TRPV4 in Porcine Lens Epithelium Regulates Hyposmotic Stress‐Induced ATP Release and Na, K‐ATPase Activity

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.

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Hyposmotic stress causes ATP release and stimulates Na,K-ATPase activity in porcine lens

Journal of Cellular Physiology ◽

10.1002/jcp.22858 ◽

2012 ◽

Vol 227 (4) ◽

pp. 1428-1437 ◽

Cited By ~ 29

Author(s):

M. Shahidullah ◽

A. Mandal ◽

C. Beimgraben ◽

N.A. Delamere

Keyword(s):

Atpase Activity ◽

Atp Release ◽

Hyposmotic Stress

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Dynamic regulation of extracellular ATP in Escherichia coli

Biochemical Journal ◽

10.1042/bcj20160879 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1395-1416 ◽

Cited By ~ 7

Author(s):

Cora Lilia Alvarez ◽

Gerardo Corradi ◽

Natalia Lauri ◽

Irene Marginedas-Freixa ◽

María Florencia Leal Denis ◽

...

Keyword(s):

Escherichia Coli ◽

Atpase Activity ◽

Atp Release ◽

Membrane Vesicles ◽

Fold Increase ◽

Extracellular Atp ◽

Outer Membrane Vesicles ◽

Dynamic Regulation ◽

E Coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

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Hyposmotic stress triggers ATP release from porcine lens via connexin and pannexin hemichannels

Acta Ophthalmologica ◽

10.1111/j.1755-3768.2011.3121.x ◽

2011 ◽

Vol 89 (s248) ◽

pp. 0-0

Author(s):

NA DELAMERE ◽

C BEIMGRABEN ◽

A MANDAL ◽

M SHAHIDULLAH

Keyword(s):

Atp Release ◽

Hyposmotic Stress

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Colocalization of ATP Release Sites and Ecto-ATPase Activity at the Extracellular Surface of Human Astrocytes

Journal of Biological Chemistry ◽

10.1074/jbc.m302680200 ◽

2003 ◽

Vol 278 (26) ◽

pp. 23331-23342 ◽

Cited By ~ 149

Author(s):

Sheldon M. Joseph ◽

Marisa R. Buchakjian ◽

George R. Dubyak

Keyword(s):

Atpase Activity ◽

Atp Release ◽

Human Astrocytes

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The Influence of SRC-Family Tyrosine Kinases on Na,K-ATPase Activity in Lens Epithelium

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.04-0809 ◽

2005 ◽

Vol 46 (2) ◽

pp. 618 ◽

Cited By ~ 17

Author(s):

Larry D. Bozulic ◽

William L. Dean ◽

Nicholas A. Delamere

Keyword(s):

Atpase Activity ◽

Tyrosine Kinases ◽

Lens Epithelium ◽

Src Family Tyrosine Kinases

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The influence of Lyn kinase on Na,K-ATPase in porcine lens epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.00174.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. C90-C96 ◽

Cited By ~ 12

Author(s):

Larry D. Bozulic ◽

William L. Dean ◽

Nicholas A. Delamere

Keyword(s):

Atpase Activity ◽

Western Blot ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Tyrosine Phosphatase ◽

Lens Epithelium ◽

Membrane Material ◽

Lyn Kinase ◽

Multiple Protein ◽

Lens Cells

Na,K-ATPase is essential for the regulation of cytoplasmic Na+and K+levels in lens cells. Studies on the intact lens suggest activation of tyrosine kinases may inhibit Na,K-ATPase function. Here, we tested the influence of Lyn kinase, a Src-family member, on tyrosine phosphorylation and Na,K-ATPase activity in membrane material isolated from porcine lens epithelium. Western blot studies indicated the expression of Lyn in lens cells. When membrane material was incubated in ATP-containing solution containing partially purified Lyn kinase, Na,K-ATPase activity was reduced by ∼38%. Lyn caused tyrosine phosphorylation of multiple protein bands. Immunoprecipitation and Western blot analysis showed Lyn treatment causes an increase in density of a 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase α1polypeptide. Incubation with protein tyrosine phosphatase 1B (PTP-1B) reversed the Lyn-dependent tyrosine phosphorylation increase and the change of Na,K-ATPase activity. The results suggest that Lyn kinase treatment of a lens epithelium membrane preparation is able to bring about partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase α1catalytic subunit.

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Regional Distribution of Na,K-ATPase Activity in Porcine Lens Epithelium

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.03-0287 ◽

2003 ◽

Vol 44 (10) ◽

pp. 4395 ◽

Cited By ~ 34

Author(s):

Shigeo Tamiya ◽

William L. Dean ◽

Christopher A. Paterson ◽

Nicholas A. Delamere

Keyword(s):

Atpase Activity ◽

Regional Distribution ◽

Lens Epithelium

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Influence of amphotericin B on the sodium pump of porcine lens epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.2.c465 ◽

1996 ◽

Vol 270 (2) ◽

pp. C465-C473 ◽

Cited By ~ 8

Author(s):

N. A. Delamere ◽

W. L. Dean ◽

J. M. Stidam ◽

A. E. Moseley

Keyword(s):

Atpase Activity ◽

Amphotericin B ◽

Plasma Membranes ◽

Sodium Pump ◽

Sodium Concentration ◽

Lens Epithelium ◽

Fiber Cells ◽

Cell Plasma ◽

Lens Cell ◽

Sodium And Potassium

Active transport by Na(+)-K(+)-ATPase in the monolayer of lens epithelium is vital for the regulation of sodium and potassium levels within the mass of fiber cells that make up the bulk of the lens. In this study, experiments were conducted using porcine lenses to test whether Na(+)-K(+)-ATPase activity in the epithelium is altered when the permeability of lens cell plasma membranes is increased by the ionophore amphotericin B. After 24 h, sodium was significantly (P < 0.01) elevated in lenses exposed to 5 or 10 microM amphotericin B. Amphotericin B stimulated 86Rb uptake, probably through an increase of cytoplasmic sodium concentration due to increased inward sodium leak; the rate of ouabain-sensitive potassium (86Rb) uptake by intact lenses was significantly increased by amphotericin B at 5 microM (P < 0.05) and 10 microM (P < 0.01). After 24 h, the epithelium from lenses exposed to amphotericin B had an Na(+)-K(+)-ATPase activity that was more than twofold higher (P < 0.01) than the Na(+)-K(+)-ATPase activity in control lenses. By immunoblot, there was an increase in Na(+)-K(+)-ATPase catalytic (alpha) subunit immunoreactive polypeptide in the epithelium of lenses exposed to amphotericin B. The increase stemmed from a marked increase of Na(+)-K(+)-ATPase alpha 2-immunoreactive polypeptide but little change in the amount of alpha 1-immunoreactive protein. As judged by immunoblot experiments, the amount of Na(+)-K(+)-ATPase beta 1-immunoreactive polypeptide also appeared to be higher in the epithelium of amphotericin B-treated lenses compared with control lenses. In summary, these results suggest that in response to a permeability challenge with amphotericin B, the porcine lens epithelium is able to increase the activity of Na(+)-K(+)-ATPase. The same permeability challenge also appears to stimulate the biosynthesis of Na(+)-K(+)-ATPase catalytic subunit as well as glycoprotein subunit polypeptides.

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