scholarly journals Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells

1988 ◽  
Vol 256 (3) ◽  
pp. 959-963 ◽  
Author(s):  
F Di Virgilio ◽  
C Fasolato ◽  
T H Steinberg
Keyword(s):  
Cell Line ◽  
Pc12 Cells ◽  
Transport System ◽  
Free Acid ◽  
Organic Anion ◽  
Cell Types ◽  
Organic Anions ◽  
Transport Systems ◽  
Fura 2 ◽  
N2a Cells

The neuroblastoma-like cell line N2A and the pheochromocytoma-like cell line PC12 excrete about 20-25% of the intracellular fluorescent Ca2+ indicator fura-2 during 10 min of incubation at 37 degrees C. The drug probenecid, known to inhibit membrane systems for the transport of organic anions [Cunningham, Israili & Dayton (1981) Clin. Pharmacol. 6, 135-151], inhibited fura-2 excretion in both cell types. However, probenecid also had untoward effects on intracellular Ca2+ homeostasis in N2A and PC12 cells. We therefore tested the drug sulphinpyrazone, another known inhibitor of organic-anion transport systems. Sulphinpyrazone fully inhibited excretion of fura-2 at 250 microM, a concentration one order of magnitude lower than that of probenecid. At this concentration and for incubation times up to 20 min, sulphinpyrazone had no untoward effects on cell viability and metabolic functions. Fura-2 was also loaded into the cytoplasm of N2A cells by permeabilization of the plasma membrane with extracellular ATP. In this case as well, the dye was rapidly released from the cells and the efflux was blocked by sulphinpyrazone. These findings suggest that N2A and PC12 cells possess a membrane system for the transport of the free-acid form of fura-2. This transport system is probably responsible for the excretion of fura-2 from these cells. Incubation of N2A and PC12 cells with sulphinpyrazone may help overcome problems arising in the investigation of [Ca2+]i homeostasis in these cell types.

Contraluminal transport systems in the proximal renal tubule involved in secretion of organic anions

1988 ◽  
Vol 254 (4) ◽  
pp. F453-F462 ◽  
Author(s):  
K. J. Ullrich ◽  
G. Rumrich
Keyword(s):  
Transport System ◽  
Driving Forces ◽  
Organic Anion ◽  
Organic Anions ◽  
Transport Systems ◽  
Exchange System ◽  
Ionic Charge ◽  
Anion Transporters ◽  
Directional Movement ◽  
Partial Charges

The transport of organic anions in the proximal tubule occurs primarily through the epithelial cells. This process involves movement across both the luminal and contraluminal membranes via specialized transport systems. Although some of the organic anions are taken up into the cell from the lumen, they can also be accumulated in tubule cells from the interstitial compartment by a variety of transporters. The relative affinities of anions for the different luminal and contraluminal transporters in concert with their conjugate driving forces determine the net directional movement, i.e., organic anion absorption or secretion. By use of the approach of stopped-flow microperfusion, it has been possible to characterize the contraluminal anion transporters in the rat. The following three different systems have been identified: 1) an exchange system for sulfate and oxalate; 2) a cotransport system for Na+ and dicarboxylates; and 3) an exchange system (the so-called p-aminohippuric acid transport system) for hydrophobic anions and long-chain fatty acids. By use of a wide variety of different analogues, the substrate specificities for these different systems were determined. Substrates with two negative ionic charges or with one negative ionic charge and one or more negative partial charges interact with all three systems, depending on the distance between the two charged groups. Polyhalogenated substrates are preferred by the dicarboxylate system. Those substrates which interact only with the p-aminohippurate transport system possess a hydrophobic area and one negative ionic charge or two negative partial charges.

Biliary secretion in elasmobranchs. II. Hepatic uptake and biliary excretion of organic anions

1976 ◽  
Vol 230 (4) ◽  
pp. 974-981 ◽  
Author(s):  
JL Boyer ◽  
J Schwarz ◽  
N Smith
Keyword(s):  
Biliary Excretion ◽  
Binding Proteins ◽  
Organic Anion ◽  
Sodium Taurocholate ◽  
Hepatic Clearance ◽  
Hepatic Uptake ◽  
Organic Anions ◽  
Transport Systems ◽  
Raja Erinacea ◽  
Low Concentrations

[35S]Bromosulfophthalein ([35C]BSP), [14C]sodium taurocholate ([14C]NaTC), AND 10 MG OF UNLABELED BSP.and of phenol-3,6-dibromophthalein disulfonate (DBSP) per kilogram body weight were injected in the caudal artery of free-swimming dogfish sharks (Squalus acanthias) and small skates (Raja erinacea). Twenty-four hours later, 85.8 +/- 15.7% of [35S]BSP was recovered in bile and liver in dogfish and 78.4 +/- 9.9% in skates. Similar results were obtained for [14C]NaTC. Unlabeled BSP or DBSP (10 mg/kg body wt) were also selectively excreted in bile over a 4-day period and at comparable rates in both species. More than 85% of [35S]BSP, BSP, and DBSP in bile was in unconjugated form. Selective hepatic clearance of BSP occurred despite nonselective binding to liver homogenates and very low concentrations of binding proteins in liver cytosol. Analysis of the organic anion plasma disappearance curves suggest that the clearance of anions into bile in elasmobranchs is delayed disproportionately relative to hepatic uptake. Albumin-BSP infusions did not prevent selective hepatic uptake of [35S]BSP, although biliary excretion was delayed further. These studies demonstrate that transport systems for biliary excretion of organic anions evolved prior to migration of marine life from the sea and relatively independently of intrahepatic conjugation and organic anion-binding proteins.

scholarly journals Characterization of an organic anion transport system in a placental cell line

2003 ◽  
Vol 285 (5) ◽  
pp. E1103-E1109 ◽  
Author(s):  
Fanfan Zhou ◽  
Kunihiko Tanaka ◽  
Michael J. Soares ◽  
Guofeng You
Keyword(s):  
Cell Line ◽  
Transport System ◽  
Apical Membrane ◽  
Organic Anion ◽  
Phosphodiesterase Inhibitor ◽  
Anion Transport ◽  
Organic Anion Transport ◽  
Estrone Sulfate ◽  
Anion Transporters ◽  
Anion Transport System

Transporters within the placenta play a crucial role in the distribution of nutrients and xenobiotics across the maternal-fetal interface. An organic anion transport system was identified on the apical membrane of the rat placenta cell line HRP-1, a model for the placenta barrier. The apical uptake of 3H-labeled organic anion estrone sulfate in HRP-1 cells was saturable ( Km = 4.67 μM), temperature and Na+ dependent, Li+ tolerant, and pH sensitive. The substrate specificity of the transport system includes various steroid sulfates, such as β-estradiol 3,17-disulfate, 17β-estradiol 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEAS) but does not include taurocholate, p-aminohippuric acid (PAH), and tetraethylammonium. Preincubation of HRP-1 cells with 8-bromo-cAMP (a cAMP analog) and forskolin (an adenylyl cyclase activator) acutely stimulated the apical transport activity. This stimulation was further enhanced in the presence of IBMX (a phosphodiesterase inhibitor). Together these data show that the apical membrane of HRP-1 cells expresses an organic anion transport system that is regulated by cellular cAMP levels. This transport system appears to be different from the known taurocholate-transporting organic anion-transporting polypeptides and PAH-transporting organic anion transporters, both of which also mediate the transport of estrone sulfate and DHEAS.

ATP-dependent multispecific organic anion transport system in rat erythrocyte membrane vesicles

1992 ◽  
Vol 262 (1) ◽  
pp. C104-C110 ◽  
Author(s):  
M. Heijn ◽  
R. P. Oude Elferink ◽  
P. L. Jansen
Keyword(s):  
Bile Acids ◽  
Transport System ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Organic Anions ◽  
Anion Transport ◽  
Maximal Velocity ◽  
Organic Anion Transport ◽  
Anion Transporter ◽  
Anion Transport System

The uptake of oxidized glutathione (GSSG) into inside-out membrane vesicles of Wistar rat erythrocytes was studied. Uptake was ATP dependent, into an osmotically active space, and saturable. Analysis of saturable ATP-dependent GSSG uptake showed two affinities for GSSG [concentration for half-maximal velocity (K1/2 1), 26 microM; K 1/2 2, 4 mM; maximum transport rate (Vmax 1), 100 pmol.mg-1.min-1; Vmax 2, 360 pmol.mg-1.min-1]. Interactions of the high-affinity system with different organic compounds were studied. Leukotriene C4, bromosulfophthalein-S-glutathione, and 2,4-dinitrophenyl-S-glutathione were effective inhibitors. In addition, anionic nonglutathione conjugates, like indocyanine green, rose bengal, dibromosulfophthalein, and sulfated or glucuronidated (divalent) bile acids inhibited GSSG transport. Monovalent bile acids had no influence on GSSG transport. Inhibition by 2,4-dinitrophenyl-S-glutathione [inhibition constant (Ki) = 2.6 microM] and sulfated glycolithocholic acid (Ki = 2.9 microM) was purely competitive. The use of adenosinetriphosphatase (ATPase) inhibitors suggested a resemblance with E1E2-type ATPase. Vesicles of erythrocytes isolated from the TR- rat, a mutant rat strain with a defective biliary secretion of organic anions, have an impaired uptake of GSSG (Vmax was decreased 2-fold). In conclusion, erythrocytes have an ATP-dependent organic anion transport system that can be inhibited by a broad range of organic anions. This system is very similar if not identical to the hepatocanalicular ATP-dependent organic anion transporter.

Interactions of organic anions with the organic cation transporter in renal BBMV

1988 ◽  
Vol 254 (1) ◽  
pp. F56-F61 ◽  
Author(s):  
P. H. Hsyu ◽  
L. G. Gisclon ◽  
A. C. Hui ◽  
K. M. Giacomini
Keyword(s):  
Proximal Tubule ◽  
Organic Cation ◽  
Organic Anion ◽  
Organic Cation Transporter ◽  
Organic Anions ◽  
Renal Proximal Tubule ◽  
Ph Gradient ◽  
Transport Systems ◽  
Organic Cations ◽  
Initial Uptake

It is generally assumed that the organic cation transport system in the renal proximal tubule is specific for organic cations and the transport of organic cations is not affected by organic anions. However, there are also data in the literature demonstrating that probenecid, a classical inhibitor of organic anion transport systems, inhibits the transport of an organic cation, cimetidine, in the renal proximal tubule. In this study we investigated the effects of probenecid and furosemide on the transport of N'-methylnicotinamide (NMN) the classical substrate of the organic cation transporter, in brush-border membrane vesicles prepared from rabbit renal cortex. In the presence of a pH gradient, both probenecid (10 mM) and furosemide reduced the initial uptake of NMN. Probenecid reduced the initial uptake of NMN to 12.1% of the control values (1.19 +/- 0.26 pmol/mg) and furosemide reduced the initial uptake of NMN to 39.2%. Probenecid (10 mM) also decreased the initial transport of NMN in the absence of a pH gradient. Inhibition of the transport of NMN by probenecid was concentration dependent, with the concentration of probenecid resulting in 50% inhibition of the transport of NMN equal to 2.31 +/- 1.18 mM in the presence of a pH gradient. Probenecid appeared to be a competitive inhibitor of NMN transport. The apparent Km (mean +/- SE) of NMN transport (2.01 +/- 0.78 mM) was increased to 18.7 +/- 10 mM (P less than 0.05) by probenecid (10 mM), whereas the Vmax was not changed (125 +/- 19.2 pmol.s-1.mg-1 vs. 186 +/- 94 pmol.s-1.mg-1, P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity.

1985 ◽  
Vol 101 (4) ◽  
pp. 1281-1287 ◽  
Author(s):  
B H Gibbons ◽  
W J Tang ◽  
I R Gibbons
Keyword(s):  
Atpase Activity ◽  
Organic Anion ◽  
Beat Frequency ◽  
Organic Anions ◽  
Transport Systems ◽  
Bend Angle ◽  
Crossbridge Cycle ◽  
Vanadate Inhibition ◽  
Increased Sensitivity ◽  
Motility Of Sperm

Substitution of any of a variety of organic anions, including acetate, propionate, lactate, gluconate, and succinate, for chloride in the reactivation medium improves the motility of demembranated sperm of Tripneustes gratilla. At the optimum concentration of 0.20 N, all of these anions improve the duration of motility, with lactate and gluconate being the best. The Michaelis constant for beat frequency (Kmf) is lower (0.11-0.14 mM at 22 degrees C) in most of the organic anions than it is in Cl- (0.20 mM), and the minimum ATP concentration required to support oscillatory beating is reduced from 10 microM in chloride to 2 microM in acetate, which together indicate a greater affinity of the axonemal ATPase for MgATP2- in the organic anions media. The maximal beat frequency, fmax, is as high as 42 Hz in 0.2 N succinate compared to 31 Hz in Cl-, whereas the mean bend angle averages 2.8 rad in acetate compared to 2.4 rad in Cl-; these values give a calculated average velocity of tubule sliding of approximately 15 micron/s in acetate and succinate, which is approximately 30% greater than the value of 11 micron/s observed in chloride. The reactivated sperm are sixfold more sensitive to vanadate inhibition in 0.2 M acetate than they are in 0.15 M Cl-. The specific ATPase activity of soluble dynein 1, which increases more than 15-fold between 0 and 1.0 N Cl-, undergoes only a twofold activation over the same range of organic anion concentration, and, like the reactivated motility, is up to 50-fold more sensitive to vanadate. This greater apparent mechanochemical efficiency and the increased sensitivity to vanadate inhibition in the organic anions suggest that they, unlike chloride, do not promote the spontaneous dissociation of ADP and PO4(3-) from the dynein-ADP-PO4 kinetic intermediate in the dynein crossbridge cycle. The use of organic anion media may lead to significant improvements in reactivation of other motile and transport systems.

scholarly journals PC12 Cell Line: Cell Types, Coating of Culture Vessels, Differentiation and Other Culture Conditions

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 958 ◽  
Author(s):  
Benita Wiatrak ◽  
Adriana Kubis-Kubiak ◽  
Agnieszka Piwowar ◽  
Ewa Barg
Keyword(s):  
Cell Line ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Cell Types ◽  
Culture Conditions ◽  
Adherent Cells ◽  
Suspension Cells ◽  
Pc12 Cell Line ◽  
Collagen Coating ◽  
Coated Surfaces

The PC12 cell line is one of the most commonly used in neuroscience research, including studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis. Two types of this line are available in the ATCC collection: traditional PC12 cells grown in suspension and well-attached adherent phenotype. PC12 cells grown in suspension tend to aggregate and adhere poorly to non-coated surfaces. Therefore, it is necessary to modify the surface of culture vessels. This paper aims to characterise the use of two distinct variants of PC12 cells as well as describe their differentiation and neuronal outgrowth with diverse NGF concentrations (rat or human origin) on various surfaces. In our study, we evaluated cell morphology, neurite length, density and outgrowth (measured spectrofluorimetrically), and expression of neuronal biomarkers (doublecortin and NeuN). We found that the collagen coating was the most versatile method of surface modification for both cell lines. For adherent cells, the coating was definitely less important, and the poly-d-lysine surface was as good as collagen. We also demonstrated that the concentration of NGF is of great importance for the degree of differentiation of cells. For suspension cells, we achieved the best neuronal characteristics (length and density of neurites) after 14 days of incubation with 100 ng/mL NGF (change every 48 h), while for adherent cells after 3–5 days, after which they began to proliferate. In the PC12 cell line, doublecortin (DCX) expression in the cytoplasm and NeuN in the cell nucleus were found. In turn, in the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell (both in the nucleus and cytoplasm). Only the traditional PC12 line grown in suspension after differentiation with NGF should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has also been noted in the cytoplasm of adherent cells, is well understood.

scholarly journals Rat hepatoma cells express novel transport systems for glutamine and glutamate in addition to those present in normal rat hepatocytes

1998 ◽  
Vol 330 (1) ◽  
pp. 255-260 ◽  
Author(s):  
D. John MGIVAN
Keyword(s):  
Cell Line ◽  
Transport System ◽  
Hepatoma Cell ◽  
Rat Hepatocytes ◽  
Hepatoma Cells ◽  
Transport Systems ◽  
Hepatoma Cell Line ◽  
High Affinity ◽  
Rat Hepatoma ◽  
Rat Hepatoma Cells

The rat hepatoma cell line H4-II-E was found to express much higher activities of Na+-dependent glutamine and aspartate transport than those observed in normal cultured hepatocytes, in agreement with previous work of others on human hepatocytes. Na+-dependent glutamine transport in rat hepatoma cells could be resolved into two components. One was pH-dependent, tolerated Li+ for Na+ substitution and was inhibited only by asparagine and histidine; characteristics similar to those of transport System N in hepatocytes. The other transport system had a similar Km for glutamine but was pH independent, did not accept Li+ ions and was completely inhibited by excess concentrations of lysine, histidine, leucine, serine and cysteine, but not by methyl-aminoisobutyrate or phenylalanine. This pattern of inhibition is distinct from that of any transporter occurring in normal hepatocytes and may indicate the presence of a new transporter isoform. Similar results were obtained with the cell line HTC. Na+-dependent aspartate transport in H4 hepatoma cells was mediated by a high-affinity system (Km 5 μM) and was inhibited by D-aspartate and L-glutamate but not by D-glutamate - properties characteristic of the high-affinity glutamate transporter EAAC1. C-terminal antibodies to the EAAC1 protein recognized a single band of 58 kDa in hepatocyte membranes, but an additional strong band of 60 kDa was present in H4 hepatoma cells. These results provide further evidence for the view that tumour cells may express additional isoforms of amino acid transport systems which are not present in non-transformed cells.

scholarly journals Induction of the high-affinity Na+-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1

1995 ◽  
Vol 310 (2) ◽  
pp. 689-692 ◽  
Author(s):  
A Ferrer-Martinez ◽  
A Felipe ◽  
B Nicholson ◽  
J Casado ◽  
M Pastor-Anglada ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Transport System ◽  
Glutamate Transport ◽  
Transport Systems ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Hypertonic Stress ◽  
High Affinity ◽  
Renal Epithelial Cell Line

The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself.

Sign in / Sign up

Export Citation Format

Share Document