68 results on '"Peter K. Lauf"'
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2. Surface-Enhanced Raman Spectroscopy (SERS) Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures
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Kevin M. Dorney, Ioana E.P. Sizemore, Tariq Alqahtani, Norma C. Adragna, and Peter K. Lauf
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Chelerythrine ,Human Lens Epithelia ,Surface-Enhanced Raman Spectroscopy ,Cellular Distribution ,Na+/K+ Pump ,Physiology ,QP1-981 ,Biochemistry ,QD415-436 - Abstract
Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na+/K+ pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Methods: Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.
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- 2013
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3. Sea Water Acidification Affects Osmotic Swelling, Regulatory Volume Decrease and Discharge in Nematocytes of the Jellyfish Pelagia noctiluca
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Rossana Morabito, Angela Marino, Peter K. Lauf, Norma C. Adragna, and Giuseppa La Spada
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Sea water acidification ,RVD ,Discharge ,Nematocytes ,Jellyfish ,Pelagia noctiluca ,Physiology ,QP1-981 ,Biochemistry ,QD415-436 - Abstract
Background: Increased acidification/PCO2 of sea water is a threat to the environment and affects the homeostasis of marine animals. In this study, the effect of sea water pH changes on the osmotic phase (OP), regulatory volume decrease (RVD) and discharge of the jellyfish Pelagia noctiluca (Cnidaria, Scyphozoa) nematocytes, collected from the Strait of Messina (Italy), was assessed. Methods: Isolated nematocytes, suspended in artificial sea water (ASW) with pH 7.65, 6.5 and 4.5, were exposed to hyposmotic ASW of the same pH values and their osmotic response and RVD measured optically in a special flow through chamber. Nematocyte discharge was analyzed in situ in ASW at all three pH values. Results: At normal pH (7.65), nematocytes subjected to hyposmotic shock first expanded osmotically and then regulated their cell volume within 15 min. Exposure to hyposmotic ASW pH 6.5 and 4.5 compromised the OP and reduced or totally abrogated the ensuing RVD, respectively. Acidic pH also significantly reduced the nematocyte discharge response. Conclusion: Data indicate that the homeostasis and function of Cnidarians may be altered by environmental changes such as sea water acidification, thereby validating their use as novel bioindicators for the quality of the marine environment.
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- 2013
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4. Canonical Bcl-2 Motifs of the Na+/K+ Pump Revealed by the BH3 Mimetic Chelerythrine: Early Signal Transducers of Apoptosis?
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Peter K. Lauf, Judith Heiny, Jarek Meller, Michael A. Lepera, Leonid Koikov, Gerald M. Alter, Thomas L. Brown, and Norma C. Adragna
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Chelerythrine ,Na+/K+ Pump-ATPase ,NKCC1 ,KCC ,K+ Channels ,Bcl-2 Proteins ,Apoptosis ,Human Lens Epithelia ,Physiology ,QP1-981 ,Biochemistry ,QD415-436 - Abstract
Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. Methods: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. Results: CET inhibited NKP and NKCC by >90% (IC50 values ∼35 and ∼15 µM, respectively) without significant KCC activity change, and stimulated K+ loss by ∼35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. Conclusion: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.
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- 2013
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5. Regulated phosphorylation of the K-Cl cotransporter KCC3 at dual C-terminal threonines is a potent switch of intracellular potassium content and cell volume homeostasis
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Norma C. Adragna, Nagendra Babu Ravilla, Peter K. Lauf, Gulnaz eBegum, Arjun R. Khanna, Dandan eSun, and Kristopher T Kahle
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Chloride Channels ,GABA Modulators ,KCC2 ,neuropathic pain ,Autism Spectrum Disorders ,NKCC1 ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K+ and Cl– efflux via the activation of K+ channels, volume-regulated anion channels (VRACs), and the K+-Cl– cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na+-K+-2Cl– cotransporter isoform 1 (NKCC1). This results in a rapid (90 %) reduction in intracellular K+ content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent (DCPIB [VRAC inhibitor]-sensitive) pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in the KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.
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- 2015
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6. Assessment of Silver-Nanoparticles-Induced Erythrocyte Cytotoxicity through Ion Transport Studies
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Arathi S L Paluri, Norma C. Adragna, Praveen Kumar Alla, Ioana E Pavel-Sizmore, Jerome L. Yaklic, and Peter K. Lauf
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0301 basic medicine ,Erythrocytes ,Silver ,Absorption spectroscopy ,Physiology ,Potassium ,Metal Nanoparticles ,chemistry.chemical_element ,lcsh:Physiology ,Silver nanoparticle ,Rubidium ,Ion ,lcsh:Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Extracellular ,Humans ,lcsh:QD415-436 ,Particle Size ,Cells, Cultured ,Ion transporter ,Ion Transport ,lcsh:QP1-981 ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Hemoglobin ,Nuclear chemistry - Abstract
Background/aims Silver nanoparticles (AgNPs) are the most frequently used nanomaterials in industrial and biomedical applications. Their functionalization significantly impacts their properties and potential applications. Despite the need to produce, investigate and apply them, not much is known about the toxicity of silver nanoparticles to and their interaction with blood components, such as erythrocytes. Here, we report on the effect of two negatively charged AgNPs (Creighton, and Lee-Meisel) on ion transport in human red blood cells (HRBCs). Methods HRBCs were obtained from blood of adult donors, which was either expired, fresh or refrigerated for variable lengths of time, and from fresh or refrigerated cord blood. Rb+ and K+ ions were measured by atomic emission and absorption spectrophotometry, respectively. Erythrocyte hemoglobin optical density (Hbc OD), was determined at 527 nm to estimate RBC volume in the same tubes in which Rb+ and K+ were measured. Cellular Rb+ uptake and intracellular K+ concentrations, [K]i, were calculated in mmol/L of original cells (LOC) per time. Rubidium, a potassium ion (K+) congener used to measure K+ influx, [K]i, and Hbc ODs were determined in the presence and absence of several concentrations (0-150 µg mL-1) of spherical AgNPs of an average diameter of 10 nm, at different time points (0-60 min). Results Creighton AgNPs inhibited Rb+ influx and depleted the cells of K+ independently of the source and in a time and dose-dependent manner. In contrast, Lee-Meisel AgNPs caused ~ 50 % Rb+ influx inhibition and ~ 15 % K+ loss with larger interindividual variability than Creighton AgNPs. The loss of K+ from HRBCs was entirely accounted for by an increase in extracellular K+ concentration, [K]o. Enhanced dark field optical microscopy in conjunction with CytoViva® hyperspectral imaging helped visualize AgNPs internalized by HRBCs, thus pointing to a potential cause for their cytotoxic effects. Conclusion These findings indicate that HRBC K+ homeostasis is an early and sensitive biomarker for AgNPs toxicity and is a function of their surface functionalization.
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- 2019
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7. Cytosolic Internalization of the Na/K ATPase Ouabain Receptor Complex (NORC)
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Norma C. Adragna, Peter K. Lauf, and Joshua L. Stricker
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Receptor complex ,Chemistry ,media_common.quotation_subject ,Biochemistry ,Ouabain ,Cytosol ,Genetics ,Biophysics ,medicine ,Na+/K+-ATPase ,Internalization ,Molecular Biology ,Biotechnology ,media_common ,medicine.drug - Published
- 2018
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8. Large Gold Nanorods Affect Glutathione but not K + Metabolism in Human Red Blood Cells
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Jerome C. Yaklic, Adam de la Zerda, Praveen Kumar Alla, Peter K. Lauf, Norma C. Adragna, Elliot D. SoRelle, Nedu C. Ihezurike, and Ioana E. Pavel‐Sizemore
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chemistry.chemical_compound ,Chemistry ,Genetics ,Biophysics ,Nanorod ,Metabolism ,Glutathione ,Affect (psychology) ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2018
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9. Not all silver nanoparticles are equal: Effect of functionalization on K homeostasis in human red blood cells
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Peter K. Lauf, Praveen Kumar Alla, Ioana E. Sizemore, Jerome L. Yaklic, Norma C. Adragna, and Arathi S L Paluri
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Chemistry ,Genetics ,Biophysics ,Surface modification ,K homeostasis ,Molecular Biology ,Biochemistry ,Silver nanoparticle ,Biotechnology - Published
- 2017
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10. Surface-Enhanced Raman Spectroscopy (SERS) Tracking of Chelerythrine, a Na+/K+Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures
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Tariq Alqahtani, Peter K. Lauf, Kevin M. Dorney, Ioana E. Sizemore, and Norma C. Adragna
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Silver ,Lysis ,Surface Properties ,Physiology ,Metal Nanoparticles ,Spectrum Analysis, Raman ,lcsh:Physiology ,Silver nanoparticle ,Surface-Enhanced Raman Spectroscopy ,lcsh:Biochemistry ,chemistry.chemical_compound ,Cytosol ,Lens, Crystalline ,Humans ,lcsh:QD415-436 ,Chelerythrine ,Na+/K+-ATPase ,Cells, Cultured ,Benzophenanthridines ,Membrane potential ,Chromatography ,Na+/K+ Pump ,lcsh:QP1-981 ,Cell Membrane ,Epithelial Cells ,Cellular Distribution ,Surface-enhanced Raman spectroscopy ,Membrane ,chemistry ,Biochemistry ,Human Lens Epithelia ,Sodium-Potassium-Exchanging ATPase - Abstract
Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na+/K+ pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Methods: Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.
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- 2013
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11. Evidence for Aquaporin-mediated Water Transport in Nematocytes of the Jellyfish Pelagia noctiluca
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Giuseppina La Spada, Peter K. Lauf, Norma C. Adragna, Angela Marino, and Rossana Morabito
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Tetraethylammonium ,Water transport ,Osmotic shock ,biology ,Physiology ,Aquaporin ,Anatomy ,Pelagia noctiluca ,biology.organism_classification ,chemistry.chemical_compound ,Hypotonic Shock ,chemistry ,Biophysics ,Nematocyst ,Cnidocyte - Abstract
Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K+, Cl- and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca2+ method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl2 (0.1-25 µM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO3 (1 µM) and TEA (tetraethylammonium, 100 µM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ∼10-7 cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.
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- 2011
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12. Mechanisms of Hyposmotic Volume Regulation in Isolated Nematocytes of the Anthozoan Aiptasia diaphana
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Rossana Morabito, Angela Marino, Peter K. Lauf, Norma C. Adragna, and Giuseppina La Spada
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Water transport ,Thiocyanate ,biology ,Physiology ,ved/biology ,Potassium ,ved/biology.organism_classification_rank.species ,Acontia ,Aiptasia diaphana ,chemistry.chemical_element ,Okadaic acid ,biology.organism_classification ,Potassium channel ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Cnidocyte - Abstract
The nature and role of potassium (K) and water transport mediating hyposmotically-induced regulatory volume decrease (RVD) were studied in nematocytes dissociated with 605 mM thiocyanate from acontia
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- 2010
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13. Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells
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Peter K. Lauf, Norma C. Adragna, Ameet A. Chimote, and Sandeep Misri
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Osmosis ,Cell Membrane Permeability ,Physiology ,Acetates ,Sodium Potassium Chloride Symporter Inhibitors ,Voltage-Dependent Anion Channels ,Clotrimazole ,Ouabain ,Bumetanide ,Symporters ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Niflumic Acid ,K+ conductance ,Intermediate-Conductance Calcium-Activated Potassium Channels ,Immunohistochemistry ,medicine.anatomical_structure ,Indenes ,Phloretin ,Biochemistry ,Lens (anatomy) ,Indans ,Sodium-Potassium-Exchanging ATPase ,Ion Channel Gating ,Sodium-Potassium-Chloride Symporters ,Blotting, Western ,Cyclopentanes ,Cell Line ,Ion ,Chloride Channels ,Lens, Crystalline ,Potassium Channel Blockers ,medicine ,Humans ,Cell Size ,Dose-Response Relationship, Drug ,Spectrophotometry, Atomic ,Epithelial Cells ,Cell Biology ,Rubidium ,Epithelium ,Kinetics ,Cell culture ,Nitrobenzoates ,Potassium ,Biophysics ,Pyrazoles ,Cotransporter - Abstract
This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain ± bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media ∼90% of the total Rb influx occurred through the Na-K pump and NKCC and ∼10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased Kc by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 ∼25 μM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.
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- 2008
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14. Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis
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Kristopher T. Kahle, Dandan Sun, Arjun Khanna, Gulnaz Begum, Norma C. Adragna, Peter K. Lauf, and Nagendra Babu Ravilla
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Osmotic shock ,Chemistry ,KCC3 ,cell volume homeostasis ,neurodegeneration ,SPAK ,Cell biology ,Cellular and Molecular Neuroscience ,cerebral edema ,Biochemistry ,Chloride channel ,NKCC1 ,regulatory volume decrease ,Phosphorylation ,Cotransporter ,K-Cl cotransporters ,Cell volume homeostasis ,Homeostasis ,Intracellular ,Ion transporter ,Original Research ,Neuroscience - Abstract
The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K(+) and Cl(-) efflux via activation of K(+) channels, volume-regulated anion channels (VRACs), and the K(+)-Cl(-) cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1). This results in a rapid (10 min) and significant (90%) reduction in intracellular K(+) content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.
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- 2015
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15. On the Interaction of the Na/K ‐Pump ATPase (NAKPA) with Pro‐Survival and Pro‐Apoptotic Bcl‐2 Proteins
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Peter K. Lauf, Chandra Kumar Maharjan, Norma C. Adragna, Karin Flues, and Tariq Alqahtani
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biology ,Apoptosis ,Chemistry ,ATPase ,Genetics ,biology.protein ,Na+/K+-ATPase ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2015
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16. The Phosphorylation State of KCC3 Encodes a Potent Molecular Switch of Transporter Activity, Cell Volume, and K + Content
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Arjun Khanna, Kristopher K. Kahle, Peter K. Lauf, Dandan Sun, Gulnaz Begum, Nagendra Babu Ravilla, and Norma C. Adragna
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Molecular switch ,Cell swelling ,Cell volume ,Cellular functions ,Transporter ,Biology ,Biochemistry ,Cell biology ,Genetics ,medicine ,Phosphorylation ,Swelling ,medicine.symptom ,Molecular Biology ,Homeostasis ,Biotechnology - Abstract
Defense of cell volume against excessive shrinkage or swelling is a requirement for multiple essential cellular functions and organismal survival. Cell swelling triggers a homeostatic counter-respo...
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- 2015
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17. Cloning and Expression of Sheep Renal K-CI Cotransporter-1
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Kenneth B. Gagnon, Jin J Zhang, Peter K. Lauf, Robert E.W. Fyffe, Sandeep Misri, and Norma C. Adragna
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DNA, Complementary ,Physiology ,Molecular Sequence Data ,Gene Expression ,In Vitro Techniques ,Biology ,Kidney ,Cell Line ,Mice ,Chlorides ,Species Specificity ,Complementary DNA ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Cloning ,Sheep ,Base Sequence ,Sequence Homology, Amino Acid ,Symporters ,urogenital system ,Molecular biology ,Recombinant Proteins ,Open reading frame ,medicine.anatomical_structure ,NIH 3T3 Cells ,Potassium ,Cotransporter - Abstract
Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding an approximately 122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH2-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated approximately 150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide.
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- 2005
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18. Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells
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Jing Zhang, Norma C. Adragna, and Peter K. Lauf
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medicine.medical_specialty ,Vascular smooth muscle ,Platelet-derived growth factor ,Physiology ,medicine.medical_treatment ,Myocytes, Smooth Muscle ,Cycloheximide ,Culture Media, Serum-Free ,Muscle, Smooth, Vascular ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Internal medicine ,medicine ,Animals ,Myocyte ,Receptors, Platelet-Derived Growth Factor ,RNA, Messenger ,Cells, Cultured ,Platelet-Derived Growth Factor ,Dose-Response Relationship, Drug ,Symporters ,biology ,Growth factor ,Cell Membrane ,Proteins ,Cell Biology ,Rats ,Cell biology ,Ion homeostasis ,Endocrinology ,chemistry ,Mitogen-activated protein kinase ,biology.protein ,Platelet-derived growth factor receptor - Abstract
Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.
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- 2003
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19. GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells
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Kazunari Higa, Peter K. Lauf, Tomomi Kanemaru, Norma C. Adragna, Hiroshi Fujise, and Miwa Fukuda
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Erythrocytes ,Physiology ,Blood cell ,chemistry.chemical_compound ,Dogs ,Chlorides ,Dinitrochlorobenzene ,medicine ,Animals ,Humans ,Ion transporter ,Cell Size ,Diamide ,Nitrates ,biology ,Sulfhydryl Reagents ,Fissipedia ,Cell Biology ,Glutathione ,Membrane transport ,biology.organism_classification ,In vitro ,Red blood cell ,medicine.anatomical_structure ,Biochemistry ,chemistry ,Potassium ,Biophysics ,Indicators and Reagents ,Cotransporter ,Oxidation-Reduction - Abstract
Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO2)-mediated oxidation, diazene dicarboxylic acid bis- N, N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO2 > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO2 and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.
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- 2001
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20. Protein Kinase G Regulates Potassium Chloride Cotransporter-3 Expression in Primary Cultures of Rat Vascular Smooth Muscle Cells
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Peter K. Lauf, Mauricio Di Fulvio, Norma C. Adragna, and Thomas M. Lincoln
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Messenger RNA ,medicine.medical_specialty ,Vascular smooth muscle ,Kidney metabolism ,Endogeny ,Cell Biology ,Transfection ,Biology ,Biochemistry ,Nitric oxide ,Cell biology ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Internal medicine ,Symporter ,cardiovascular system ,medicine ,Molecular Biology ,cGMP-dependent protein kinase - Abstract
K-Cl cotransport (KCC) is activated by nitric oxide donors and appears to be regulated by the cGMP signaling pathway. Expression of KCC mRNAs (KCC1–KCC4) in rat vascular smooth muscle cells (VSMCs) is unknown. We have reported the presence of KCC1 and KCC3 mRNAs in primary cultures of VSMCs by specific reverse transcription-polymerase chain reaction. KCC2 mRNA appeared at extremely low levels. KCC4 mRNA was undetectable. Semiquantitative reverse transcription-polymerase chain reaction revealed a 2:1 KCC1/KCC3 mRNA ratio in VSMCs. Depletion of protein kinase G (PKG)-1 from VSMCs did not change KCC3 mRNA expression. Analogous results were obtained with PKG-1-catalytic domain- and vector only-transfected VSMCs lacking endogenous PKG, suggesting no involvement of PKG-1 in the maintenance of basal KCC3 mRNA expression. However, 8-bromo-cGMP, a PKG stimulator, acutely increased KCC3 mRNA expression in a concentration- and time-dependent fashion; this effect was blocked by the PKG inhibitor KT5823 but not by actinomycin D. These findings show that VSMCs express mainly two mRNA isoforms, KCC1 and KCC3, and suggest that PKG participates post-transcriptionally in the acute KCC3 mRNA regulation. The role of KCC3 on cell volume and electrolyte homeostasis in response to PKG modulators remains to be determined.
- Published
- 2001
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21. Swelling-Induced K+ Fluxes in Vascular Smooth Muscle Cells are Mediated by Charybdotoxin-Sensitive K+ Channels
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Yana Anfinogenova, Ryszard Grygorczyk, Peter K. Lauf, Sergei N. Orlov, Xavier Rodriguez, Pavel Hamet, and Norma C. Adragna
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chemistry.chemical_compound ,Vascular smooth muscle ,Charybdotoxin ,chemistry ,Physiology ,medicine ,Biology ,Swelling ,medicine.symptom ,Cotransporter ,Cell biology ,K channels - Abstract
This study examines the relative contributions of K-Cl cotransport and K+ channels to swelling-induced K+ fluxes in vascular smooth muscle cells (VSMC). DIOA known as a potent in
- Published
- 2001
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22. Speaker Abstracts
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Peter K. Lauf, Kenneth B. E. Gagnon, Norma C. Adragna, and Robert E.W. Fyffe
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0303 health sciences ,03 medical and health sciences ,0302 clinical medicine ,Physiology ,Chemistry ,Cell culture ,030220 oncology & carcinogenesis ,Biophysics ,Cotransporter ,C6 glioma ,030304 developmental biology ,Ion - Published
- 2000
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23. Two Operational Modes of K-Cl Cotransport in Low K+ Sheep Red Blood Cells
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Lisa Carnes, Olga Ortiz-Carranza, Peter K. Lauf, and Norma C. Adragna
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Physiology ,Kinase ,Phosphatase ,Spermine ,Biology ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Biophysics ,Efflux ,Cytoskeleton ,Cotransporter ,Flux (metabolism) ,Calyculin - Abstract
The present study further characterizes the two operational modes of K-Cl cotransport in low K+ sheep red blood cells previously described and proposed by us: (i) a membrane-bound cotransport entity devoid of modulation by putative kinases/phosphatases, and (ii) an ATP- and Mg-modulated activity. We now report that quinine, spermine, and 300 µM Cai2+ inhibit (40-100%) K-Cl efflux in ATP- and Mgi2+-depleted cells and hence interact directly with the cotransporter moiety. The sensitivity of K-Cl cotransport to calyculin, however, was modulated by the ATP content of the cells: after depletion of both ATP and Mgi2+ the flux was insensitive to the inhibitor, whereas at about 120 µmol ATP/L cells calyculine inhibited K-Cl efflux by 95-100% in Mgi2+-free cells. The K-Cl cotransporter itself, free of modulation by kinases/phosphatases, ‘senses’ changes in cell volume perhaps at the membrane/cytoskeleton level. Cellular ATP then switches the co-transporter’s operational mode so that under physiological conditions the rate of K-Cl efflux is under the control of a putative kinases/phosphatases pathway.
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- 1997
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24. Apelin regulation of potassium chloride cotransport (KCC) in vascular smooth muscle cells (VSMCs): Relation to Cardiovascular Disease (CVD)
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Neelima Sharma, Peter K. Lauf, and Norma C. Adragna
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medicine.medical_specialty ,Vascular smooth muscle ,Chemistry ,Potassium ,chemistry.chemical_element ,Disease ,Biochemistry ,Apelin ,Endocrinology ,Internal medicine ,Genetics ,medicine ,Cotransporter ,Molecular Biology ,Biotechnology - Published
- 2013
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25. The K‐Cl cotransporter KCC3 is a catalytic switch of cellular K and volume homoestasis
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Nagendra Babu Ravilla, Peter K. Lauf, Norma C. Adragna, and Kristopher T. Kahle
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Volume (thermodynamics) ,Chemistry ,Genetics ,Biophysics ,Cotransporter ,Molecular Biology ,Biochemistry ,Biotechnology ,Catalysis - Published
- 2013
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26. Novel Mechanism of Na/K Pump Inhibition by Chelerythrine (CET), a PKC Inhibitor, Uncovers Potential Early Signal Transducers of Apoptosis
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Peter K. Lauf, Norma C. Adragna, Judith A. Heiny, Gerald M. Alter, Tom Brown, and Jarek Meller
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chemistry.chemical_compound ,Chelerythrine ,Chemistry ,Apoptosis ,Mechanism (biology) ,Genetics ,Biophysics ,Na+/K+-ATPase ,Molecular Biology ,Biochemistry ,Signal ,Protein kinase C ,Biotechnology - Published
- 2013
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27. Rb influx in K‐Cl cotransporter 3 (KCC3)‐transfected human embryonic kidney 293 (HEK293) cells and effect of external Na
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Kristopher T. Kahle, Peter K. Lauf, Norma C. Adragna, and Nagendra Babu Ravilla
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Kidney ,medicine.anatomical_structure ,Chemistry ,HEK 293 cells ,Genetics ,medicine ,Transfection ,Cotransporter ,Molecular Biology ,Biochemistry ,Embryonic stem cell ,Biotechnology ,Cell biology - Published
- 2013
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28. A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium
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Norma C. Adragna and Peter K. Lauf
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Erythrocytes ,Physiology ,Intracellular pH ,Potassium ,education ,Inorganic chemistry ,Drug Resistance ,Ionophore ,chemistry.chemical_element ,4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ,Ouabain ,Divalent ,Chlorides ,Reference Values ,Electrochemistry ,medicine ,Animals ,Magnesium ,Ions ,chemistry.chemical_classification ,Membrane potential ,Nitrates ,Sheep ,Symporters ,Water ,Articles ,Hydrogen-Ion Concentration ,Rubidium ,chemistry ,Thermodynamics ,Carrier Proteins ,Cotransporter ,medicine.drug - Abstract
Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K-Cl cotransport. The anion exchange inhibitor 4,4'diisothiocyanato-2,2'disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.
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- 1996
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29. Chelerythrine, a protein kinase C inhibitor, abolishes K flux through Na/K pump and Na‐K‐2Cl cotransporter‐1, and stimulates SK channels in human lens epithelial cells
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Michael A. Lepera, Peter K. Lauf, and Norma C. Adragna
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Chemistry ,Kinase ,A protein ,Biochemistry ,Molecular biology ,SK channel ,chemistry.chemical_compound ,Chelerythrine ,medicine.anatomical_structure ,Lens (anatomy) ,Genetics ,medicine ,Biophysics ,Na+/K+-ATPase ,Cotransporter ,Molecular Biology ,Biotechnology - Published
- 2012
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30. Sea water acidification alters cell volume regulation and discharge in jellyfish
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Rossana Morabito, Peter K. Lauf, Norma C. Adragna, Angela Marino, and Giuseppina La Spada
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Fishery ,Jellyfish ,biology ,biology.animal ,Cell volume ,Genetics ,Environmental science ,Seawater ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2012
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31. KCC2a expression in a human fetal lens epithelial cell line
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Peter K. Lauf, Norma C. Adragna, Vinita Srivastava, Mauricio Di Fulvio, and Neelima Sharma
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Gene isoform ,Physiology ,ATPase ,Molecular Sequence Data ,Biology ,Immunofluorescence ,Cell Line ,Western blot ,Lens, Crystalline ,medicine ,Humans ,Protein Isoforms ,RNA, Messenger ,medicine.diagnostic_test ,Base Sequence ,Symporters ,Epithelial Cells ,Molecular biology ,Immunohistochemistry ,Epithelium ,Blot ,medicine.anatomical_structure ,Real-time polymerase chain reaction ,Microscopy, Fluorescence ,Cell culture ,Mutation ,biology.protein ,Sodium-Potassium-Exchanging ATPase - Abstract
The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin.
- Published
- 2012
32. Evidence for a Na/K pump in nematocytes isolated from sea anemones
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Peter K. Lauf, Rossana Morabito, Giuseppina La Spada, Angela Marino, and Norma C. Adragna
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Chemistry ,Genetics ,Biophysics ,Cnidocyte ,Na+/K+-ATPase ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2012
33. Hydroxyurea affects cell morphology, cation transport, and red blood cell adhesion in cultured vascular endothelial cells
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Peter Fonseca, Peter K. Lauf, and Norma C. Adragna
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Endothelium ,Membrane permeability ,Cell adhesion molecule ,Immunology ,Cell Biology ,Hematology ,Biology ,Cell morphology ,Biochemistry ,Molecular biology ,Endothelial stem cell ,Red blood cell ,medicine.anatomical_structure ,Cell culture ,medicine ,Cation transport - Abstract
Hydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, approximately 200%) and cell K (maximum, approximately 50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr- loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.
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- 1994
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34. Eryptotic red blood cell adhesion to vascular endothelium: CXCL16/SR-PSOX, a pathological amplifier. Focus on 'Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX'
- Author
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Peter K. Lauf
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Chemokine ,Erythrocytes ,Physiology ,Phosphatidylserines ,chemistry.chemical_compound ,medicine ,Human Umbilical Vein Endothelial Cells ,Humans ,Scavenger receptor ,Receptor ,CXCL16 ,Receptors, Scavenger ,biology ,Cell Membrane ,Cell Biology ,Phosphatidylserine ,Chemokine CXCL16 ,Transmembrane protein ,Cell biology ,Red blood cell ,medicine.anatomical_structure ,chemistry ,biology.protein ,Calcium ,Endothelium, Vascular ,Chemokines, CXC ,Proto-oncogene tyrosine-protein kinase Src - Abstract
THE STUDY BY LANG AND COLLEAGUES (2), published in this issue, shows that red blood cells (RBCs), undergoing suicidal death or “eryptosis,” attach to human umbilical vascular endothelial cells (HUVECs) under experimental flow conditions via their CXC ligand 16 (CXCL16)/SR-PSOX transmembrane ligands with the potential to greatly impact the microvascular circulation. CXCL16, a chemokine with a cysteine-X-cysteine motif, occurs either in soluble form or as a transmembrane receptor (16) that can be tyrosine phosphorylated and bind Src homology 2-containing proteins; it is synonymous with SR-PSOX, the scavenger receptor for phosphatidylserine (PS) and oxidized low-density lipoproteins (oxLDL) (19). Here, I review developments that underlie this discovery (2), emphasizing the complex cellular mechanisms of erythrocyte-augmented/induced vascular occlusion in conjunction with their suicidal death (eryptosis). After 120 days in the circulation, 3 10 9 or one-third milliliter human RBCs/day undergo demise within the reticuloendothelial system by macrophage-mediated phagocytosis. Macrophages, which trap and digest old (but not intact mature) erythrocytes, recognize “senescent” antigens (SCA) of 62 kDa suggested to be related to band-3 proteins involved in Cl/HCO3 exchange (7). An alternate hypothesis considers autoantibodies formed against SCA epitopes derived from band-3 aggregation (13). Simultaneously, another concept emerged: reduction of the number of sialic acid residues (10 9 /cell) would uncover the presence of Thomsen (T)
- Published
- 2011
35. K Influx Parameters in Rat Aortic Vascular Smooth Muscle Cells (RASMCs)
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Peter K. Lauf, Neelima Sharma, and Norma C. Adragna
- Subjects
medicine.medical_specialty ,Endocrinology ,Vascular smooth muscle ,Chemistry ,Internal medicine ,Genetics ,medicine ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2011
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36. Evidence for aquaporin-mediated water tran sport in nematocytes of the jellyfish Pelagia noctiluca
- Author
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Angela, Marino, Rossana, Morabito, Giuseppina, La Spada, Norma C, Adragna, and Peter K, Lauf
- Subjects
Nematocytes ,Volume regulation ,Tetraethylammonium ,Water ,Biological Transport ,Anthozoa ,Aquaporins ,Water transport, Aquaporins,Osmotic swelling, Volume regulation,Nematocytes, Jellyfish,Pelagia noctiluca ,Dithiothreitol ,Nematocyst ,Osmotic Pressure ,Mercuric Chloride ,Osmotic swelling ,Animals ,Silver Nitrate ,Jellyfish ,Cell Size ,Water transport ,Pelagia noctiluca - Abstract
Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K(+), Cl(-) and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca(2+) method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl(2) (0.1-25 μM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO(3) (1 μM) and TEA (tetraethylammonium, 100 μM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ~10(-7) cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.
- Published
- 2011
37. Mechanisms of Hyposmotic Volume Regulationin Isolated Nematocytes of the Anthozoan Aiptasiadiaphana
- Author
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Angela, Marino, Rossana, Morabito, Giuseppina, La Spada, Norma C, Adragna, and Peter K, Lauf
- Subjects
Hyposmotic cell volume regulation ,Nematocytes ,Okadaic acid ,Aiptasia diaphana ,Potassium channels ,Sea Anemones ,Nematocyst ,Barium ,Chloride Channels ,Ethylmaleimide ,Osmotic Pressure ,Water channels ,Potassium-chloride cotransport ,Potassium Channel Blockers ,N-ethylmaleimide ,Animals ,Cell Size - Abstract
The nature and role of potassium (K) and water transport mediating hyposmotically-induced regulatory volume decrease (RVD) were studied in nematocytes dissociated with 605 mM thiocyanate from acontia of the Anthozoan Aiptasia diaphana. Cell volume and hence RVD were calculated from the inverse ratios of the cross sectional areas of nematocytes (A/A(o)) measured before (A(o)) and after (A) challenge with 65% artificial sea water (ASW). To distinguish between K channels and K-Cl cotransport (KCC), external sodium (Na) and chloride (Cl) were replaced by K and nitrate (NO(3)), respectively. Inhibitors were added to identify K channels (barium, Ba), and putative kinase (N-ethylmaleimide, NEM) and phosphatase (okadaic acid, OA) regulation of KCC. In 65% NaCl ASW, nematocytes displayed a biphasic change in A/A(o), peaking within 4 min due to osmotic water entry and thereafter declining within 6 min due to RVD. Changing NaCl to KCl or NaNO(3) ASW did not affect the osmotic phase but attenuated RVD, consistent with K channel and KCC mechanisms. Ba (3 mM) inhibited RVD. NEM and OA, applied separately, inhibited the osmotic phase and muted RVD suggesting primary action on water transport (aquaporins). NEM and OA together reduced the peak A/A(o) ratio during the osmotic phase whereas RVD was inhibited when OA preceded NEM. Thus, both K channels and KCC partake in the nematocyte RVD, the extent of which is determined by functional thiols and dephosphorylation of putative aquaporins facilitating the preceding osmotic water shifts.
- Published
- 2010
38. Erythrocytes Constitute the Main Blood Compartment for Aluminum
- Author
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Jodee Collins, Bernardo Arevalo, Peter K. Lauf, and Norma C. Adragna
- Subjects
Chemistry ,Genetics ,Biophysics ,Compartment (pharmacokinetics) ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2009
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39. Role of Rh (rhesus) complex in K‐Cl cotransport (KCC) regulation in red blood cells (RBCs) of Rh and Rh‐associated glycoprotein (RhAg) knockout (KO) mice
- Author
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Dominique Goossens, Peter K. Lauf, Jean-Pierre Cartron, and Norma C. Adragna
- Subjects
chemistry.chemical_classification ,biology ,Chemistry ,RHAG ,Genetics ,biology.protein ,Cotransporter ,Glycoprotein ,Molecular Biology ,Biochemistry ,Molecular biology ,Biotechnology - Published
- 2009
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40. Further characteristics of hyposmotically activated K loss in human lens epithelial cells (hLECs)
- Author
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Peter K. Lauf, Ameet A. Chimote, and Norma C. Adragna
- Subjects
medicine.anatomical_structure ,Optics ,business.industry ,Chemistry ,Lens (anatomy) ,Genetics ,medicine ,business ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2008
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41. Li fluxes reveal presence of Na‐Cl Cotransport (NCC) in Human Lens Epithelial Cells (LECs)
- Author
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Peter K. Lauf, Norma C. Adragna, and Ameet A. Chimote
- Subjects
medicine.anatomical_structure ,Chemistry ,Lens (anatomy) ,Na cl cotransport ,Genetics ,medicine ,Biophysics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2008
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42. K-Cl cotransport function and its potential contribution to cardiovascular disease
- Author
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Peter K. Lauf and Norma C. Adragna
- Subjects
Biochemistry ,Kinase ,Physiology (medical) ,Second messenger system ,Cellular homeostasis ,Transporter ,Biology ,Cotransporter ,Neuroscience ,Homeostasis ,Intracellular ,Function (biology) ,Pathology and Forensic Medicine - Abstract
K–Cl cotransport is the coupled electroneutral movement of K and Cl ions carried out by at least four protein isoforms, KCC1-4. These transporters belong to the SLC12A family of coupled cotransporters and, due to their multiple functions, play an important role in the maintenance of cellular homeostasis. Significant information exists on the overall function of these transporters, but less is known about the role of the specific isoforms. Most functional studies were done on K–Cl cotransport fluxes without knowing the molecular details, and only recently attention has been paid to the isoforms and their individual contribution to the fluxes. This review summarizes briefly and updates the information on the overall functions of this transporter, and offers some ideas on its potential contribution to the pathophysiological basis of cardiovascular disease. By virtue of its properties and the cellular ionic distribution, K–Cl cotransport participates in volume regulation of the nucleated and some enucleated cells studied thus far. One of the hallmarks in cardiovascular disease is the inability of the organism to maintain water and electrolyte balance in effectors and/or target tissues. Oxidative stress is another compounding factor in cardiovascular disease and of great significance in our modern life styles. Several functions of the transporter are modulated by oxidative stress, which in turn may cause the transporter to operate in either "overdrive" with the purpose to counteract homeostatic changes, or not to respond at all, again setting the stage for pathological changes leading to cardiovascular disease. Intracellular Mg, a second messenger, acts as an inhibitor of K–Cl cotransport and plays a crucial role in regulating the activity of protein kinases and phosphatases, which, in turn, regulate a myriad of cellular functions. Although the role of Mg in cardiovascular disease has been dealt with for several decades, this chapter is evolving nowadays at a faster pace and the relationships between Mg, K–Cl cotransport, and cardiovascular disease is an area that awaits further experimentation. We envision that further studies on the role of K–Cl cotransport, and ideally on its specific isoforms, in mammalian cells will add missing links and help to understand the cellular mechanisms involved in the pathophysiology of cardiovascular disease.
- Published
- 2007
43. Characterization of glial cell K-Cl cotransport
- Author
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Kenneth B. E. Gagnon, Peter K. Lauf, Robert E.W. Fyffe, and Norma C. Adragna
- Subjects
Physiology ,Sodium-Potassium-Chloride Symporters ,Ouabain ,Cell Line ,chemistry.chemical_compound ,Chlorides ,Furosemide ,medicine ,Animals ,Protein Isoforms ,Solute Carrier Family 12, Member 2 ,RNA, Messenger ,Ion transporter ,Bumetanide ,DNA Primers ,Ion Transport ,Base Sequence ,Symporters ,Reverse Transcriptase Polymerase Chain Reaction ,N-Ethylmaleimide ,Rubidium ,Molecular biology ,Rats ,Kinetics ,chemistry ,Hypotonic Solutions ,Cell culture ,Ethylmaleimide ,Symporter ,Potassium ,Cotransporter ,Neuroglia ,Intracellular ,medicine.drug - Abstract
The molecular mechanism of K-Cl cotransport (KCC) consists of at least 4 isoforms, KCC 1, 2, 3, and 4 which, in multiple combinations, exist in most cells, including erythrocytes and neuronal cells.We utilized reverse-transcriptase-polymerase chain reaction (RT-PCR) and ion flux studies to characterize KCC activity in an immortalized in vitro cell model for fibrous astrocytes, the rat C6 glioblastoma cell. Isoform-specific sets of oligonucleotide primers were synthesized for NKCC1, KCC1, KCC2, KCC3, KCC4, and also for NKCC1 and actin. K-Cl cotransport activity was determined by measuring either the furosemide-sensitive, or the Cl(-)-dependent bumetanide-insensitive Rb(+) (a K(+) congener) influx in the presence of the Na/K pump inhibitor ouabain. Rb(+) influx was measured at a fixed external Cl concentrations, [Cl(-)](e), as a function of varying external Rb concentrations, [Rb(+)](e), and at a fixed [Rb(+)](e) as a function of varying [Cl(-)](e), and with equimolar Cl replacement by anions of the chaotropic series.RT-PCR of C6 glioblastoma (C6) cells identified mRNA for three KCC isoforms (1, 3, and 4). NKCC1 mRNA was also detected. The apparent K(m) for KCC-mediated Rb(+) influx was 15 mM [Rb(+)](e), and V(max) 12.5 nmol Rb(+) * mg protein(-1) * minute(-1). The calculated apparent K(m) for external Cl(-) was 13 mM and V(max) 14.4 nmol Rb(+) * mg protein(-1) * minute(-1). The anion selectivity sequence of the furosemide-sensitive Rb(+) influx was Cl(-)Br-=NO(3)(-)I(-)=SCN(-)Sfm(-) (sulfamate). Established activators of K-Cl cotransport, hyposmotic shock and N-ethylmaleimide (NEM) pretreatment, stimulated furosemide-sensitive Rb(+) influx. A ñ50% NEM-induced loss of intracellular K(+) was prevented by furosemide.We have identified by RT-PCR the presence of three distinct KCC isoforms (1, 3, and 4) in rat C6 glioblastoma cells, and functionally characterized the anion selectivity and kinetics of their collective sodium-independent cation-chloride cotransport activity.
- Published
- 2007
44. Functional regulation of K‐Cl cotransport (COT) by the nitric oxide (NO) pathway, vasoconstrictors (VCs) and inhibitors of the contractile apparatus (ICA) in vascular smooth muscle cells (VSMCs)
- Author
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Peter K. Lauf and Norma C. Adragna
- Subjects
medicine.medical_specialty ,Vascular smooth muscle ,Contractile apparatus ,Biochemistry ,Nitric oxide ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Internal medicine ,Genetics ,medicine ,Cotransporter ,Molecular Biology ,Biotechnology - Published
- 2007
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45. Chloride determination in human lens epithelial cells (B3) by a fluorescent dye
- Author
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Peter K. Lauf, Norma C. Adragna, and Ameet A. Chimote
- Subjects
Chemistry ,Genetics ,medicine ,Biophysics ,Lens (geology) ,Molecular Biology ,Biochemistry ,Chloride ,Fluorescence ,Biotechnology ,medicine.drug - Published
- 2007
- Full Text
- View/download PDF
46. Properties of hyposmotically activated K channels in human lens epithelial cells (FHL124 LECs)
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Sandeep Misri, Peter K. Lauf, Norma C. Adragna, Sameer Ali, Pooia Fattahi, and Ameet A. Chimote
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Physics ,medicine.anatomical_structure ,Lens (anatomy) ,Genetics ,Biophysics ,medicine ,Channel (broadcasting) ,Molecular Biology ,Biochemistry ,Biotechnology ,K channels - Abstract
FHL24 LECs (a gift of Dr. John Reddan, Oakland University), when exposed to hyposmotic media (150mOsM) exhibit regulatory volume decrease (RVD) mediated primarily by a K-selective channel (Lauf et ...
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- 2007
- Full Text
- View/download PDF
47. NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells
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Peter K. Lauf, Mauricio Di Fulvio, Shalin Shah, and Norma C. Adragna
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medicine.medical_specialty ,Vascular smooth muscle ,Physiology ,Gene Expression ,Receptors, Cytoplasmic and Nuclear ,Biology ,Nitric Oxide ,Muscle, Smooth, Vascular ,Nitric oxide ,chemistry.chemical_compound ,Soluble Guanylyl Cyclase ,Downregulation and upregulation ,Physiology (medical) ,Internal medicine ,Gene expression ,medicine ,Animals ,Nitric Oxide Donors ,RNA, Messenger ,Receptor ,Cells, Cultured ,Messenger RNA ,Symporters ,Cell biology ,Rats ,Endocrinology ,chemistry ,Mechanism of action ,Guanylate Cyclase ,medicine.symptom ,Triazenes ,Cardiology and Cardiovascular Medicine ,Soluble guanylyl cyclase ,Nitroso Compounds - Abstract
Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.
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- 2003
48. Nitric oxide signaling pathway regulates potassium chloride cotransporter-1 mRNA expression in vascular smooth muscle cells
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Peter K. Lauf, Norma C. Adragna, and Mauricio Di Fulvio
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Nitroprusside ,medicine.medical_specialty ,Vascular smooth muscle ,Indoles ,Time Factors ,Carbazoles ,Cycloheximide ,Biology ,Nitric Oxide ,Biochemistry ,Muscle, Smooth, Vascular ,Nitric oxide ,chemistry.chemical_compound ,Alkaloids ,Internal medicine ,medicine ,Animals ,RNA, Messenger ,Enzyme Inhibitors ,RNA Processing, Post-Transcriptional ,Protein kinase A ,Molecular Biology ,Cyclic GMP ,Cells, Cultured ,Messenger RNA ,Dose-Response Relationship, Drug ,Symporters ,Reverse Transcriptase Polymerase Chain Reaction ,Biological Transport ,Cell Biology ,Cell biology ,Rats ,Endocrinology ,chemistry ,cardiovascular system ,Aminoquinolines ,Dactinomycin ,Sodium nitroprusside ,Soluble guanylyl cyclase ,Cotransporter ,medicine.drug ,Signal Transduction - Abstract
Rat vascular smooth muscle cells (VSMCs) express at least two mRNAs for K-Cl cotransporters (KCC): KCC1 and KCC3. cGMP-dependent protein kinase I regulates KCC3 mRNA expression in these cells. Here, we show evidence implicating the nitric oxide (NO)/cGMP signaling pathway in the expression of KCC1 mRNA, considered to be the major cell volume regulator. VSMCs, expressing soluble guanylyl cyclase (sGC) and PKG-I isoforms showed a time- and concentration-dependent increase in KCC1 mRNA levels after treatment with sodium nitroprusside as demonstrated by semiquantitative RT-PCR. sGC-dependent regulation of KCC1 mRNA expression was confirmed using YC-1, a NO-independent sGC stimulator. The sGC inhibitor LY83583 blocked the effects of sodium nitroprusside and YC-1. Moreover, 8-Br-cGMP increased KCC1 mRNA expression in a concentration- and time-dependent fashion. The 8-Br-cGMP effect was partially blocked by KT5823 but not by actinomycin D. However, actinomycin D and cycloheximide increased basal KCC1 mRNA in an additive manner, suggesting different mechanisms of action for both drugs. These findings suggest that in VSMCs, the NO/cGMP-signaling pathway participates in KCC1 mRNA regulation at the post-transcriptional level.
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- 2001
49. K-Cl cotransport: immunohistochemical and ion flux studies in human embryonic kidney (HEK293) cells transfected with full-length and C-terminal-domain-truncated KCC1 cDNAs
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Kenneth B. Gagnon, Jin Zhang, Robert E.W. Fyffe, Eric Delpire, Peter K. Lauf, and Norma C. Adragna
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Gene isoform ,DNA, Complementary ,Physiology ,Biology ,Kidney ,Transfection ,Chlorides ,Furosemide ,Humans ,Oxazoles ,Ion transporter ,Ion Transport ,Microscopy, Confocal ,Symporters ,HEK 293 cells ,Osmolar Concentration ,Kidney metabolism ,Hydrogen-Ion Concentration ,Rubidium ,Molecular biology ,Genistein ,Immunohistochemistry ,Cell biology ,Ion homeostasis ,Microscopy, Fluorescence ,Ethylmaleimide ,Symporter ,Potassium ,Cotransporter ,Carrier Proteins - Abstract
Coupled K and Cl movements are mediated by several isoforms of the K-Cl cotransporter (COT) encoded by the KCC genes. The ubiquitous KCC1 isoform, important for cell volume and ion homeostasis, has 12 transmembrane domains (Tmds), and cytoplasmic N- and C-terminal domains (Ntd and Ctd). This study investigates the cellular localization of KCC1 by confocal microscopy, activation of K-Cl COT by various non-osmotic and osmotic interventions with net unidirectional K and Rb fluxes at 37( degrees )C, and the effect of Ctd deletion on K-Cl COT regulation. Human embryonic kidney (HEK293) cells were transfected with full-length (fl) rabbit (rb)KCC1 and - CtdKCC1 cDNAs obtained after truncation at nucleotide 2011. Normal cells exposed to polyclonal anti-Ctd antibodies against Ctd epitopes within a 77 amino acid sequence (a.a.943-1020) revealed granular membrane and cytoplasmic immunostaining, presumably endogenous KCC1. Additional diffuse membrane and cytoplasmic immunofluorescence in flKCC1-transfected cells was absent in -CtdKCC1-transfected cells. Monoclonal antibodies against a c-myc epitope at the protein Ntd showed both membrane and cytosolic fluorescence. Basal and N-ethylmaleimide (NEM)-stimulated Rb influxes through K-Cl COT, calculated as Cl-dependent Rb fluxes, were 2-3-fold higher in flKCC1-transfected than in normal cells. NEM stimulation of K-Cl COT was highest in flKCC1-transfected cells, significantly lower in stably and abrogated in transiently -CtdKCC1-transfected cells. Furosemide, calyculin and genistein inhibited basal and NEM-stimulated K-Cl COT in normal and transfected cells. Staurosporine and hydroxylamine were ineffective stimulators. No effect of pH(0) changes (6.3-8.4) was observed in basal or NEM-stimulated K-Cl COT, in both normal and transfected cells. However, inhibition by NEM occurred at pH(0) 8.4. Furthermore, in a Cl-independent manner, NEM lowered cell K content by30% and hypotonicity (210-70mOsM) stimulated furosemide-sensitive Rb influx and K loss. Thus, in cultured normal and KCC1-transfected cells, K-Cl COT shows significant differences from erythrocytes, and NEM and cell swelling open furosemide-sensitive and Cl-independent K/Rb channels. Failure of K-Cl COT in cells transfected with Ctd-truncated KCC1 to respond to NEM suggests a role of the Ctd for signal transduction.
- Published
- 2001
50. K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation
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Richard E. White, Sergei N. Orlov, Peter K. Lauf, and Norma C. Adragna
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Vascular smooth muscle ,Erythrocytes ,Indoles ,Physiology ,Swine ,Vasodilator Agents ,Vasodilation ,Pharmacology ,Isosorbide Dinitrate ,environment and public health ,Muscle, Smooth, Vascular ,chemistry.chemical_compound ,Enzyme Inhibitors ,Oxazoles ,Aorta ,Cells, Cultured ,Symporters ,Chemistry ,Hydralazine ,Coronary Vessels ,Genistein ,medicine.anatomical_structure ,Biochemistry ,Ethylmaleimide ,Sodium nitroprusside ,medicine.drug ,inorganic chemicals ,Nitroprusside ,Endothelium ,Carbazoles ,Nitric Oxide ,Nitric oxide ,Alkaloids ,Chlorides ,Isometric Contraction ,medicine ,Animals ,Ion transporter ,Sheep ,urogenital system ,Biological Transport ,Cell Biology ,Rubidium ,Rats ,Propylene Glycols ,Vasoconstriction ,Potassium ,Marine Toxins ,Carrier Proteins ,cGMP-dependent protein kinase - Abstract
K-Cl cotransport, the electroneutral-coupled movement of K and Cl ions, plays an important role in regulatory volume decrease. We recently reported that nitrite, a nitric oxide derivative possessing potent vasodilation properties, stimulates K-Cl cotransport in low-K sheep red blood cells (LK SRBCs). We hypothesized that activation of vascular smooth muscle (VSM) K-Cl cotransport by vasodilators decreases VSM tension. Here we tested this hypothesis by comparing the effects of commonly used vasodilators, hydralazine (HYZ), sodium nitroprusside, isosorbide mononitrate, and pentaerythritol, on K-Cl cotransport in LK SRBCs and in primary cultures of rat VSM cells (VSMCs) and of HYZ-induced K-Cl cotransport activation on relaxation of isolated porcine coronary rings. K-Cl cotransport was measured as the Cl-dependent K efflux or Rb influx in the presence and absence of inhibitors for other K/Rb transport pathways. All vasodilators activated K-Cl cotransport in LK SRBCs and HYZ in VSMCs, and this activation was inhibited by calyculin and genistein, two inhibitors of K-Cl cotransport. KT-5823, a specific inhibitor of protein kinase G, abolished the sodium nitroprusside-stimulated K-Cl cotransport in LK SRBCs, suggesting involvement of the cGMP pathway in K-Cl cotransport activation. Hydralazine, in a dose-dependent manner, and sodium nitroprusside relaxed (independently of the endothelium) precontracted arteries when only K-Cl cotransport was operating and other pathways for K/Rb transport, including the Ca-activated K channel, were inhibited. Our findings suggest that K-Cl cotransport may be involved in vasodilation.
- Published
- 2000
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