Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of the regulation by muscarinic acetylcholine receptors (mAChRs) of intracellular cAMP levels and cAMP-regulated gene expression. Treatment with the muscarinic agonist carbachol results in an increase in luciferase activity in JEG-3 cells transiently transfected with mouse m1 (8-10-fold) and chick m4 (3-5-fold) mAChRs. Control experiments indicate that these responses are not due to a calcium-mediated pathway and are dependent upon a functional protein kinase A. The m1 and m4 responses are not sensitive to pertussus toxin and the m4 response was potentiated by it. Thus, these responses do not result from direct stimulation of adenylate cyclase by beta gamma subunits released from pertussis toxin-sensitive G-proteins. Atropine treatment of cells transfected with high levels of m4 mAChR, but not m1, causes an elevation in basal levels of cAMP response element-mediated luciferase expression in the absence of agonist. This suggests that the m4 receptor is spontaneously active and can cause constitutive inhibition of adenylyl cyclase that is relieved by atropine treatment. Surprisingly, the m4 receptor exhibits little if any agonist-induced inhibition of luciferase expression at either low or high levels of receptor expression. JEG-3 cells express Gi alpha-1 and Gi alpha-3 but not Gi alpha-2. Cotransfection of Gi alpha subunits with m4 demonstrates that the m4 receptor requires Gi alpha-2 for optimal agonist-mediated inhibition. Even in the presence of Gi alpha-2, high levels of receptor increased luciferase expression at high concentrations of agonist. Thus, the m4 mAChR can undergo a switch in functional coupling from inhibition to stimulation of adenylyl cyclase. This switch is dependent on the level of receptor expression, the subtypes of G-proteins coexpressed with the receptor, and the concentration of agonist. Furthermore, we demonstrate that the Gi alpha-2 G-protein alpha subunit preferentially couples the m4 mAChR to inhibition of adenylyl cyclase in JEG-3 cells.
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PMID:Differential regulation of cAMP-mediated gene transcription by m1 and m4 muscarinic acetylcholine receptors. Preferential coupling of m4 receptors to Gi alpha-2. 814 70

The baculovirus expression vector system utilizing the strong polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) was used for high level expression of the two alpha 2-adrenoceptor subtypes alpha 2A-C10 and alpha 2B-C2 in Spodoptera frugiperda (Sf-9) insect cells. For rapid screening of recombinant viruses the luciferase gene was expressed under the early ETL-promoter (early transcript large) in the same plasmid. Both receptor subtypes showed the same rank order of binding affinity for four agonists tested: dexmedetomidine > l-medetomidine = clonidine > noradrenaline. For the alpha 2A-C10 subtype, these agonists inhibited forskolin stimulated cAMP production through pertussis toxin sensitive G-proteins. In contrast, for the alpha 2B-C2 subtype the agonists stimulated both basal and forskolin stimulated cAMP production.
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PMID:Two human alpha 2-adrenoceptor subtypes alpha 2A-C10 and alpha 2B-C2 expressed in Sf9 cells couple to transduction pathway resulting in opposite effects on cAMP production. 857 36

We have characterized the interaction of endothelin (ET) with cultured neonatal rat ventricular myocytes. Binding studies indicate a single population of ETA receptors [53,000 sites/cell, apparent dissociation constant (Kd) for ET-1 approximately 0.07 nM]. Analysis of mRNA levels for ET receptors using 35 cycles of reverse transcriptase-polymerase chain reaction demonstrates the presence of only ETA-receptor message. Studies with ET-1 and a variety of congeners and antagonists indicate that ETA receptors couple to both the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase. In myocytes transfected with an atrial natriuretic factor (ANF) promoter linked to a luciferase reporter gene, ET-1 stimulates luciferase expression through an ETA receptor. These data indicate that the ETA receptor is the exclusive receptor on neonatal ventricular myocytes and that this receptor couples to both phosphoinositide hydrolysis and adenylyl cyclase. ET-1 also induces a threefold increase in mitogen-activated protein kinase (MAPK) activity, an effect that is not sensitive to pertussis toxin (PTx). By contrast, ET-stimulated ANF-luciferase expression is partially inhibited by treatment of cells with PTx, suggesting that both PTx-sensitive (Gi) and PTx-insensitive (Gq) pathways mediate the effects of ET-1 on ANF gene expression in neonatal myocytes and that hormonal regulation of ANF expression may utilize pathways in addition to the activation of MAPK.
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PMID:Endothelin ETA receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways. 903 31

We reported previously that somatostatin inhibits the expression of the immediate early gene c-fos. Accordingly, we characterized the molecular mechanisms by which somatostatin inhibits c-fos gene expression. Because growth factors activate c-fos through a region of its promoter known as the serum response element [SRE; base pairs (bp) -357 to -276] we transfected rat pituitary adenoma cells (GH3) with plasmids containing the SRE or the SRE core fragment (bp -320 to -298) upstream of the luciferase reporter gene. Epidermal growth factor (EGF) stimulated SRE-luciferase activity, and this effect was inhibited by somatostatin and by the analog MK-678. Identical results were obtained with the SRE core plasmid, demonstrating that the sequence between bp -320 and -298 of the c-fos promoter is a somatostatin response element. Because the extracellular signal-regulated protein kinases (ERKs) induce the SRE via phosphorylation of transcription factors such as Elk-1, we examined the effect of somatostatin on ERK phosphorylation and activation. EGF stimulated tyrosine phosphorylation of ERK2, and MK-678 attenuated this effect. In experiments using in-gel kinase assays, MK-678 also inhibited EGF-stimulated ERK activity via a pertussis toxin sensitive pathway, and this effect resulted in inhibition of Elk-1 transcriptional activity. Our data suggest that one mechanism of somatostatin action involves inhibition of ERK activity, Elk-1 phosphorylation and transcriptional activation, and ultimately c-fos gene transcription.
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PMID:Molecular mechanisms for somatostatin inhibition of c-fos gene expression. 914 1

In cultured rat hepatocytes, the gluconeogenic key enzyme, phosphoenolpyruvate carboxykinase (PCK), is induced by glucagon via elevation of cyclic 3',5' adenosine monophosphate (cAMP). The proinflammatory cytokine, interleukin-6 (IL-6), which in the liver together with IL-1beta and tumor necrosis factor alpha triggers the acute-phase response, had been shown to attenuate the glucagon-induced increase in PCK gene transcription, messenger (mRNA) levels, and enzyme activity. The molecular mechanism of this inhibition was investigated in the present study. Glucagon increased cyclic cAMP and PCK mRNA levels to a transient maximum twofold and fivefold, respectively. The increases were attenuated by IL-6. Forskolin, which stimulates adenylate cyclase activity, increased cAMP and PCK mRNA levels 1.6-fold and fivefold, respectively. However, IL-6 attenuated the forskolin-stimulated increase in PCK mRNA but not the increase in cAMP. This showed that IL-6 inhibited PCK mRNA increase in part by the attenuation of cAMP increase, but also beyond cAMP formation. This was confirmed in experiments in which PCK mRNA levels were increased by the nonhydrolyzable cAMP-analogue, chlorophenylthio (CPT)-cAMP. The increase in PCK mRNA was again attenuated by IL-6. In pertussis toxin- and in isobutylmethylxanthine-treated hepatocytes, IL-6 still inhibited the glucagon-stimulated increase in cAMP, indicating that IL-6 did not activate an inhibitory G-protein or phosphodiesterase, which could cause the impairment of cAMP increase. To demonstrate whether the inhibition of PCK gene expression by IL-6 beyond cAMP might be caused by the inhibition of the activation of the PCK gene promoter by cAMP, cultured rat hepatocytes were transfected with a luciferase reporter gene construct under the control of a PCK gene promoter fragment (base -979 to base +32). Luciferase activity was determined after stimulation of the cells with CPT-cAMP in the absence or presence of IL-6. CPT-cAMP increased luciferase activity by 1.7-fold, which was inhibited in the presence of IL-6. It is concluded that IL-6 had a dual inhibitory effect on the stimulation of PCK gene expression by glucagon. It inhibited the increase in cAMP at a site before cAMP formation by adenylate cyclase and at a site after cAMP formation, the activation of the PCK gene promoter by cAMP.
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PMID:Mechanism of the impairment of the glucagon-stimulated phosphoenolpyruvate carboxykinase gene expression by interleukin-6 in rat hepatocytes: inhibition of the increase in cyclic 3',5' adenosine monophosphate and the downstream cyclic 3',5' adenosine monophosphate action. 921 54

A cyclic AMP-responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose-dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein-coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5-HT) receptor agonist ligands caused an inhibition of forskolin-stimulated luciferase expression in the rank order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin. The response to 5-HT was reversed by the 5-HT1 receptor antagonists cyanopindolol and pindolol, but not the 5-HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene-related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8-37). These results were consistent with the presence of 5-HT(1B-like) and calcitonin (C1a-like) receptors in CHO cells, with the responses to 5-HT and CGRP being pertussis and cholera toxin-sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein-coupled receptors linked to adenylyl cyclase.
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PMID:Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene. 928 53

We have previously observed that gastrin has a cholecystokinin B (CCK-B) receptor-mediated growth-promoting effect on the AR42J rat pancreatic acinar cell line and that this effect is paralleled by induction of expression of the early response gene c-fos. We undertook these experiments to elucidate the mechanism for induction of c-fos and the linkage of this action to the trophic effects of gastrin. Gastrin (0.1-10 nM) dose dependently induced luciferase activity in AR42J cells transfected with a construct consisting of a luciferase reporter gene coupled to the serum response element (SRE) of the c-fos promoter. This effect was blocked by the specific CCK-B receptor antagonist D2 but not by the specific CCK-A receptor antagonist L-364,718 or by pertussis toxin, indicating that gastrin targets the SRE via specific CCK-B receptors through a mechanism independent of Gi. Inhibition of protein kinase C (PKC) either by prolonged (24 h) exposure of the cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (100 nM) or by incubation with the selective inhibitor GF-109203X (3.5 microM) resulted in an 80% reduction in luciferase activity. Similar results were observed in the presence of the specific extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD-98059 (50 microM). We measured ERK2 activity in AR42J cells via in-gel kinase assays and observed that gastrin (1 pM-100 nM) induced ERK2 enzyme activity in a dose-dependent manner. Addition of GF-109203X and PD-98059, either alone or in combination, produced, respectively, partial and total inhibition of gastrin-induced ERK2 activity. Gastrin induction of ERK2 activity also resulted in a threefold increase in the transcriptional activity of Elk-1, a factor known to bind to the c-fos SRE and to be phosphorylated and activated by ERK2. PD-98059 blocked the growth-promoting effect of gastrin on the AR42J cells, demonstrating that this effect depends on activation of MEK. Our data lead us to conclude that the trophic actions of gastrin are mediated by ERK2-induced c-fos gene expression via PKC-dependent and -independent pathways.
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PMID:Molecular mechanisms for the growth factor action of gastrin. 935 32

A Chinese hamster ovary (CHO) cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was stably transfected with the long form of the rat D2 dopamine receptor. Saturation binding analysis using [3H]spiperone showed that the receptor was expressed at low levels (Bmax = 96.5+/-15.8 fmol/mg), but with an affinity characteristic of the D2 receptor (Kd = 21.5+/-3.7 pM). Luciferase expression in this cell line was modified in a dose dependent manner with dopamine receptor agonists (N-propylapomorphine > apomorphine > quinpirole > dopamine) and antagonists (spiperone > (+)-butaclamol > D0710 > (-)-sulpiride > tiapride > remoxipride), according to their rank order of potency in binding and cAMP accumulation studies. Dopamine-mediated inhibition of forskolin-stimulated luciferase expression was pertussis toxin sensitive. This demonstrated the efficiency of the luciferase reporter gene assay for the functional testing of D2 dopamine receptors, which are negatively coupled to the adenylyl cyclase signaling pathway, when heterogously expressed at low levels in CHO cells.
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PMID:Functional analysis of the D2L dopamine receptor expressed in a cAMP-responsive luciferase reporter cell line. 969 85

The pineal hormone, melatonin, inhibits proliferation of estrogen receptor (ER)-positive MCF-7 human breast cancer cells, modulates both ER mRNA and protein expression, and appears to be serum dependent, indicating interaction between melatonin and serum components. To examine the effects of melatonin on ER activity, ER transactivation assays were performed by transiently transfecting MCF-7 cells with an ERE-luciferase reporter construct. MCF-7 cells pre-treated with melatonin for as little as 5 min followed by either epidermal growth factor (EGF) or insulin resulted in the estrogen-independent transactivation of the ER. None of the compounds when used alone transactivated the ER. The ability of melatonin and EGF to transactivate the ER was abolished by the addition of the antiestrogen, ICI 164384, suggesting that melatonin and EGF co-operate to transactivate the ER. The modulation of ER transactivation was associated with changes in mitogen activated protein kinase activity and ER phosphorylation. This ER transactivation was blocked by pertussis toxin, a Galpha i-protein-coupled receptor inhibitor, suggesting cross talk between the G-protein-coupled melatonin receptor pathway and the EGF/insulin tyrosine kinase receptor pathways in modulating ER transactivation. Exactly how the ability of melatonin in combination with EGF to transactivate the ER relates to melatonin's observed growth suppressive effects is not clear. It is possible that, although melatonin and EGF transactivate the ER, this transactivation does not result in the full transcription of estrogen-responsive genes, but rather, makes the ER refractory to activation by estradiol, thus, blocking the mitogenic actions of estradiol.
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PMID:Estrogen receptor transactivation in MCF-7 breast cancer cells by melatonin and growth factors. 972 86

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric Gi/Go protein-coupled and Gq/G11 protein-coupled receptors. The aims of the current study were: (i) to investigate whether the Gi/Go protein-coupled adenosine A1 receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor. 2. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1+/-0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 microM; 89+/-4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block Gi/Go-dependent pathways). 3. Adenosine A1 receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 microM; 6+/-10% of control). In contrast, daidzein (100 microM), the inactive analogue of genistein had no significant effect (96+/-12 of control). MAP kinase responses to CPA (1 microM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55+/-8% inhibition) and LY 294002 (30 microM; 40+/-5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 microM). 4. Activation of the endogenous P2Y2 purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50=1.6+/-0.3 microM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67+/-3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 microm; 45+/-5% inhibition), indicating the possible involvement of both Gi/Go protein and Gq protein-dependent pathways in the overall response to UTP. 5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting. 6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 microM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 microM). 7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression. 8. In conclusion, our studies have shown that the transfected adenosine A1 receptor and the endogenous P2Y2 purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y2 purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.
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PMID:Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells. 972 63


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