Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway

2007 ◽  
Vol 293 (2) ◽  
pp. C790-C796 ◽  
Author(s):  
Shigeo Tamiya ◽  
Mansim C. Okafor ◽  
Nicholas A. Delamere
Keyword(s):  
Atpase Activity ◽  
Tyrosine Kinases ◽  
Short Term ◽  
Signaling Mechanism ◽  
Src Tyrosine Kinase ◽  
Immunoreactive Band ◽  
Rabbit Lens ◽  
Nonreceptor Tyrosine Kinases ◽  
Dependent Pathway

The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium (86Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium (86Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at ∼60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium (86Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens.

Short-term effect of aldosterone on NEM-sensitive ATPase in rat collecting tubule

1989 ◽  
Vol 257 (2) ◽  
pp. F177-F181 ◽  
Author(s):  
C. Khadouri ◽  
S. Marsy ◽  
C. Barlet-Bas ◽  
A. Doucet
Keyword(s):  
Atpase Activity ◽  
Affinity Constant ◽  
Short Term ◽  
Collecting Tubule ◽  
Lag Period ◽  
In Vitro Effects ◽  
Collecting Tubules

Because previous studies indicated that in the collecting tubule, N-ethylmaleimide (NEM)-sensitive ATPase, the biochemical equivalent of the proton pump, is controlled by mineralocorticoids in the long term, the present study was designed to investigate whether such control also exists in the short term. Therefore we investigated the in vivo and in vitro effects of aldosterone on the enzyme activity in cortical and outer medullary collecting tubules (CCT and MCT, respectively) from adrenalectomized rats. Administration of aldosterone (10 micrograms/kg body wt) markedly stimulated NEM-sensitive ATPase activity in the CCT and MCT within 3 h. Similarly, incubating CCT or MCT for 3 h in the presence of 10(-8) M aldosterone enhanced NEM-sensitive ATPase activity up to values similar to those previously measured in the corresponding nephron segments of normal rats. In vitro stimulation of NEM-sensitive ATPase was dose dependent in regard to aldosterone (apparent affinity constant approximately 10(-9) M), appeared after a 30-min lag period, and reached its maximum after 2-2.5 h. Finally, actinomycin D and cycloheximide totally abolished the in vitro action of aldosterone, demonstrating the involvement of protein synthesis in this process.

Optimisation of EGFR TKI efficiency in the therapeutic scheme of EGFR wild-type lung cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e18532-e18532
Author(s):  
Mathilde Cabart ◽  
Judith Raimbourg ◽  
Lisenn Lalier ◽  
Jaafar Bennouna ◽  
Francois Vallette
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Wild Type ◽  
Short Term ◽  
Egfr Ligands ◽  
Egfr Mutated ◽  
Egfr Tki

e18532 Background: EGFR tyrosine kinase inhibitors (EGFR TKI) have improved the therapeutic care of lung cancer patients but only a small sub-population of patients, namely those harboring EGFR-mutated tumors, benefit from this therapy. The observation that EGFR TKI enhance prognosis and quality of life in all patients when used as second line or maintenance treatment impelled us into the search of potential markers of the optimal introduction kinetics of EGFR TKI in the therapeutic scheme. Methods: We used lung cancer cell lines harboring either wild-type or mutated EGFR (NCI-H1650, NCI-H1975) to study the consequences of cisplatin treatment in vitro on the consecutive sensitivity to erlotinib. Results: Sub-lethal cisplatin pretreatment (3µM) enhances erlotinib toxicity in EGFR wild-type, but not EGFR mutated cells (A549 IC50 drops from 28 to 15µM for short-term or 12µM for long-term exposure). This correlates with EGFR activation following short-term or prolonged cisplatin treatment through the secretion of EGFR ligands. This activation of EGFR is concomitant to the decrease in other receptor tyrosine kinases phosphorylation including Met. Conclusions: The sensitivity of EGFR wild-type lung cancer cells to erlotinib in vitro is enhanced by cisplatin pretreatment. We identified potential markers of this sensitization, namely EGFR ligands, which serum level might be predicitive of the optimal efficiency of EGFR TKI. In vivo validation of these markers is under investigation. The concomitant decrease in other receptor tyrosine kinases phosphorylation suggests that the targeting of other receptor tyrosine kinases might potentiate EGFR TKI efficiency.

scholarly journals Uroguanylin inhibits H-ATPase activity and surface expression in renal distal tubules by a PKG-dependent pathway

2014 ◽  
Vol 307 (6) ◽  
pp. C532-C541 ◽  
Author(s):  
Vanessa da Silva Lima ◽  
Renato O. Crajoinas ◽  
Luciene R. Carraro-Lacroix ◽  
Alana N. Godinho ◽  
João L. G. Dias ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Distal Tubule ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Camp Content ◽  
Bicarbonate Reabsorption ◽  
Guanylate Cyclase C ◽  
Dependent Pathway

Cumulative evidence suggests that guanylin peptides play an important role on electrolyte homeostasis. We have previously reported that uroguanylin (UGN) inhibits bicarbonate reabsorption in a renal distal tubule. In the present study, we tested the hypothesis that the bicarbonaturic effect of UGN is at least in part attributable to inhibition of H+-ATPase-mediated hydrogen secretion in the distal nephron. By in vivo stationary microperfusion experiments, we were able to show that UGN inhibits H+-ATPase activity by a PKG-dependent pathway because KT5823 (PKG inhibitor) abolished the UGN effect on distal bicarbonate reabsorption and H89 (PKA inhibitor) was unable to prevent it. The in vivo results were confirmed by the in vitro experiments, where we used fluorescence microscopy to measure intracellular pH (pHi) recovery after an acid pulse with NH4Cl. By this technique, we observed that UGN and 8 bromoguanosine-cGMP (8Br-cGMP) inhibited H+-ATPase-dependent pHi recovery and that the UGN inhibitory effect was abolished in the presence of the PKG inhibitor. In addition, by using RT-PCR technique, we verified that Madin-Darby canine kidney (MDCK)-C11 cells express guanylate cyclase-C. Besides, UGN stimulated an increase of both cGMP content and PKG activity but was unable to increase the production of cellular cAMP content and PKA activity. Furthermore, we found that UGN reduced cell surface abundance of H+-ATPase B1 subunit in MDCK-C11 and that this effect was abolished by the PKG inhibitor. Taken together, our data suggest that UGN inhibits H+-ATPase activity and surface expression in renal distal cells by a cGMP/PKG-dependent pathway.

scholarly journals The v-Src SH3 domain binds phosphatidylinositol 3'-kinase.

1993 ◽  
Vol 13 (9) ◽  
pp. 5225-5232 ◽  
Author(s):  
X Liu ◽  
L E Marengere ◽  
C A Koch ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Chicken Embryo ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

p60src and p72Syk Tyrosine Kinases Activate Both Lw and CD44 to Mediate Sickle Red Blood Cell Adhesion to Endothelium

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3223-3223
Author(s):  
Edward Chiou ◽  
Rahima Zennadi
Keyword(s):  
Shear Stress ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Selective Inhibitor ◽  
Intermittent Flow ◽  
Αvβ3 Integrin ◽  
Src Tyrosine Kinase

Abstract Abstract 3223 The vaso-occlusive process in patients with sickle cell disease is complex and involves interactions between hemoglobin S red blood cells (SSRBCs) and vascular endothelial cells (ECs). However, the pathophysiologic triggers inducing SSRBC adhesion and vaso-occlusion are poorly understood. Elucidation of these mechanisms at the molecular level would allow development of new preventive and treatment strategies to abrogate vaso-occlusive events. Because non-receptor tyrosine kinases in non-erythroid cells are known to mediate cell-cell interactions, we have now investigated the role of non-receptor tyrosine kinases in modulating SSRBC adherence to cultured ECs, identified the kinases involved, and defined both the receptors on activated SSRBCs and the ligands on ECs involved in these interactions. Less than 20% of non-treated SSRBCs were able to adhere to non-stimulated ECs in vitro in intermittent flow conditions at a shear stress of 2 dynes/cm2. However, treatment of SSRBCs with sodium orthovanadate (Na3VO4), a potent broad spectrum inhibitor of protein tyrosine phosphatases, was able to significantly up-regulate RBC adhesion to cultured ECs by 4.6±1-fold over baseline adhesion in vitro. Na3VO4 in contrast, completely failed to increase adhesion of normal RBCs to cultured ECs. The increased SSRBC adhesion induced by Na3VO4 was significantly inhibited with piceatannol (p<0.001), which inhibits non-receptor tyrosine kinases p72Syk and p56Lck, and PP1 (p<0.001), an src-selective tyrosine p56Lck, p59Fyn and p60src kinase inhibitor. However, genistein, a broad range inhibitor of tyrosine kinases, and damnacanthal, a highly potent and selective inhibitor of p56Lck, completely failed to inhibit the effect of Na3VO4 on SSRBC adhesion. In addition, phenylarsine oxide, which specifically activates p56Lck tyrosine kinase, did not affect SSRBC adhesion to ECs. Together, these data suggest that SSRBC adhesion to ECs can be upregulated via activation of at least p72Syk and p60src tyrosine kinases, but not via the src-tyrosine kinase p56Lck. We further confirmed that increased SSRBC adhesion by Na3VO4 treatment is indeed a result of the activation of p72Syk and p60src tyrosine kinases. Slight basal phosphorylation of p72Syk and p60src was detected in all SSRBC samples tested. However, Na3VO4-treatment of SSRBCs significantly enhanced phosphorylation of both p60src and p72Syk kinases over basal phosphorylation (p<0.05), and this effect induced by Na3VO4 was completely blocked with the src-selective inhibitor PP1 or PP2, and piceatannol, respectively, suggesting that p72Syk and p60src undergo enhanced activation and are involved in up-regulation of SSRBC adhesion to endothelium. Moreover, at a shear stress of 2 dynes/cm2, both anti-LW (ICAM-4) and anti-CD44 antibodies individually inhibited adhesion of activated SSRBCs to ECs, by up to 83% and 78%, respectively. Recombinant soluble LW (srLW) and CD44 (srCD44) proteins also completely abolished adhesion of activated SSRBCs to ECs, identifying LW and CD44 as the RBC receptors involved in this interaction. The EC ligands for activated SSRBCs were also identified using antibody inhibition studies, as the αvβ3 integrin, a ligand previously shown to be important to SSRBC adhesion to activated ECs in vivo, and the endothelial CD44. These data demonstrate that activation of p72Syk and p60src-dependent pathways can act to activate LW- and CD44-mediated SSRBC adhesion to endothelial αvβ3 integrin and CD44, respectively, suggesting that this mechanism may initiate or exacerbate vaso-occlusion by increasing SSRBC adhesion to the endothelium. RBC CD44 is also the first adhesion molecule shown to be involved in SSRBC adhesion to endothelium. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tissue culture of sockeye salmon intestine: functional response of Na+-K+-ATPase to cortisol

2005 ◽  
Vol 288 (6) ◽  
pp. R1598-R1605 ◽  
Author(s):  
Philip A. Veillette ◽  
Graham Young
Keyword(s):  
Atpase Activity ◽  
Sockeye Salmon ◽  
Culture Medium ◽  
Slow Release ◽  
Short Term ◽  
Direct Target ◽  
Dose Dependent ◽  
Short Term Culture

A method to culture tissue explants of the intestine from freshwater-adapted sockeye salmon ( Oncorhynchus nerka) was developed to assess possible direct effects of cortisol on Na+-K+-ATPase activity. As judged by several criteria, explants from pyloric ceca and the posterior region of the intestine remained viable during short-term (6-day) culture, although Na+-K+-ATPase activity declined and basolateral components of the enterocytes were observed to be partially degraded. Addition of cortisol to the culture medium maintained Na+-K+-ATPase activity (over 2–12 days) above that of control explants and, in some cases, was similar to levels before culture. The response to cortisol was dose dependent (0.001–10 μg/ml). Within the physiological range, the response was specific for cortisol and showed the following hierarchy: dexamethasone ≥ cortisol > 11-deoxycortisol > cortisone. Insulin maintained Na+-K+-ATPase activity over controls in explants of ceca but not posterior intestine. To compare in vivo and in vitro responses, slow-release implants of cortisol (50 μg/g) were administered to salmon for 7 days. This treatment elevated plasma cortisol levels and stimulated Na+-K+-ATPase activity in both intestinal regions. The results demonstrate that the teleost intestine is a direct target of cortisol, this corticosteroid protects in vitro functionality of Na+-K+-ATPase, and explants retain cortisol responsiveness during short-term culture.

scholarly journals Regulation of Fibroblast Cell Polarity by Src Tyrosine Kinase

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 135
Author(s):  
Kazuo Katoh
Keyword(s):  
Cell Polarity ◽  
Cell Elongation ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Fibroblast Cell ◽  
Dominant Negative ◽  
Src Tyrosine Kinase ◽  
Src Inhibitor ◽  
Src Gene ◽  
Nonreceptor Tyrosine Kinases

Src protein tyrosine kinases (SFKs) are a family of nonreceptor tyrosine kinases that are localized beneath the plasma membrane and are activated during cell adhesion, migration, and elongation. Due to their involvement in the activation of signal transduction cascades, SFKs have been suggested to play important roles in the determination of cell polarity during cell extension and elongation. However, the mechanism underlying Src-mediated polarity formation remains unclear. The present study was performed to investigate the mechanisms underlying Src-induced cell polarity formation and cell elongation using Src knockout fibroblasts (SYFs) together with an inhibitor of Src. Normal and Src knockout fibroblasts were also transfected with a wild-type c-Src, dominant negative c-Src, or constitutively active c-Src gene to analyze the changes in cell morphology. SYF cells cultured on a glass substrate elongated symmetrically into spindle-shaped cells, with the formation of focal adhesions at both ends of the cells. When normal fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, they elongated into symmetrical spindle-shaped cells, similar to SYF cells. These results suggest that cell polarity during extension and elongation may be regulated by SFKs and that the expression and regulation of Src are important for the formation of polarity during cell elongation.

The v-Src SH3 domain binds phosphatidylinositol 3'-kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5225-5232
Author(s):  
X Liu ◽  
L E Marengere ◽  
C A Koch ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Chicken Embryo ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

scholarly journals Phosphorylation of synthetic peptides containing Tyr-Met-X-Met motifs by nonreceptor tyrosine kinases in vitro.

1993 ◽  
Vol 268 (33) ◽  
pp. 25146-25151
Author(s):  
P Garcia ◽  
S E Shoelson ◽  
S T George ◽  
D A Hinds ◽  
A R Goldberg ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Synthetic Peptides ◽  
Nonreceptor Tyrosine Kinases

scholarly journals H2O2 alters rat cardiac sarcomere function and protein phosphorylation through redox signaling

2010 ◽  
Vol 299 (3) ◽  
pp. H723-H730 ◽  
Author(s):  
Benjamin S. Avner ◽  
Aaron C. Hinken ◽  
Chao Yuan ◽  
R. John Solaro
Keyword(s):  
Atpase Activity ◽  
Troponin I ◽  
Force Production ◽  
Redox Signaling ◽  
Response To Treatment ◽  
Sarcomeric Proteins ◽  
Signaling Mechanism ◽  
Organ Systems ◽  
Cardiac Myofilament

ROS, such as H2O2, are a component of pathological conditions in many organ systems and have been reported to be elevated in cardiac pathophysiology. The experiments presented here test the hypothesis that H2O2 induces alterations in cardiac myofilament function by the posttranslational modification of sarcomeric proteins indirectly through PKC signaling. In vitro assessment of actomyosin Mg2+-ATPase activity of myofibrillar fractions showed blunted relative ATP consumption in the relaxed state (pCa 8.0) in response to treatment with 0.5 mM H2O2 before myofilament isolation. The effect was attributable to downstream “redox signaling,” inasmuch as the direct application of H2O2 to isolated myofibrils did not alter Mg2+-ATPase activity. Ca2+-ATPase activity, which was used as a measure of myofibrillar myosin function, was unaffected by H2O2. Functional experiments using rat cardiac trabeculae treated with 0.5 or 5 mM H2O2 followed by detergent extraction of membranes demonstrated increased Ca2+ sensitivity of force production, a faster rate of force redevelopment, and (for 5 mM) decreased maximum tension. Biochemical analysis of myocardial samples treated with 0.5 mM H2O2 demonstrated increased phosphorylation of two sarcomeric proteins: cardiac troponin I and myosin-binding protein-C. These changes were eliminated by a general PKC inhibitor. However, H2O2 and the general PKC activator PMA induced different phosphorylation patterns in cardiomyocytes in which PKC-δ was elevated by viral infection. These data provide evidence that PKC-dependent redox signaling affects the function of cardiac myofilaments and indicate modification of specific proteins through this signaling mechanism.

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