2 phosphorylation that was prevented by PKC and, in part, PKA inhibition. In conclusion, orexins may influence adrenal steroidogenesis by differential regulation of the expression of steroidogenic enzymes involving Ca(2+), as well as PKC-ERK1/2 signaling.
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PMID:Hypocretin/orexin increases the expression of steroidogenic enzymes in human adrenocortical NCI H295R cells. 1979 50


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