scholarly journals Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient

2005 ◽  
Vol 393 (2) ◽  
pp. 583-589 ◽  
Author(s):  
Rafael Romero-Calderón ◽  
David E. Krantz
Keyword(s):  
Mammalian Cells ◽  
Organic Anion ◽  
S2 Cells ◽  
Transport Activity ◽  
Molecular Identity ◽  
Polyamine Transport ◽  
Drosophila S2 Cells ◽  
Solute Carrier ◽  
Polyamine Uptake

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl−- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1′-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4′-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity.

scholarly journals A Store-operated Calcium Channel in Drosophila S2 Cells

2004 ◽  
Vol 123 (2) ◽  
pp. 167-182 ◽  
Author(s):  
Andriy V. Yeromin ◽  
Jack Roos ◽  
Kenneth A. Stauderman ◽  
Michael D. Cahalan
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Noise Analysis ◽  
Reversal Potential ◽  
S2 Cells ◽  
Pipette Solution ◽  
Crac Channels ◽  
Drosophila S2 Cells ◽  
Cell Recording ◽  
Inward Currents

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

scholarly journals Use of Drosophila S2 Cells as a Model for Studying Ehrlichia chaffeensis Infections

2008 ◽  
Vol 74 (6) ◽  
pp. 1886-1891 ◽  
Author(s):  
Alison Luce-Fedrow ◽  
Tonia Von Ohlen ◽  
Daniel Boyle ◽  
Roman R. Ganta ◽  
Stephen K. Chapes
Keyword(s):  
Mammalian Cells ◽  
Genetic Manipulation ◽  
Rrna Gene ◽  
Ehrlichia Chaffeensis ◽  
S2 Cells ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Mammalian Cell Lines ◽  
Drosophila S2 Cells ◽  
Human Monocytic Ehrlichiosis

ABSTRACT Ehrlichia chaffeensis is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that Drosophila S2 cells are permissive for the growth of E. chaffeensis. We saw morulae (aggregates of bacteria) by microscopy, detected the E. chaffeensis 16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect E. chaffeensis in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for E. chaffeensis through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of E. chaffeensis. The use of a Drosophila system has the potential to serve as a model system for studying Ehrlichia due to its completed genome, ease of genetic manipulation, and the availability of mutants.

Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

2010 ◽  
Vol 72 (1) ◽  
pp. 87-94 ◽  
Author(s):  
Morten G. Rasch ◽  
Ida K. Lund ◽  
Martin Illemann ◽  
Gunilla Høyer-Hansen ◽  
Henrik Gårdsvoll
Keyword(s):  
Full Length ◽  
S2 Cells ◽  
Protease Domain ◽  
Purification And Characterization ◽  
Drosophila S2 Cells

scholarly journals Levels of Polyamines and Kinetic Characterization of Their Uptake in the Soybean Pathogen Phytophthora sojae

2006 ◽  
Vol 72 (5) ◽  
pp. 3350-3356 ◽  
Author(s):  
M. Constantine Chibucos ◽  
Paul F. Morris
Keyword(s):  
Developmental Stages ◽  
Phytophthora Sojae ◽  
Biologically Active ◽  
Potential Control ◽  
High Affinity ◽  
Polyamine Transport ◽  
Wide Range ◽  
Soybean Roots ◽  
Polyamine Uptake

ABSTRACT Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-14C]putrescine and [1,4-14C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.

scholarly journals ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

2020 ◽  
pp. jbc.RA120.013908
Author(s):  
Norin Nabil Hamouda ◽  
Chris Van den Haute ◽  
Roeland Vanhoutte ◽  
Ragna Sannerud ◽  
Mujahid Azfar ◽  
...  
Keyword(s):  
Transport System ◽  
Mammalian Cells ◽  
Polyamine Biosynthesis ◽  
Polyamine Transport System ◽  
Polyamine Transport ◽  
Mrna And Protein Expression ◽  
Transport Inhibitors ◽  
Pulmonary Arterial ◽  
Polyamine Uptake ◽  
P Type

Polyamines, such as putrescine, spermidine and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system (PTS) using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor MGBG (methylglyoxal bis- (guanylhydrazone)), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in wild-type cells induced a CHO-MG phenotype, demonstrating a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3 as a major component of the mammalian PTS that confers sensitivity to MGBG and that has been previously genetically linked with pulmonary arterial hypertension.

Characterization of lysophosphatidylcholine-induced changes of intracellular calcium in Drosophila S2 cells

Life Sciences ◽  
2015 ◽  
Vol 131 ◽  
pp. 57-62 ◽  
Author(s):  
Pan Wang ◽  
Qi Wang ◽  
Lin Yang ◽  
Qi-Lian Qin ◽  
Yi-Jun Wu
Keyword(s):  
Intracellular Calcium ◽  
S2 Cells ◽  
Drosophila S2 Cells ◽  
Induced Changes

scholarly journals Heterologous Expression and Functional Characterization of a Mouse Renal Organic Anion Transporter in Mammalian Cells

1999 ◽  
Vol 274 (3) ◽  
pp. 1519-1524 ◽  
Author(s):  
Kogo Kuze ◽  
Peter Graves ◽  
Amy Leahy ◽  
Patricia Wilson ◽  
Heidi Stuhlmann ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Organic Anion ◽  
Functional Characterization ◽  
Organic Anion Transporter ◽  
Anion Transporter

scholarly journals Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells

1994 ◽  
Vol 299 (1) ◽  
pp. 19-22 ◽  
Author(s):  
J L Mitchell ◽  
G G Judd ◽  
A Bareyal-Leyser ◽  
S Y Ling
Keyword(s):  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Feedback Inhibition ◽  
Mammalian Tissue ◽  
Polyamine Biosynthesis ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Polyamine Transport ◽  
A Cell ◽  
Polyamine Uptake

Antizyme, a spermidine-induced protein that binds and stimulates ornithine decarboxylase degradation, is now shown also to mediate the rapid feedback inhibition of polyamine uptake into mammalian cells. Using a cell line (HZ7) transfected with truncated antizyme cDNA, and mutant ornithine decarboxylase cell lines, we demonstrate that this newly discovered action of antizyme is distinct from its role in modulating polyamine biosynthesis.

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