Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway

2002 ◽  
Vol 283 (4) ◽  
pp. G893-G899 ◽  
Author(s):  
Monica C. Chen ◽  
Travis E. Solomon ◽  
Eduardo Perez Salazar ◽  
Robert Kui ◽  
Enrique Rozengurt ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Paracellular Permeability ◽  
Maximal Response ◽  
Src Tyrosine Kinase

Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr416 was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 μM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr416 in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this Gs-coupled receptor in primary epithelial cells.

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

scholarly journals Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets

Blood ◽  
2005 ◽  
Vol 106 (2) ◽  
pp. 550-557 ◽  
Author(s):  
Swaminathan Murugappan ◽  
Haripriya Shankar ◽  
Surya Bhamidipati ◽  
Robert T. Dorsam ◽  
Jianguo Jin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Time Dependent ◽  
Dependent Manner ◽  
Src Tyrosine Kinase ◽  
Functional Implications

Abstract Thrombin has been known to cause tyrosine phosphorylation of protein kinase C δ (PKCδ) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCδ and the role of this event in platelet function. PKCδ was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCδ did not occur in Gαq-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5′-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ′, N ′-tetraacetic acid), suggesting a role for Gαq pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCδ. Inhibition of tyrosine phosphorylation or the kinase activity of PKCδ dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCδ in a calcium- and Src-family kinase–dependent manner in platelets, with functional implications in thromboxane A2 generation.

The Tyrosine Kinase Inhibitor Adaphostin (NSC 680410), but Not Imatinib Mesylate, Inhibits Survival and Src Tyrosine Kinase Family- Triggered Signaling Pathways of MM Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3352-3352
Author(s):  
Klaus Podar ◽  
Melissa Simoncini ◽  
Yu-Tzu Tai ◽  
Martin Sattler ◽  
Kenji Ishitsuka ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Signaling Pathways ◽  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Parp Cleavage ◽  
Src Tyrosine Kinase ◽  
Kinase Family ◽  
Long Term Treatment

Abstract The tyrosine kinase inhibitor adaphostin is a member of the tyrophostin family of small molecules that interfere with peptide binding rather, than targeting the kinase ATP-binding site. Adaphostin has therefore been examined as an alternative to the 2-phenylaminopyrimidine derivate imatinib mesylate, with remarkable efficacy in the treatment of chronic myeloic leukemia (CML). Previous studies show that adaphostin induces apoptosis: (1) in Bcr/Abl+ cells more rapidly than imatinib mesylate; (2) in imatinib mesylate resistant cells; and (3) in Bcr/ Abl - cells. Imatinib mesylate has minimal, if any activity in MM; the efficacy of adaphostin in multiple myeloma (MM) is unknown. Here we compare the effects of adaphostin and imatinib mesylate against human MM cells. Our results show concentration-dependent apoptosis in MM.1S, U266, OPM-2, INA-6, RPMI8226 and RPMI-Dox40 MM cells after treatment with adaphostin, but not with imatinib mesylate. Imatinib mesylate induced more than 50% apoptosis in K562 cells using concentrations as low as 1mM, which served as a positive control. Moreover, adaphostin, but not imatinib mesylate, induced caspase-9, caspase-8, and PARP cleavage, as well as downregulation of Mcl-1, in MM cells. Further results demonstrated that adaphostin induces peroxide production and DNA strand breaks after long-term treatment. Importantly MM cell proliferation induced by MM cell binding to BMSCs was abrogated by adaphostin- treatment. IL-6 and IGF-1 signaling and sequelae triggered by these cytokines are important growth, survival, and drug resistance factors in MM; conversely, adaphostin but not imatinib mesylate, inhibited phosphorylation of Src tyrosine kinase family, Akt-1, and ERK. Taken together, our studies in MM cells show that (1) adaphostin- inhibits IGF-1- and IL-6- triggered signaling pathways as well as (2) induces reactive oxygen species and apoptosis. These studies therefore provide the preclinical framework for its clinical evaluation to improve patient outcome in MM.

EFFECT OF SRC TYROSINE KINASE INHIBITOR, PP1, ON THE CYTOKINE RESPONSE INDUCED BY LIPOPOLYSACCHARIDE.

2005 ◽  
Vol 33 ◽  
pp. A139
Author(s):  
Sandra D Buttram ◽  
Patrick M Kochanek ◽  
Delbert G Gillespie ◽  
Zaichuan Mi ◽  
Edwin K Jackson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Cytokine Response ◽  
Src Tyrosine Kinase

scholarly journals Transforming growth factor-α increases tyrosine phosphorylation of microtubule-associated protein kinase in a small intestinal crypt cell line (IEC-6)

1994 ◽  
Vol 303 (2) ◽  
pp. 455-460 ◽  
Author(s):  
B L Oliver ◽  
R I Sha'afi ◽  
J J Hajjar
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Tyrosine Phosphorylation ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Intestinal Crypt ◽  
Small Intestinal ◽  
Intestinal Crypt Cell

The small intestinal crypt cell line (IEC-6) is an undifferentiated, untransformed, mitotically active cell used in this study to determine the effect of transforming growth factor-alpha (TGF-alpha) on tyrosine phosphorylation levels of cellular proteins. Thymidine incorporation increased maximally after addition of 2 ng/ml TGF-alpha for 24 h. At the same dose, TGF-alpha induced the tyrosine phosphorylation of proteins with approximate molecular masses of 42, 44, 52, 80, 150 and 175 kDa as shown by Western blots treated with anti-phosphotyrosine antibody. The most intense phosphorylation was seen in the 42 kDa (p42) and 44 kDa (p44) proteins, which were identified as two isoforms of microtubule-associated protein kinase (MAPK). This phosphorylation was seen as early as 5 min post stimulation and was dose dependent. Both p42 and p44 were found in the nucleus after stimulation, although a basal level of unphosphorylated protein was present before stimulation. The observed tyrosine phosphorylation of p42 and p44 was inhibited by genistein, a tyrosine kinase inhibitor, and tyrphostin 23, an epidermal growth factor receptor tyrosine kinase inhibitor. We conclude that MAPK is tyrosine phosphorylated in response to TGF-alpha stimulation of IEC-6 cells.

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