Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1

The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.

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Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+Channel Function

Anesthesiology ◽

10.1097/aln.0b013e318197941e ◽

2009 ◽

Vol 110 (3) ◽

pp. 582-590 ◽

Cited By ~ 44

Author(s):

Wei Ouyang ◽

Karl F. Herold ◽

Hugh C. Hemmings

Keyword(s):

Rank Order ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Na Channel ◽

Dependent Manner ◽

Fast Inactivation ◽

Inhaled Anesthetics ◽

Voltage Gated ◽

Channel Inhibition ◽

Voltage Dependent

Background Inhibition of voltage-gated Na channels (Na(v)) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane, and desflurane) on Na(v)1.4, a well-characterized pharmacological model for Na(v) effects. Methods Na currents (I(Na)) from rat Na(v)1.4 alpha-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording. Results Halogenated inhaled anesthetics reversibly inhibited Na(v)1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equianesthetic concentrations, peak I(Na) was inhibited with a rank order of desflurane > halothane approximately enflurane > isoflurane approximately sevoflurane from a physiologic holding potential (-80 mV). This suggests that the contribution of Na channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (-120 mV), peak I(Na) was inhibited with a rank order of potency for tonic inhibition of peak I(Na) of halothane > isoflurane approximately sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane. Conclusions Five halogenated inhaled anesthetics all inhibit a voltage-gated Na channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.

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Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

Journal of Endocrinology ◽

10.1677/joe-08-0104 ◽

2008 ◽

Vol 198 (3) ◽

pp. 561-569 ◽

Cited By ~ 72

Author(s):

Wenbin Shang ◽

Ying Yang ◽

Libin Zhou ◽

Boren Jiang ◽

Hua Jin ◽

...

Keyword(s):

Insulin Receptor ◽

Glucose Uptake ◽

Signaling Pathway ◽

Glucose Transport ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

C2c12 Myotubes ◽

Maximal Effect ◽

Dependent Manner ◽

Active Constituent

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb1 has been shown to regulate peroxisome proliferator-activated receptor γ activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb1 on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb1 significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 μM and a time of 3 h. In adipocytes, Rb1 promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb1 increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb1-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb1 stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.

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Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini

Biochemical Journal ◽

10.1042/bj3040531 ◽

1994 ◽

Vol 304 (2) ◽

pp. 531-536 ◽

Cited By ~ 16

Author(s):

H Ohnishi ◽

T Mine ◽

I Kojima

Keyword(s):

Cyclic Amp ◽

G Protein ◽

Pertussis Toxin ◽

Dose Response ◽

Response Curve ◽

Dose Response Curve ◽

Amylase Secretion ◽

Ionophore A23187 ◽

Dependent Manner ◽

Pancreatic Acini

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

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Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun.

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7352 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7352-7362 ◽

Cited By ~ 209

Author(s):

E Cano ◽

C A Hazzalin ◽

L C Mahadevan

Keyword(s):

Protein Kinases ◽

Map Kinases ◽

Early Gene ◽

Intracellular Signals ◽

Protein Map ◽

Mitogen Activated Protein ◽

Serine Kinases ◽

Translational Arrest ◽

Epidermal Growth ◽

Nuclear Responses

Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.

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Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7352-7362.1994 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7352-7362 ◽

Cited By ~ 1

Author(s):

E Cano ◽

C A Hazzalin ◽

L C Mahadevan

Keyword(s):

Protein Kinases ◽

Map Kinases ◽

Early Gene ◽

Intracellular Signals ◽

Protein Map ◽

Mitogen Activated Protein ◽

Serine Kinases ◽

Translational Arrest ◽

Epidermal Growth ◽

Nuclear Responses

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

AJP Cell Physiology ◽

10.1152/ajpcell.1994.267.4.c1130 ◽

1994 ◽

Vol 267 (4) ◽

pp. C1130-C1135 ◽

Cited By ~ 45

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.

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Induction of MAP Kinase Homologues during Growth and Morphogenetic Development of Karnal Bunt (Tilletia indica) under the Influence of Host Factor(s) from Wheat Spikes

The Scientific World JOURNAL ◽

10.1100/2012/539583 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

Atul K. Gupta ◽

J. M. Seneviratne ◽

G. K. Joshi ◽

Anil Kumar

Keyword(s):

Map Kinase ◽

Map Kinases ◽

Fungal Growth ◽

Host Factor ◽

Host Factors ◽

Karnal Bunt ◽

Dependent Manner ◽

Tilletia Indica ◽

Mitogen Activated Protein

Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) in response to certain environmental conditions, play important role in mating type switching (Fus3) and pathogenicity (Pmk1) in many fungi. In order to determine the roles of such regulatory genes inTilletia indica, the causal pathogen of Karnal bunt (KB) of wheat, semi-quantitative and quantitative RT-PCR was carried out to isolate and determine the expression of MAP kinase homologues during fungal growth and development underin vitroculture. Maximum expression of TiFus3 and TiPmk1 genes were observed at 14th and 21st days of culture and decreased thereafter. To investigate whether the fungus alters the expression levels of same kinases upon interaction with plants, cultures were treated with 1% of host factors (extracted from S-2 stage of wheat spikes). Such treatment induced the expression of MAPks in time dependent manner compared to the absence of host factors. These results suggest that host factor(s) provide certain signal(s) which activate TiFus3 and TiPmk1 during morphogenetic development ofT. indica. The results also provides a clue about the role of host factors in enhancing the disease potential due to induction of MAP kinases involved in fungal development and pathogenecity.

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Recombinant human GM-CSF induces leukocytosis and activates peripheral blood polymorphonuclear neutrophils in nonhuman primates

Blood ◽

10.1182/blood.v70.1.206.bloodjournal701206 ◽

1987 ◽

Vol 70 (1) ◽

pp. 206-213 ◽

Cited By ~ 1

Author(s):

P Mayer ◽

C Lam ◽

H Obenaus ◽

E Liehl ◽

J Besemer

Keyword(s):

Chinese Hamster Ovary Cells ◽

White Blood Cells ◽

Chinese Hamster ◽

Coli Strain ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Ovary Cells ◽

Gm Csf ◽

Cell Counts

The in vivo efficacy of glycosylated and nonglycosylated recombinant human granulocyte macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and Escherichia coli respectively was studied in rhesus monkeys following a daily subcutaneous (SC; three times) or intravenous (IV; over six hours) dose for seven consecutive days. The monkeys responded to the rh GM-CSF with a prompt (within 24 hours) rise in circulating white blood cells (WBCs). Thereafter the total cell counts increased steadily in a dose- dependent manner with repeated dosing to numbers six times over the pretreatment levels. Overall, granulocyte counts increased fivefold, lymphocytes twofold to fourfold, and monocytes threefold to fourfold. Platelets and erythrocytes were unaffected. Within 1 week after the end of treatment the leukocytosis had disappeared. Of the two routes of treatment, SC (three times daily)-administered rh GM-CSF was more effective than the same dose given by a six-hour IV infusion. In addition to inducing leukocytosis, parenterally administered rh GM-CSF primed mature circulating granulocytes for enhanced oxidative metabolism and killing of an E coli strain. These results show that exogenously administered glycosylated or nonglycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the phagocytic function of mature granulocytes in monkeys.

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iGIST - a kinetic bioassay for pertussis toxin based on its effect on inhibitory GPCR signaling

10.1101/2020.09.29.318451 ◽

2020 ◽

Cited By ~ 1

Author(s):

Valeriy M. Paramonov ◽

Cecilia Sahlgren ◽

Adolfo Rivero-Müller ◽

Arto T. Pulliainen

Keyword(s):

Pertussis Toxin ◽

Chinese Hamster Ovary Cells ◽

Cluster Formation ◽

Somatostatin Receptor ◽

Chinese Hamster ◽

Adenosine Monophosphate ◽

Hek293 Cells ◽

Sensitivity Threshold ◽

Gpcr Signaling

ABSTRACTDetection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require annually thousands of mice. Here, we describe iGIST (Interference in Gαi-mediated Signal Transduction) - an animal-free kinetic bioassay for detection of PTX by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory α-subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells co-expressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high affinity agonist octreotide, and a luminescent 3’5’-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in picogram/ml range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e. a commercial PTX- toxoid containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer-independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.

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Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.25.2.854-864.2005 ◽

2005 ◽

Vol 25 (2) ◽

pp. 854-864 ◽

Cited By ~ 90

Author(s):

Sandrine Marchetti ◽

Clotilde Gimond ◽

Jean-Claude Chambard ◽

Thomas Touboul ◽

Danièle Roux ◽

...

Keyword(s):

Map Kinases ◽

Fluorescent Protein ◽

Proteasomal Degradation ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Double Mutation ◽

Extracellular Signal ◽

Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

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