Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Ortiz-Acevedo, Alejandro; Rigor, Robert R.; Maldonado, Héctor; Cala, Peter M

doi:10.1152/ajpcell.00160.2008

Cited by 4 publications

(9 citation statements)

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“…We previously demonstrated that activation of NHE1-mediated Na + transport activity in osmotically shrunken atRBCs is dependent upon a rate-limiting phosphorylation-dependent biochemical event [31] . The behavior of this rate-limiting event is consistent with that of a simple kinase and phosphatase pair, where the NHE1-inactivating phosphatase activity is inhibited by treatment with the protein phosphatase inhibitor CLA.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to mammalian cells, NHE1 transport activity is virtually nonexistent in quiescent atRBCs. Following suspension in hyperosmotic media, at RBCs exhibit an increase in NHE1-mediated Na + flux activity that is nominally 2-orders of magnitude greater than that of cells at normal resting volume in isosmotic medium [31] , [32] , [33] . In addition, the at RBC NHE1 homolog ( at NHE1) is 79% identical to human NHE1 at the amino acid level, retains the hallmark housekeeping characteristics of mammalian NHE1: cell pH and volume regulation, and is expressed in abundance in at RBCs compared to cell types known to over-express NHE1 (e.g., tumor cells) [33] , [34] .…”

Section: Introductionmentioning

confidence: 99%

“…This model is based on analysis of relaxation kinetics describing activation and inactivation of NHE1 transport activity, in response to acute osmotic shrinkage or re-swelling after OCS, respectively. The notion that phosphorylation is involved in control of NHE1 activity during OCS is based on the observation that NHE1 inactivation is prevented under various osmotic conditions by treatment with the protein phosphatase (PP1/PP2A) inhibitor calyculin-A (CLA) [31] . These functional studies provide a detailed kinetic description of the rate-limiting biochemical events in NHE1 activation and inactivation during osmotic cell volume perturbation, and while strongly suggestive of a phosphorylation-dependent mechanism, do not firmly establish a role for phosphorylation in NHE1 activation by OCS, or that CLA treatment affects the same cell signaling events as OCS.…”

Section: Introductionmentioning

confidence: 99%

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Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

et al. 2011

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The Na+/H + Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

et al. 2011

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“…K + ‐free solutions were prepared by replacing KCl with N ‐methyl‐ D ‐glucamine (NMDG) as well as replacing KH 2 PO 4 with NaH 2 PO 4 . Na + /K + ‐free solutions contained 10 mM of D‐glucose, 136.89 mM of NMDG, 140 mM of HCl, 1 mM of MgCl 2 , and 10 mM of HEPES 22,23 . Neutrophils were labeled with BCECF/AM for 30 minutes, and then, treated with DMSO or MVBR‐28 for 5 minutes followed by activation by fMLF.…”

Section: Methodsmentioning

confidence: 99%

A synthesized heterocyclic chalcone inhibits neutrophilic inflammation through K⁺‐dependent pH regulation

et al. 2020

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Human neutrophils have a vital role in host defense and inflammatory responses in innate immune systems. Growing evidence shows that the overproduction of reactive oxygen species and granular proteolytic enzymes from activated neutrophils is linked to the pathogenesis of acute inflammatory diseases. However, adequate therapeutic targets are still lacking to regulate neutrophil functions. Herein, we report that MVBR-28, synthesized from the Mannich bases of heterocyclic chalcone, has antineutrophilic inflammatory effects through regulation of intracellular pH. MVBR-28 modulates neutrophil functions by attenuating respiratory burst, degranulation, and migration. Conversely, MVBR-28 has no antioxidant effects and fails to alter elastase activity in cell-free systems. The anti-inflammatory effects of MVBR-28 are not seen through cAMP pathways. Significantly, MVBR-28 potently inhibits extracellular Ca 2+ influx in N-formyl-methionyl-leucyl-phenylalanine (fMLF)-and thapsigarginactivated human neutrophils. Notably, MVBR-28 attenuates fMLF-induced intracellular alkalization in a K + -dependent manner, which is upstream of Ca 2+ pathways. |

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“…As described previously (26), venous blood was drawn from adult Amphiuma tridactylum into 12-ml syringes containing 0.1 ml of heparin (10 kU/ml) as approved by IACUC Protocol 12754 (Shein Pharmaceutical, Florham Park, NJ) and separated from the plasma by gentle centrifugation (1,000 g) in a Clay Adams "Dynac" centrifuge (Clay Adams, Parsippany, NJ). Subsequently, RBCs were washed three times in 10 -15 volumes of isosmotic Ringer (IR) solution matched to the animal's plasma osmolarity (220 -250 mosM).…”

Section: Generalmentioning

confidence: 99%

Coordinated control of volume regulatory Na⁺/H⁺ and K⁺/H⁺ exchange pathways in Amphiuma red blood cells

Ortiz-Acevedo

Rigor

Maldonado

et al. 2010

American Journal of Physiology-Cell Physiology

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The Na+/H+ and K+/H+ exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H+ exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na+/H+ and K+/H+ exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na+/H+ and K+/H+ exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na+/H+ exchange activation increases with cell shrinkage, whereas the rate of Na+/H+ exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K+/H+ exchange activation increases with cell swelling, whereas the rate of K+/H+ exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K+/H+ exchange are reciprocally related to those controlling Na+/H+ exchange.

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Activation of Na⁺/H⁺ and K⁺/H⁺ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Abstract: Ortiz-Acevedo A, Rigor RR, Maldonado HM, Cala PM. Activation of Na ϩ /H ϩ and K ϩ /H ϩ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity.

Cited by 4 publications

References 52 publications

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

A synthesized heterocyclic chalcone inhibits neutrophilic inflammation through K⁺‐dependent pH regulation

Coordinated control of volume regulatory Na⁺/H⁺ and K⁺/H⁺ exchange pathways in Amphiuma red blood cells

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Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Abstract: Ortiz-Acevedo A, Rigor RR, Maldonado HM, Cala PM. Activation of Na ϩ /H ϩ and K ϩ /H ϩ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity.

Cited by 4 publications

References 52 publications

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

A synthesized heterocyclic chalcone inhibits neutrophilic inflammation through K+‐dependent pH regulation

Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells

Contact Info

Product

Resources

About

Activation of Na⁺/H⁺ and K⁺/H⁺ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

A synthesized heterocyclic chalcone inhibits neutrophilic inflammation through K⁺‐dependent pH regulation

Coordinated control of volume regulatory Na⁺/H⁺ and K⁺/H⁺ exchange pathways in Amphiuma red blood cells