Publications by authors named "Peter J S Smith"

48 Publications

Parkinson's disease protein DJ-1 regulates ATP synthase protein components to increase neuronal process outgrowth.

Cell Death Dis 2019 06 13;10(6):469. Epub 2019 Jun 13.

Department of Internal Medicine (Endocrinology), Yale University, New Haven, CT, USA.

Familial Parkinson's disease (PD) protein DJ-1 mutations are linked to early onset PD. We have found that DJ-1 binds directly to the FF ATP synthase β subunit. DJ-1's interaction with the β subunit decreased mitochondrial uncoupling and enhanced ATP production efficiency while in contrast mutations in DJ-1 or DJ-1 knockout increased mitochondrial uncoupling, and depolarized neuronal mitochondria. In mesencephalic DJ-1 KO cultures, there was a progressive loss of neuronal process extension. This was ameliorated by a pharmacological reagent, dexpramipexole, that binds to ATP synthase, closing a mitochondrial inner membrane leak and enhancing ATP synthase efficiency. ATP synthase c-subunit can form an uncoupling channel; we measured, therefore, ATP synthase F (β subunit) and c-subunit protein levels. We found that ATP synthase β subunit protein level in the DJ-1 KO neurons was approximately half that found in their wild-type counterparts, comprising a severe defect in ATP synthase stoichiometry and unmasking c-subunit. We suggest that DJ-1 enhances dopaminergic cell metabolism and growth by its regulation of ATP synthase protein components.
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http://dx.doi.org/10.1038/s41419-019-1679-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565618PMC
June 2019

The mitochondrial complex V-associated large-conductance inner membrane current is regulated by cyclosporine and dexpramipexole.

Mol Pharmacol 2015 Jan 20;87(1):1-8. Epub 2014 Oct 20.

Department of Internal Medicine (K.N.A., P.Z., S.S., H.L., E.A.J.) and Department of Neurobiology (E.A.J.), Yale University School of Medicine, New Haven, Connecticut; Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom (K.N.A.); Department of Neuroscience, Imaging and Clinical Sciences, University G.d'Annunzio of Chieti-Pescara, Chieti-Pescara, Italy (L.B.); Knopp Biosciences LLC, Pittsburgh, Pennsylvania (S.I.D., A.P.S., V.K.G.); and Biocurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts (P.J.S.S.)

Inefficiency of oxidative phosphorylation can result from futile leak conductance through the inner mitochondrial membrane. Stress or injury may exacerbate this leak conductance, putting cells, and particularly neurons, at risk of dysfunction and even death when energy demand exceeds cellular energy production. Using a novel method, we have recently described an ion conductance consistent with mitochondrial permeability transition pore (mPTP) within the c-subunit of the ATP synthase. Excitotoxicity, reactive oxygen species-producing stimuli, or elevated mitochondrial matrix calcium opens the channel, which is inhibited by cyclosporine A and ATP/ADP. Here we show that ATP and the neuroprotective drug dexpramipexole (DEX) inhibited an ion conductance consistent with this c-subunit channel (mPTP) in brain-derived submitochondrial vesicles (SMVs) enriched for F1FO ATP synthase (complex V). Treatment of SMVs with urea denatured extramembrane components of complex V, eliminated DEX- but not ATP-mediated current inhibition, and reduced binding of [(14)C]DEX. Direct effects of DEX on the synthesis and hydrolysis of ATP by complex V suggest that interaction of the compound with its target results in functional conformational changes in the enzyme complex. [(14)C]DEX bound specifically to purified recombinant b and oligomycin sensitivity-conferring protein subunits of the mitochondrial F1FO ATP synthase. Previous data indicate that DEX increased the efficiency of energy production in cells, including neurons. Taken together, these studies suggest that modulation of a complex V-associated inner mitochondrial membrane current is metabolically important and may represent an avenue for the development of new therapeutics for neurodegenerative disorders.
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http://dx.doi.org/10.1124/mol.114.095661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279080PMC
January 2015

Paper-based colorimetric enzyme linked immunosorbent assay fabricated by laser induced forward transfer.

Biomicrofluidics 2014 May 19;8(3):036502. Epub 2014 May 19.

Optoelectronics Research Centre, University of Southampton, Highfield, Southampton SO17 1BJ, United Kingdom.

We report the Laser Induced Forward Transfer (LIFT) of antibodies from a liquid donor film onto paper receivers for application as point-of-care diagnostic sensors. To minimise the loss of functionality of the active biomolecules during transfer, a dynamic release layer was employed to shield the biomaterial from direct exposure to the pulsed laser source. Cellulose paper was chosen as the ideal receiver because of its inherent bio-compatibility, liquid transport properties, wide availability and low cost, all of which make it an efficient and suitable platform for point-of-care diagnostic sensors. Both enzyme-tagged and untagged IgG antibodies were LIFT-printed and their functionality was confirmed via a colorimetric enzyme-linked immunosorbent assay. Localisation of the printed antibodies was exhibited, which can allow the creation of complex 2-d patterns such as QR codes or letters for use in a final working device. Finally, a calibration curve was determined that related the intensity of the colour obtained to the concentration of active antibodies to enable quantitative assessment of the device performance. The motivation for this work was to implement a laser-based procedure for manufacturing low-cost, point-of-care diagnostic devices on paper.
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http://dx.doi.org/10.1063/1.4878696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032417PMC
May 2014

Expression profiling of primary and metastatic ovarian tumors reveals differences indicative of aggressive disease.

PLoS One 2014 14;9(4):e94476. Epub 2014 Apr 14.

Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.

The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094476PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986100PMC
January 2015

Quantitative exploration of the contribution of settlement, growth, dispersal and grazing to the accumulation of natural marine biofilms on antifouling and fouling-release coatings.

Biofouling 2014 Feb 13;30(2):223-36. Epub 2014 Jan 13.

a Department of Marine Chemistry and Geochemistry , Woods Hole Oceanographic Institution , Woods Hole , MA , USA.

The accumulation of microbial biofilms on ships' hulls negatively affects ship performance and efficiency while also playing a role in the establishment of even more detrimental hard-fouling communities. However, there is little quantitative information on how the accumulation rate of microbial biofilms is impacted by the balance of the rates of cell settlement, in situ production (ie growth), dispersal to surrounding waters and mortality induced by grazers. These rates were quantified on test panels coated with copper-based antifouling (AF) or polymer-based fouling-release (FR) coatings by using phospholipids as molecular proxies for microbial biomass. The results confirmed the accepted modes of efficacy of these two types of coatings. In a more extensive set of experiments with only the FR coatings, it was found that seasonally averaged cellular production rates were 1.5 ± 0.5 times greater than settlement and the dispersal rates were 2.7 ± 0.8 greater than grazing. The results of this study quantitatively describe the dynamic balance of processes leading to the accumulation of microbial biofilm on coatings designed for ships' hulls.
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http://dx.doi.org/10.1080/08927014.2013.861422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3935016PMC
February 2014

Identification of ovarian cancer metastatic miRNAs.

PLoS One 2013 12;8(3):e58226. Epub 2013 Mar 12.

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA.

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2-4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058226PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595263PMC
September 2013

Effects of dexpramipexole on brain mitochondrial conductances and cellular bioenergetic efficiency.

Brain Res 2012 Mar 28;1446:1-11. Epub 2012 Jan 28.

Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.

Cellular stress or injury can result in mitochondrial dysfunction, which has been linked to many chronic neurological disorders including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Stressed and dysfunctional mitochondria exhibit an increase in large conductance mitochondrial membrane currents and a decrease in bioenergetic efficiency. Inefficient energy production puts cells, and particularly neurons, at risk of death when energy demands exceed cellular energy production. Here we show that the candidate ALS drug dexpramipexole (DEX; KNS-760704; ((6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine) and cyclosporine A (CSA) inhibited increases in ion conductance in whole rat brain-derived mitochondria induced by calcium or treatment with a proteasome inhibitor, although only CSA inhibited calcium-induced permeability transition in liver-derived mitochondria. In several cell lines, including cortical neurons in culture, DEX significantly decreased oxygen consumption while maintaining or increasing production of adenosine triphosphate (ATP). DEX also normalized the metabolic profile of injured cells and was protective against the cytotoxic effects of proteasome inhibition. These data indicate that DEX increases the efficiency of oxidative phosphorylation, possibly by inhibition of a CSA-sensitive mitochondrial conductance.
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http://dx.doi.org/10.1016/j.brainres.2012.01.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746080PMC
March 2012

The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: role of NADH and consequences for insulin secretion.

Toxicol Appl Pharmacol 2012 Jan 15;258(2):216-25. Epub 2011 Nov 15.

Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA 02543, United States.

Pancreatic β-cells release insulin in response to elevation of glucose from basal (4-7mM) to stimulatory (8-16mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H(2)O(2)), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H(2)O(2) inhibit insulin secretion. Menadione, which produces H(2)O(2) via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H(2)O(2) production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1-10μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H(2)O(2) formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H(2)O(2) and menadione on insulin secretion.
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http://dx.doi.org/10.1016/j.taap.2011.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259196PMC
January 2012

Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.

Nat Cell Biol 2011 Sep 18;13(10):1224-33. Epub 2011 Sep 18.

Department of Internal Medicine, Yale University, New Haven, Connecticut 06520, USA.

Anti-apoptotic Bcl2 family proteins such as Bcl-x(L) protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-x(L) enhances the efficiency of energy metabolism. Our evidence indicates that Bcl-x(L)interacts directly with the β-subunit of the F(1)F(O) ATP synthase, decreasing an ion leak within the F(1)F(O) ATPase complex and thereby increasing net transport of H(+) by F(1)F(O) during F(1)F(O) ATPase activity. By patch clamping submitochondrial vesicles enriched in F(1)F(O) ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-x(L) activity increases the membrane leak conductance. In addition, recombinant Bcl-x(L) protein directly increases the level of ATPase activity of purified synthase complexes, and inhibition of endogenous Bcl-x(L) decreases the level of F(1)F(O) enzymatic activity. Our findings indicate that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-x(L)-expressing neurons.
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http://dx.doi.org/10.1038/ncb2330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186867PMC
September 2011

Catechol metabolites of endogenous estrogens induce redox cycling and generate reactive oxygen species in breast epithelial cells.

Carcinogenesis 2011 Aug 10;32(8):1285-93. Epub 2011 Jun 10.

Department of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, 170 Frelinghuysen Road, Piscataway, NJ 08854, USA.

Estrogens are major risk factors for the development of breast cancer; they can be metabolized to catechols, which are further oxidized to DNA-reactive quinones and semiquinones (SQs). These metabolites are mutagenic and may contribute to the carcinogenic activity of estrogens. Redox cycling of the SQs and subsequent generation of reactive oxygen species (ROS) is also an important mechanism leading to DNA damage. The SQs of exogenous estrogens have been shown to redox cycle, however, redox cycling and the generation of ROS by endogenous estrogens has never been characterized. In the present studies, we determined whether the catechol metabolites of endogenous estrogens, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone and 2-hydroxyestriol, can redox cycle in breast epithelial cells. These catechol estrogens, but not estradiol, estrone, estriol or 2-methoxyestradiol, were found to redox cycle and generate hydrogen peroxide (H(2)O(2)) and hydroxyl radicals in lysates of three different breast epithelial cell lines: MCF-7, MDA-MB-231 and MCF-10A. The generation of ROS required reduced nicotinamide adenine dinucleotide phosphate as a reducing equivalent and was inhibited by diphenyleneiodonium, a flavoenzyme inhibitor, indicating that redox cycling is mediated by flavin-containing oxidoreductases. Using extracellular microsensors, catechol estrogen metabolites stimulated the release of H(2)O(2) by adherent cells, indicating that redox cycling occurs in viable intact cells. Taken together, these data demonstrate that catechol metabolites of endogenous estrogens undergo redox cycling in breast epithelial cells, resulting in ROS production. Depending on the localized concentrations of catechol estrogens and enzymes that mediate redox cycling, this may be an important mechanism contributing to the development of breast cancer.
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http://dx.doi.org/10.1093/carcin/bgr109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149209PMC
August 2011

Plasma membrane electron transport in pancreatic β-cells is mediated in part by NQO1.

Am J Physiol Endocrinol Metab 2011 Jul 19;301(1):E113-21. Epub 2011 Apr 19.

United States Coast Guard Academy, New London, Connecticut, USA.

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal β-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of β-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in β-cells.
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http://dx.doi.org/10.1152/ajpendo.00673.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129843PMC
July 2011

Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase.

Free Radic Biol Med 2011 Apr 4;50(7):874-82. Epub 2011 Jan 4.

Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854, USA.

Diquat and paraquat are nonspecific defoliants that induce toxicity in many organs including the lung, liver, kidney, and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In this study, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese hamster ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild-type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective at generating ROS compared to paraquat (K(M)=1.0 and 44.2μM, respectively, for H(2)O(2) generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5μM, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (V(max)≈6.0nmol H(2)O(2)/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than in CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than in CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.12.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647689PMC
April 2011

Construction, theory, and practical considerations for using self-referencing of Ca(2+)-selective microelectrodes for monitoring extracellular Ca(2+) gradients.

Methods Cell Biol 2010 ;99:91-111

BioCurrents Research Center, Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts, USA.

Ca(2+) signaling in the extra- and intracellular domains is linked to Ca(2+) transport across the plasma membrane. Noninvasive monitoring of these resulting extracellular Ca(2+) gradients with self-referencing of Ca(2+)-selective microelectrodes is used for studying Ca(2+) signaling across Kingdoms. The quantitated Ca(2+) flux enables comparison with changes to intracellular [Ca(2+)] measured with other methods and determination of Ca(2+) transport stoichiometry. Here, we review the construction of Ca(2+)-selective microelectrodes, their physical characteristics, and their use in self-referencing mode to calculate Ca(2+) flux. We also discuss potential complications when using them to measure Ca(2+) gradients near the boundary layers of single cells and tissues.
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http://dx.doi.org/10.1016/B978-0-12-374841-6.00004-9DOI Listing
March 2011

Windows to cell function and dysfunction: signatures written in the boundary layers.

Bioessays 2010 Jun;32(6):514-23

BioCurrents Research Center, Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA 02543, USA.

The medium surrounding cells either in culture or in tissues contains a chemical mix varying with cell state. As solutes move in and out of the cytoplasmic compartment they set up characteristic signatures in the cellular boundary layers. These layers are complex physical and chemical environments the profiles of which reflect cell physiology and provide conduits for intercellular messaging. Here we review some of the most relevant characteristics of the extracellular/intercellular space. Our initial focus is primarily on cultured cells but we extend our consideration to the far more complex environment of tissues, and discuss how chemical signatures in the boundary layer can or may affect cell function. Critical to the entire essay are the methods used, or being developed, to monitor chemical profiles in the boundary layers. We review recent developments in ultramicro electrochemical sensors and tailored optical reporters suitable for the task in hand.
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http://dx.doi.org/10.1002/bies.200900173DOI Listing
June 2010

Physiological and pharmacological characterizations of the larval Anopheles albimanus rectum support a change in protein distribution and/or function in varying salinities.

Comp Biochem Physiol A Mol Integr Physiol 2010 Sep 10;157(1):55-62. Epub 2010 May 10.

The Whitney Laboratory for Marine Bioscience, University of Florida, 9505 OceanShore Boulevard, St. Augustine, FL 32080, USA.

Ion regulation is a biological process crucial to the survival of mosquito larvae and a major organ responsible for this regulation is the rectum. The recta of anopheline larvae are distinct from other subfamilies of mosquitoes in several ways, yet have not yet been characterized extensively. Here we characterize the two major cell types of the anopheline rectum, DAR and non-DAR cells, using histological, physiological, and pharmacological analyses. Proton flux was measured at the basal membrane of 2%- and 50%-artificial sea water-reared An. albimanus larvae using self-referencing ion-selective microelectrodes, and the two cell types were found to differ in basal membrane proton flux. Additionally, differences in the response of that flux to pharmacological inhibitors in larvae reared in 2% versus 50% ASW indicate changes in protein function between the two rearing conditions. Finally, histological analyses suggest that the non-DAR cells are structurally suited for mediating ion transport. These data support a model of rectal ion regulation in which the non-DAR cells have a resorptive function in freshwater-reared larvae and a secretive function in saline water-reared larvae. In this way, anopheline larvae may adapt to varying salinities.
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http://dx.doi.org/10.1016/j.cbpa.2010.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904869PMC
September 2010

Release and elementary mechanisms of nitric oxide in hair cells.

J Neurophysiol 2010 May 10;103(5):2494-505. Epub 2010 Mar 10.

Center for Neuroscience, Department of Anesthesiology and Pain Medicine, Program in Communication Sciences, University of California, Davis, Davis, CA 95616, USA.

The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency-current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca(2+) current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca(2+) channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca(2+)-activated K(+) currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells.
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http://dx.doi.org/10.1152/jn.00017.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867581PMC
May 2010

Glucagon-like peptide-1 induced signaling and insulin secretion do not drive fuel and energy metabolism in primary rodent pancreatic beta-cells.

PLoS One 2009 Jul 13;4(7):e6221. Epub 2009 Jul 13.

Molecular Nutrition Unit and Montreal Diabetes Research Center at the Centre de Recherche du Centre Hospitalier de l'Université de Montréal and Department of Nutrition and Biochemistry, Université de Montréal, Montréal, Quebec, Canada.

Background: Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic beta-cells, in particular cAMP, Ca(2+) and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors.

Methodology/prinicipal Findings: GLP-1 or Ex-4 at high glucose caused release (approximately 20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on beta-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca(2+)](i) and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered.

Conclusions/significance: The results indicate that GLP-1 barely affects beta-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the beta-cell, and that the beta-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a "push" (fuel substrate driven) process, rather than a "pull" mechanism secondary to enhanced insulin release as well as to Ca(2+), cAMP and PKB signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006221PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704866PMC
July 2009

Role for malic enzyme, pyruvate carboxylation, and mitochondrial malate import in glucose-stimulated insulin secretion.

Am J Physiol Endocrinol Metab 2009 Jun 17;296(6):E1354-62. Epub 2009 Mar 17.

BioCurrents Research Center, Marine Biological Laboratory, 7 MBL St., Lillie 219, Woods Hole, MA 02543, USA.

Pyruvate cycling has been implicated in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. The operation of some pyruvate cycling pathways is proposed to necessitate malate export from the mitochondria and NADP(+)-dependent decarboxylation of malate to pyruvate by cytosolic malic enzyme (ME1). Evidence in favor of and against a role of ME1 in GSIS has been presented by others using small interfering RNA-mediated suppression of ME1. ME1 was also proposed to account for methyl succinate-stimulated insulin secretion (MSSIS), which has been hypothesized to occur via succinate entry into the mitochondria in exchange for malate and subsequent malate conversion to pyruvate. In contrast to rat, mouse beta-cells lack ME1 activity, which was suggested to explain their lack of MSSIS. However, this hypothesis was not tested. In this report, we demonstrate that although adenoviral-mediated overexpression of ME1 greatly augments GSIS in rat insulinoma INS-1 832/13 cells, it does not restore MSSIS, nor does it significantly affect GSIS in mouse islets. The increase in GSIS following ME1 overexpression in INS-1 832/13 cells did not alter the ATP-to-ADP ratio but was accompanied by increases in malate and citrate levels. Increased malate and citrate levels were also observed after INS-1 832/13 cells were treated with the malate-permeable analog dimethyl malate. These data suggest that although ME1 overexpression augments anaplerosis and GSIS in INS-1 832/13 cells, it is not likely involved in MSSIS and GSIS in pancreatic islets.
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http://dx.doi.org/10.1152/ajpendo.90836.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692397PMC
June 2009

Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity.

J Neurochem 2009 Apr 16;109(2):644-55. Epub 2009 Feb 16.

Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Biomedical Research Center, Baltimore, Maryland, USA.

In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O(2) consumption, cytosolic Ca(2+) concentration ([Ca(2+)](i)), and mitochondrial membrane potential (mDeltapsi) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca(2+)](i) and mDeltapsi were monitored with Fluo-4 and TMRE(+), respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca(2+)](i) followed seconds afterwards by an increase in O(2) consumption which reached a maximum level within 1-5 min. A modest increase in mDeltapsi occurred during this time period, and then, shortly before maximal O(2) consumption was reached, the mDeltapsi, as indicated by TMRE(+) fluorescence, dissipated. Maximal O(2) consumption lasted up to 5 min and then declined together with mDeltapsi and ATP levels, while [Ca(2+)](i) further increased. mDeltapsi and [Ca(2+)](i) returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca(2+)](i) increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O(2) consumption, [Ca(2+)](i), and mDeltapsi. The data obtained using this new method are consistent with a model where Ca(2+) influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.
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http://dx.doi.org/10.1111/j.1471-4159.2009.05997.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805059PMC
April 2009

Ion trapping with fast-response ion-selective microelectrodes enhances detection of extracellular ion channel gradients.

Biophys J 2009 Feb;96(4):1597-605

BioCurrents Research Center, Cell Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.

Previously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 microm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10-55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane.
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http://dx.doi.org/10.1016/j.bpj.2008.11.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717230PMC
February 2009

Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia.

Cell 2008 Dec;135(6):1108-17

Center for Systems Biology, Program in Membrane Biology/Nephrology Division, Massachusetts General Hospital, Boston, MA 02114, USA.

Basal cells are by definition located on the basolateral side of several epithelia, and they have never been observed reaching the lumen. Using high-resolution 3D confocal imaging, we report that basal cells extend long and slender cytoplasmic projections that not only reach toward the lumen but can cross the tight junction barrier in some epithelia of the male reproductive and respiratory tracts. In this way, the basal cell plasma membrane is exposed to the luminal environment. In the epididymis, in which luminal acidification is crucial for sperm maturation and storage, these projections contain the angiotensin II type 2 receptor (AGTR2). Activation of AGTR2 by luminal angiotensin II, increases proton secretion by adjacent clear cells, which are devoid of AGTR2. We propose a paradigm in which basal cells scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells.
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http://dx.doi.org/10.1016/j.cell.2008.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646085PMC
December 2008

Imaging the electric field associated with mouse and human skin wounds.

Wound Repair Regen 2008 May-Jun;16(3):432-41

BioElectroMed Corporation, 849 Mitten Rd, Ste. 105, Burlingame, CA 94010, USA.

We have developed a noninvasive instrument called the bioelectric field imager (BFI) for mapping the electric field between the epidermis and the stratum corneum near wounds in both mouse and human skin. Rather than touching the skin, the BFI vibrates a small metal probe with a displacement of 180 mum in air above the skin to detect the surface potential of the epidermis through capacitative coupling. Here we describe our first application of the BFI measuring the electric field between the stratum corneum and epidermis at the margin of skin wounds in mice. We measured an electric field of 177+/-14 (61) mV/mm immediately upon wounding and the field lines pointed away from the wound in all directions around it. Because the wound current flows immediately upon wounding, this is the first signal indicating skin damage. This electric field is generated at the outer surface of the epidermis by the outward flow of the current of injury. An equal and opposite current must flow within the multilayered epidermis to generate an intraepidermal field with the negative pole at the wound site. Because the current flowing within the multilayered epidermis is spread over a larger area, the current density and subsequent E field generated in that region is expected to be smaller than that measured by the BFI beneath the stratum corneum. The field beneath the stratum corneum typically remained in the 150-200 mV/mm range for 3 days and then began to decline over the next few days, falling to zero once wound healing was complete. The mean wound field strength decreased by 64+/-7% following the application of the sodium channel blocker, amiloride, to the skin near the wound and increased by 82+/-21% following the application of the Cl- channel activator, prostaglandin E2.
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http://dx.doi.org/10.1111/j.1524-475X.2008.00389.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086402PMC
July 2008

Using Pharmabase to perform pharmacological analyses of cell function.

Curr Protoc Bioinformatics 2006 Mar;Chapter 14:Unit 14.2

BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts, USA.

Pharmabase is designed to form a bridge between the molecular dimension of cell transport processes and the functional manipulation of the protein players. It has as its emphasis membrane transport and related pharmacology. Several search and navigation options are available, including membrane transport, disease, and a graphic interface arranged by pathway and cell type. The level of entry to the database can be tailored to the investigator's level of expertise. The final product of Pharmabase is a Compound Record detailing the use and targets of individual compounds. Navigation routes generally fall into hierarchical keys and are cross-referenced. Pharmabase encourages input from its user community. It is maintained by the BioCurrents Research Center, an NIH resource funded through the National Center for Research Resources (NCRR).
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http://dx.doi.org/10.1002/0471250953.bi1402s13DOI Listing
March 2006

Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons.

Proc Natl Acad Sci U S A 2008 Feb 4;105(6):2169-74. Epub 2008 Feb 4.

Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.
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http://dx.doi.org/10.1073/pnas.0711647105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2542873PMC
February 2008

Characterization of optimized Na+ and Cl- liquid membranes for use with extracellular, self-referencing microelectrodes.

Anal Bioanal Chem 2008 Mar 11;390(5):1355-9. Epub 2008 Jan 11.

BioCurrents Research Center, Program in Molecular Physiology, Marine Biological Laboratory, Woods Hole, MA 02543, USA.

Self-referencing with ion-selective microelectrodes (ISMs) is a useful approach for monitoring near-real-time ion flux near single cells and across epithelia. While ISMs for H+, Ca2+, and K+ have been optimized for use with self-referencing, ISMs for two other primary inorganic ions, Na+ and Cl-, have not. In this study, we have characterized ISMs based on three Na+ ionophores (I, VI, and X) and one Cl- ionophore to assess their suitability for use with self-referencing. ISMs constructed with Na+ ionophore VI have short response times (approximately 100 ms) but possess nearly an order of magnitude less selectivity for Na+ over K+ than ISMs constructed with Na+ ionophore X. The Na+ ionophore X mixture was enhanced to give it a shorter response time while not compromising its selectivity. A Cl(-)-selective microelectrode was constructed and characterized with superior anionic selectivity compared with previously reported Cl- ISMs used with self-referencing. This Cl(-)-selective microelectrode, however, has a relatively slow response time (approximately 3 s), thus requiring changes to the self-referencing protocol. Self-referencing with these ISMs will enable near-real-time ion flux measurements for Na+ and Cl-.
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http://dx.doi.org/10.1007/s00216-007-1804-zDOI Listing
March 2008

Modulation of extracellular proton fluxes from retinal horizontal cells of the catfish by depolarization and glutamate.

J Gen Physiol 2007 Aug;130(2):169-82

Department of Biology, Indiana Wesleyan University, Marion, IN 46953, USA.

Self-referencing H(+)-selective microelectrodes were used to measure extracellular proton fluxes from cone-driven horizontal cells isolated from the retina of the catfish (Ictalurus punctatus). The neurotransmitter glutamate induced an alkalinization of the area adjacent to the external face of the cell membrane. The effect of glutamate occurred regardless of whether the external solution was buffered with 1 mM HEPES, 3 mM phosphate, or 24 mM bicarbonate. The AMPA/kainate receptor agonist kainate and the NMDA receptor agonist N-methyl-D-aspartate both mimicked the effect of glutamate. The effect of kainate on proton flux was inhibited by the AMPA/kainate receptor blocker CNQX, and the effect of NMDA was abolished by the NMDA receptor antagonist DAP-5. Metabotropic glutamate receptor agonists produced no alteration in proton fluxes from horizontal cells. Depolarization of cells either by increasing extracellular potassium or directly by voltage clamp also produced an alkalinization adjacent to the cell membrane. The effects of depolarization on proton flux were blocked by 10 microM nifedipine, an inhibitor of L-type calcium channels. The plasmalemma Ca(2+/)H(+) ATPase (PMCA) blocker 5(6)-carboxyeosin also significantly reduced proton flux modulation by glutamate. Our results are consistent with the hypothesis that glutamate-induced extracellular alkalinizations arise from activation of the PMCA pump following increased intracellular calcium entry into cells. This process might help to relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons from photoreceptor synaptic terminals. Our findings argue strongly against the hypothesis that protons released by horizontal cells act as the inhibitory feedback neurotransmitter that creates the surround portion of the receptive fields of retinal neurons.
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http://dx.doi.org/10.1085/jgp.200709737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151636PMC
August 2007

Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit.

Am J Physiol Cell Physiol 2007 Jul 28;293(1):C199-210. Epub 2007 Mar 28.

Massachusetts General Hospital, Harvard Medical School, Program in Membrane Biology, Nephrology Div., 185 Cambridge St., CPZN 8150, Boston, MA 02114-2790, USA.

An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H(+)ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1(-/-) mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1(-/-) mice compared with B1(+/+). Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1(-/-) mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1(-/-) mice as in B1(+/+) (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1(-/-) mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1(-/-) mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility.
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http://dx.doi.org/10.1152/ajpcell.00596.2006DOI Listing
July 2007

Measuring extracellular ion gradients from single channels with ion-selective microelectrodes.

Biophys J 2007 Apr 26;92(7):L52-4. Epub 2007 Jan 26.

Under many different conditions activated plasma membrane ion channels give rise to changes in the extracellular concentration of the permeant ion(s). The magnitude and duration of these changes are dependent on the electrochemical driving force(s) on the permeant ion(s) as well as conductance, open time, and channel density. We have modeled the change in the extracellular [K+] due to efflux through Ca2+-activated K+ channels, mSlo, to determine the range of parameters that would give rise to measurable signals in the surrounding media. Subsequently we have used extracellular, K+-selective microelectrodes to monitor localized changes in [K+]ext due to efflux through mSlo channels expressed in Xenopus oocytes. The rapid changes in [K+] show a close fit with the predicted model when the time response of the ion-selective microelectrode is taken into account, providing proof of the concept. Measurement of the change in extracellular ion concentration with ion-selective microelectrodes provides a noninvasive means for functional mapping of channel location and density, as well as characterizing the properties of ion channels in the plasma membrane.
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http://dx.doi.org/10.1529/biophysj.106.102947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864814PMC
April 2007

Paraquat increases cyanide-insensitive respiration in murine lung epithelial cells by activating an NAD(P)H:paraquat oxidoreductase: identification of the enzyme as thioredoxin reductase.

J Biol Chem 2007 Mar 17;282(11):7939-49. Epub 2007 Jan 17.

Department of Environmental and Occupational Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity mediating this reaction in lung cells is not completely understood. Using self-referencing microsensors, we measured the effects of paraquat on oxygen flux into murine lung epithelial cells. Paraquat (10-100 microm) was found to cause a 2-4-fold increase in cellular oxygen flux. The mitochondrial poisons cyanide, rotenone, and antimycin A prevented mitochondrial- but not paraquat-mediated oxygen flux into cells. In contrast, diphenyleneiodonium (10 microm), an NADPH oxidase inhibitor, blocked the effects of paraquat without altering mitochondrial respiration. NADPH oxidases, enzymes that are highly expressed in lung epithelial cells, utilize molecular oxygen to generate superoxide anion. We discovered that lung epithelial cells possess a distinct cytoplasmic diphenyleneiodonium-sensitive NAD(P)H:paraquat oxidoreductase. This enzyme utilizes oxygen, requires NADH or NADPH, and readily generates the reduced paraquat radical. Purification and sequence analysis identified this enzyme activity as thioredoxin reductase. Purified paraquat reductase from the cells contained thioredoxin reductase activity, and purified rat liver thioredoxin reductase or recombinant enzyme possessed paraquat reductase activity. Reactive oxygen intermediates and subsequent oxidative stress generated from this enzyme are likely to contribute to paraquat-induced lung toxicity.
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http://dx.doi.org/10.1074/jbc.M611817200DOI Listing
March 2007