Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.
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PMID:Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell-line using FLIPR. 1049 27

The orexin-orexin receptor system has been implicated in the regulation of wakefulness/sleep states. Behavioral and psycho-stimulant effects of orexins have also been shown. Mesolimbic dopamine neurons in the ventral tegmental area (VTA) are implicated in the regulation of reward and wakefulness/sleep, In the present study, we examined the effect of orexin-A on cytosolic [Ca2+]i concentration ([Ca2+]) in the isolated rat VTA dopamine neurons. Orexin-A (10-12-10-8 M) concentration dependently increased [Ca2+]i in dopamine-containing neurons. The [Ca2+]i responses to orexin-A were inhibited under Ca2+-free conditions and by blockers of voltage-gated L- and N-type [Ca2+]i channels, nitrendipine and omega-conotoxin, respectively. The [Ca2+]i responses were also abolished by a phosphatidylcholine-specific phospholipase C inhibitor, D609, and a protein kinase C (PKC) inhibitor, calphostin C. A PKC activator, TPA, mimicked orexin-A in increasing [Ca2+]i. These results indicate that orexin-A increases [Ca2+]i in VTA dopamine neurons via phosphatidylcholine-specific PLC- and PKC-mediated activation of L- and N-type Ca2+ channels. This effect may serve as the mechanism by which orexin regulates wakefulness/sleep states and exerts its behavioral and psychostimulant effects.
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PMID:Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area. 1143 17

Orexin (ORX)-A is a 33-amino acid peptide with demonstrated roles in the regulation of energy metabolism, autonomic control, and sleep. Orexin receptors (OXRs), OX1R and OX2R, and immunoreactive axons are present in the nucleus tractus solitarius (NTS). We demonstrated previously that bath application of ORX-A depolarizes NTS neurons through activation of a nonselective cationic conductance (NSCC) and inhibition of a sustained potassium current (IK). The present study examined the signaling pathways underlying the excitatory effects of ORX-A on NTS neurons using whole-cell patch-clamp recording techniques. Inclusion of guanosine 5'-O-(2-thiodiphosphate) in the internal pipette solution abolished the effects of ORX-A, confirming that the actions of ORX-A are mediated by G-protein-coupled receptors. The responses of ORX-A were also blocked by a phospholipase C (PLC) inhibitor, D609, and by a nonselective protein kinase (PK) inhibitor, H7, demonstrating the involvement of PLC and protein kinases. However, PKA appears not to play a role, because the depolarizing effects of ORX-A were still observed when the PKA inhibitor peptide (6-22) was included in the pipette solution, and bath application of 8-bromo-cAMP (a PKA agonist) was without effect on NTS neurons. In contrast, 12-O-tetradecanoylphorbol-13-acetate (a PKC agonist) depolarized NTS neurons, and bisindolylmaleimide (BIS), a PKC inhibitor, abolished the depolarizing effects of ORX-A. Finally, voltage-clamp experiments demonstrated that BIS also blocked the activation of NSCC and inhibition of IK by ORX-A in NTS neurons. These results therefore show that the excitatory effects of ORX-A on NTS neurons are mediated through activation of the PLC-PKC-NSCC and -IK signaling pathways, which probably result from OXR-coupled activation of Gq.
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PMID:Excitatory effects of orexin-A on nucleus tractus solitarius neurons are mediated by phospholipase C and protein kinase C. 1286 5

Signal transduction pathways of orexin receptors were examined using a nerve-like cell line transfected with orexin receptor type-1 (OX1R) and orexin receptor type-2 (OX2R). Forskolin-stimulated cyclic adenosine 3,5-monophosphate (cAMP) accumulation in OX2R-expressing cells was inhibited by orexin in a dose-dependent manner, and the effect was abolished by pretreatment with pertussis toxin (PTX). The inhibitory effect of orexin on forskolin-stimulated cAMP accumulation was not observed in OX1R-expressing cells. Administration of orexin to these cells resulted in a transient increase of intracellular calcium concentration ([Ca(2+)](i)). Orexin-stimulated increases in [Ca(2+)](i) in OX1R- or OX2R-expressing cells were not affected by the PTX pretreatment. These observations suggest that OX1R couples exclusively to PTX-insensitive G-proteins, while OX2R couples to both PTX-sensitive and -insensitive G-proteins. To examine the relative contributions of these G-proteins in OX2R-mediated activation of neurons, we used histaminergic tuberomammillary nucleus neurons, in which OX2R is abundantly expressed. We found that a phospholipase C (PLC)-inhibitor, U73122, inhibits orexin-mediated neuronal activation, but PTX showed no effect on it. This suggests that although OX2R couples to multiple G-proteins, activation of neurons by orexins through OX2R is mediated via a PTX-insensitive, PLC dependent pathway.
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PMID:Orexin receptor type-1 couples exclusively to pertussis toxin-insensitive G-proteins, while orexin receptor type-2 couples to both pertussis toxin-sensitive and -insensitive G-proteins. 1450 Nov 63

Orexin-A and -B (hypocretin-1 and -2) have been implicated in the stimulation of feeding. Here we show the effector neurons and signaling mechanisms for the orexigenic action of orexins in rats. Immunohistochemical methods showed that orexin axon terminals contact with neuropeptide Y (NPY)- and proopiomelanocortin (POMC)-positive neurons in the arcuate nucleus (ARC) of the rats. Microinjection of orexins into the ARC markedly increased food intake. Orexins increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the isolated neurons from the ARC, which were subsequently shown to be immunoreactive for NPY. The increases in [Ca(2+)](i) were inhibited by blockers of phospholipase C (PLC), protein kinase C (PKC) and Ca(2+) uptake into endoplasmic reticulum. The stimulation of food intake and increases in [Ca(2+)](i) in NPY neurons were greater with orexin-A than with orexin-B, indicative of involvement of the orexin-1 receptor (OX(1)R). In contrast, orexin-A and -B equipotently attenuated [Ca(2+)](i) oscillations and decreased [Ca(2+)](i) levels in POMC-containing neurons. These effects were counteracted by pertussis toxin, suggesting involvement of the orexin-2 receptor and Gi/Go subtypes of GTP-binding proteins. Orexins also decreased [Ca(2+)](i) levels in glucose-responsive neurons in the ventromedial hypothalamus (VMH), a satiety center. Leptin exerted opposite effects on these three classes of neurons. These results demonstrate that orexins directly regulate NPY, POMC and glucose-responsive neurons in the ARC and VMH, in a manner reciprocal to leptin. Orexin-A evokes Ca(2+) signaling in NPY neurons via OX(1)R-PLC-PKC and IP(3) pathways. These neural pathways and intracellular signaling mechanisms may play key roles in the orexigenic action of orexins.
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PMID:Orexins (hypocretins) directly interact with neuropeptide Y, POMC and glucose-responsive neurons to regulate Ca 2+ signaling in a reciprocal manner to leptin: orexigenic neuronal pathways in the mediobasal hypothalamus. 1506 49

Orexin-A (OR-A) and OR-B are derived from a common 130-amino acid precursor peptide, prepro-OR, by proteolytic cleavage. Orexins orchestrate their actions by binding and activating two types of G protein-coupled receptors, OR-1 receptor (OX1R) and OR-2 receptor (OX2R). Besides playing a role in the regulation of feeding and energy homeostasis in rats, ORs appear to increase sexual arousal as well as copulatory performance in rats. Furthermore, OR-A and -B immunoreactivity has been detected in rat testis. In view of these findings we investigated the expression of OR receptors and their signaling characteristics in the human male reproductive system. Using RT-PCR analysis, we were able to demonstrate that both OX1R and OX2R are expressed in the testis, epididymis, penis, and seminal vesicle, whereas prepro-OR was expressed only in the epididymis and penis. Protein expression of both OR receptors in human testis was confirmed by Western blotting analysis, with apparent molecular masses of 50 kDa for OX1R and 40 kDa for OX2R. Immunofluorescent studies revealed staining for both OX1R and OX2R in Leydig cells, myoid cells of the seminiferous tubules, and Sertoli cells. To test the ability of ORs to activate testicular phospholipase C, we determined the effects of OR-A and OR-B on inositol trisphosphate production. When membranes from testicular biopsies were incubated with OR-A or OR-B, there was a rapid inositol trisphosphate turnover. This effect appeared to be dose dependent. These data provide a novel insight into the expression and signaling characteristics of OR receptors in the human male reproductive system and potentially further implicate these peptides, acting in an autocrine/paracrine manner, in the regulation of arousal mechanisms in humans.
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PMID:Expression of human prepro-orexin and signaling characteristics of orexin receptors in the male reproductive system. 1507 Sep 69

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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PMID:OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms. 1561 Nov 18

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

Orexin-A and orexin-B are hypothalamic peptides that act via two G protein-coupled receptors, named orexin type 1 and type 2 receptors (OX1-Rs and OX2-Rs). The most studied biological functions of orexins are the central control of feeding and sleep, but in the past few years findings that orexin system modulates the hypothalamic-pituitary-adrenal (HPA) axis, acting on both its central and peripheral branches, have accumulated. Orexins and their receptors are expressed in the hypothalamic paraventricular nucleus and median eminence and orexin receptors in pituitary corticotropes, adrenal cortex, and medulla. Whereas the effects of orexins on adrenal aldosterone secretion are doubtful, compelling evidence indicates that these peptides enhance glucocorticoid production in rats and humans. This effect involves a 2-fold mechanism: 1) stimulation of the adrenocorticotropin-releasing hormone-mediated pituitary release of adrenocorticotropin, which in turn raises adrenal glucocorticoid secretion; and 2) direct stimulation of adrenocortical cells via OX1-Rs coupled to the adenylate cyclase-dependent cascade. The effects of orexins on catecholamine release from adrenal medulla are unclear and probably of minor relevance, but there are indications that orexins can stimulate in vitro secretion of human pheochromocytoma cells via OX2-Rs coupled to the phospholipase C-dependent cascade. Evidence is also available that orexins enhance the growth in vitro of adrenocortical cells, mainly acting via OX2-Rs. Moreover, findings suggest that the orexin system may favor HPA axis responses to stresses and play a role in the pathophysiology of cortisol-secreting adrenal adenomas.
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PMID:Orexins in the regulation of the hypothalamic-pituitary-adrenal axis. 1650 82

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


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