scholarly journals Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells

1996 ◽  
Vol 316 (1) ◽  
pp. 81-86 ◽  
Author(s):  
Daisuke HIRANO ◽  
Yoshiko AOKI ◽  
Hiroyuki OGASAWARA ◽  
Hisashi KODAMA ◽  
Iwao WAGA ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
A2a Receptor ◽  
Mitogen Activated Protein

Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5´-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0±5.4 nM, Bmax = 707±11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca2+-independent/ wortmannin-sensitive pathway in CHO cells.

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB

1994 ◽  
Vol 267 (4) ◽  
pp. C1130-C1135 ◽  
Author(s):  
Y. Wang ◽  
P. M. Rose ◽  
M. L. Webb ◽  
M. J. Dunn
Keyword(s):  
Protein Kinase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Thymidine Uptake ◽  
Mapk Activation ◽  
Transfected Cells ◽  
Gel Retardation ◽  
Mitogen Activated Protein

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

scholarly journals Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24

1996 ◽  
Vol 320 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Caroline POINOT-CHAZEL ◽  
Marielle PORTIER ◽  
Monsif BOUABOULA ◽  
Natalio VITA ◽  
Florence PECCEU ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cho Cells ◽  
Mapk Pathway ◽  
Chinese Hamster ◽  
Structural Features ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Transcriptional Level ◽  
Single Class ◽  
Mitogen Activated Protein

Neurotensin (NT) is a neuropeptide that is important in a variety of biological processes such as signal transduction and cell growth. NT effects are mediated by a single class of cell-surface receptors, known as neurotensin receptors (NTRs), which exhibit structural features of the G-protein-coupled receptors superfamily. We investigated NTR signalling properties with Chinese hamster ovary (CHO) cells stably transformed with human NTR (hNTR). First, we showed that NTR stimulation by NT induced the activation of the mitogen-activated protein kinases (MAPKs) in time- and dose-dependent manners. Both p42 and p44 MAPK isoforms were retarded in gel-shift assays, which was consistent with their activation by phosphorylation. In addition we showed that NT caused a prolonged activation of MAPK as measured by in-gel kinase assay. Secondly, we demonstrated that NT induced the expression of the growth-related gene Krox-24 at the protein level, as assessed by Western-blot analysis, and at the transcriptional level, as demonstrated in CHO cells transfected with hNTR and a reporter gene for Krox-24. Activation of MAPK and induction of Krox-24 were both prevented by the NTR antagonist SR 48692, confirming the specific action on NTR. Furthermore we observed coupling of NTR to a mitogenic pathway and Krox-24 induction in the human adenocarcinoma cell line HT29, which naturally expresses NTRs. Considering coupling pathways between NTR stimulation and MAPK activation, we observed a partial inhibition by pertussis toxin (PTX) and a complete blockade by the protein kinase C (PKC) inhibitor GF 109203X. Taken together, these results suggest that (1) stimulation of NTR activates the MAPK pathway by mechanisms involving dual coupling to both PTX-sensitive and PTX-insensitive G-proteins as well as PKC activation, and (2) these effects are associated with the induction of Krox-24, which might be a target of MAPK effector.

scholarly journals Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy

1996 ◽  
Vol 314 (1) ◽  
pp. 115-121 ◽  
Author(s):  
Marie A. BOGOYEVITCH ◽  
Monica B. ANDERSSON ◽  
Judith GILLESPIE-BROWN ◽  
Angela CLERK ◽  
Peter E. GLENNON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cardiac Myocyte ◽  
Mapk Pathway ◽  
Gene Promoter ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
Adrenergic Antagonist ◽  
Mitogen Activated Protein ◽  
Two Peaks

Phenylephrine and noradrenaline (α-adrenergic agonism) or isoprenaline (β-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2–4 μM and that for isoprenaline was 0.2–0.3 μM. Maximal activation of MAPK by isoprenaline was inhibited by the β-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the α1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN´N´-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that α- and β-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

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