scholarly journals Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

1994 ◽  
Vol 5 (3) ◽  
pp. 313-322 ◽  
Author(s):  
J R Apgar
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Adenosine Receptor ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Filamentous Actin ◽  
Ige Receptor ◽  
Ige Mediated ◽  
Rbl Cells ◽  
Stimulation Of

Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein.

scholarly journals Regulation of the antigen-induced F-actin response in rat basophilic leukemia cells by protein kinase C.

1991 ◽  
Vol 112 (6) ◽  
pp. 1157-1163 ◽  
Author(s):  
J R Apgar
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Induced Response ◽  
Dependent Manner ◽  
Filamentous Actin ◽  
Kinase C ◽  
Rbl Cells ◽  
Basophilic Leukemia

Multivalent antigen that is capable of binding to and crosslinking the IgE receptors on rat basophilic leukemia (RBL) cells, induces a rapid and sustained rise in the content of filamentous actin. This reorganization of the actin may be responsible for changes in cellular morphology during the degranulation process. The antigen-stimulated polymerization of actin can be blocked in a dose-dependent manner by protein kinase inhibitors which also block degranulation. Conversely, reagents such as PMA, 1,2-dioctanoyl-sn-glycerol (diC8), and 1-oleoyl-2-acetyl-glycerol (OAG) which stimulate protein kinase C (PKC) also activate the rise in F-actin, although they have no effect on degranulation by themselves. The actin response which can be stimulated by the PKC activators can also be blocked by protein kinase inhibitors indicating that the PMA- and OAG-induced response is probably through activation of a protein kinase. Depletion of PKC activity through long term (20 h) exposure of RBL cells to PMA, also inhibited the F-actin response when the cells were stimulated with either multivalent antigen or OAG. External Ca++, which is an absolute requirement for degranulation, is not necessary for the rise in F-actin, but may modulate the response. Furthermore, ionomycin, which induces a large Ca++ influx, does not stimulate the F-actin increase even at doses that cause degranulation. These results suggest that activation of a protein kinase, such as PKC, may be responsible for signaling the polymerization of actin in RBL cells and that a rise in intracellular Ca++ is neither necessary nor sufficient for this response.

scholarly journals Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig

Reproduction ◽  
2012 ◽  
Vol 144 (5) ◽  
pp. 535-546 ◽  
Author(s):  
R Prochazka ◽  
M Blaha ◽  
L Nemcova
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Kinase Inhibitors ◽  
Egf Receptor ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Protein Kinase Inhibitors ◽  
Akt Inhibitor ◽  
Cumulus Expansion

To define signaling pathways that drive FSH- and epidermal growth factor (EGF)-like peptide-induced cumulus expansion and oocyte meiotic resumption, in vitro cultured pig cumulus–oocyte complexes were treated with specific protein kinase inhibitors. We found that FSH-induced maturation of oocytes was blocked in germinal vesicle (GV) stage by protein kinase A (PKA), MAPK14, MAPK3/1, and EGF receptor (EGFR) tyrosine kinase inhibitors (H89, SB203580, U0126, and AG1478 respectively) whereas phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) inhibitor (LY294002) blocked maturation of oocytes in metaphase I (MI). Amphiregulin (AREG)-induced maturation of oocytes was efficiently blocked in GV by U0126, AG1478, and low concentrations of LY294002; H89, SB203580, and high concentrations of LY294002 allowed the oocytes to undergo breakdown of GV and blocked maturation in MI. Both FSH- and AREG-induced cumulus expansion was incompletely inhibited by H89 and completely inhibited by SB203580, U0126, AG1478, and LY294002. The inhibitors partially or completely inhibited expression of expansion-related genes (HAS2, PTGS2, and TNFAIP6) with two exceptions: H89 inhibited only TNFAIP6 expression and LY294002 increased expression of PTGS2. The results of this study are consistent with the idea that PKA and MAPK14 pathways are essential for FSH-induced transactivation of the EGFR, and synthesis of EGF-like peptides in cumulus cells and MAPK3/1 is involved in regulation of transcriptional and posttranscriptional events in cumulus cells required for meiotic resumption and cumulus expansion. PI3K/AKT signaling is important for regulation of cumulus expansion, AREG-induced meiotic resumption, and oocyte MI/MII transition. The present data also indicate the existence of an FSH-activated and PKA-independent pathway involved in regulation of HAS2 and PTGS2 expression in cumulus cells.

Lysophosphatidylcholine stimulates phospholipase D in human coronary endothelial cells: role of PKC

1996 ◽  
Vol 271 (4) ◽  
pp. H1706-H1710 ◽  
Author(s):  
D. A. Cox ◽  
M. L. Cohen
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Human Coronary Artery ◽  
Gf 109203X ◽  
Messenger System ◽  
Stimulation Of

Lysophosphatidylcholine (lyso PC) mediates multiple potentially atherogenic effects on endothelial cells, although the cellular mechanism of these effects remains unclear. Phospholipase D (PLD) has been recognized as a novel second-messenger system that may regulate cellular function. The purpose of this study was to determine the effect of lyso PC on PLD activity in human coronary artery endothelial cells (HCAEC) by measuring [3H]phosphatidylethanol production in cells labeled with [3H]myristic acid. After incubation with lyso PC (20 microM) for 40 min, PLD activity was markedly stimulated from five- to sixfold. Stimulation of PLD activity by lyso PC was concentration dependent (half-maximum effective concentration of 7.6 microM) and was not mimicked by phosphatidylcholine (20 microM). Because PLD can be regulated by protein kinases, the effect of several protein kinase inhibitors on lyso PC-stimulated PLD activity was tested. The protein kinase A inhibitor H-89 (300 nM) and the tyrosine kinase inhibitors genistein (30 microM) and tyrphostin A25 (100 microM) had no effect on the stimulation of PLD by lyso PC (20 microM). The protein kinase C (PKC) inhibitor calphostin C (10-300 nM) affected neither lyso PC (20 microM)-nor 4 beta-phorbol 12,13-dibutyrate (PDBu, 300 nM)-stimulated PLD activity, suggesting that this agent may not inhibit PKC in these cells. In contrast, the selective PKC inhibitors GF-109203X (0.3-10 microM) and chelerythrine (1-30 microM) concentration dependently inhibited lyso PC (20 microM)-stimulated PLD activity and blocked PDBu (300 nM)-stimulated PLD activity. Together, these data document that lyso PC stimulated PLD in human endothelial cells, possibly by a PKC-dependent mechanism, and provide evidence that PLD activation in human endothelium is a novel and important mechanism by which lyso PC mediates its cellular and possibly atherogenic effects.

Abstract 483: The Signaling Pathways of Nicotine-induced ERK1/2 Phosphorylation in Rat Mesangial Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ping Hua ◽  
Wenguang Feng ◽  
Gabriel Rezonzew ◽  
Phillip H Chumley ◽  
Edgar A Jaimes
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Kinase Inhibitors ◽  
Mesangial Cells ◽  
Protein Kinase Inhibitors ◽  
Biologically Active ◽  
Calcium Calmodulin Dependent ◽  
Nicotine Receptors ◽  
High Concentrations

Tobacco smoking is associated with accelerated progression of chronic kidney disease of different etiologies including diabetes and hypertension. However, the mechanisms involved are not well understood. We have previously reported that nicotine, a biologically active compound present in high concentrations in tobacco, induces cell proliferation and fibronectin production in mesangial cells which are prevented by ERK1/2 inhibition (AJP’05). In these studies we determined whether rat mesangial cells (MC) express nicotine receptors and characterized the signaling pathways that lead to ERK1/2 phosphorylation in response to nicotine. MC were grown in DMEM with 15% FBS in the presence of 0.4 mg/ml G418 and starved for 24 hours in DMEM without FBS before treatment. We first demonstrated that MC are endowed with several nicotinic Ach receptor (nAChR) subunits including α2-7 and β1-4 as assessed by western blot. Treatment of rat MC with nicotine at 10 -7 M caused a time-dependent ERK1/2 phosphorylation which peaked after 10 min of stimulation( N=3). Several protein kinase inhibitors were then used to identify the upstream kinases that mediate nicotine-induced ERK1/2 phosphorylation. The calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (10 -7 M) decreased the ERK1/2 phosphorylation level by ∼57% (0.41 of 0.95) as compared to nicotine. The PKC inhibitor Go6983 at 10 -9 M, the PKA inhibitor H89 (10 -8 M) and the EGFR inhibitor AG 1478 (10 -7 M) also inhibited ERK1/2 phosphorylation by 60% (0.38 of 0.95), 48% (0.63 of 1.20) and 68% (0.40 of 1.23) respectively as compared to nicotine. Given the role of the nicotine receptors as agonist-regulated Ca 2+ channels, we determined the effects of Ca 2+ channel blockade on nicotine induced ERK1/2 phosphorylation. Treatment of MC with the calcium channel Verapamil (10 -9 M) resulted in 33% (0.49 of 0.73) inhibition of ERK1/2 phosphorylation as compared to nicotine. In summary, we have determined in these studies that rat MC are endowed with several nAChR subunits and that ERK1/2 phosphorylation in response to nicotine requires CaMK II, PKA, PKC and EGFR. In addition, we have demonstrated that these effects require Ca 2+ consistent with the role of the nAChR as agonist-regulated Ca 2+ channels in MC.

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