scholarly journals Relative contribution of OAT and OCT transporters to organic electrolyte transport in rabbit proximal tubule

2004 ◽  
Vol 287 (5) ◽  
pp. F999-F1010 ◽  
Author(s):  
Xiaohong Zhang ◽  
Carlotta E. Groves ◽  
Andrew Bahn ◽  
Wendy M. Barendt ◽  
Marcos D. Prado ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cultured Cells ◽  
Organic Anion ◽  
Weak Acid ◽  
Organic Electrolyte ◽  
Weak Base ◽  
Estrone Sulfate ◽  
High Affinity ◽  
Relative Contribution ◽  
Relative Expression Level

We compared the characteristics of several cloned rabbit organic electrolyte (OE) transporters expressed in cultured cells with their behavior in intact rabbit renal proximal tubules (RPT) to determine the contribution of each to basolateral uptake of the weak acid ochratoxin A (OTA) and the weak base cimetidine (CIM). The activity of organic anion transporters OAT1 and OAT3 proved to be distinguishable because OAT1 had a high affinity for PAH ( Ktof 20 μM) and did not support estrone sulfate (ES) transport, whereas OAT3 had a high affinity for ES ( Ktof 4.5 μM) and a weak interaction with PAH (IC50> 1 mM). In contrast, both transporters robustly accumulated OTA. Intact RPT also accumulated OTA, with OAT1 and OAT3 each responsible for ∼50%: ES and PAH each reduced uptake by ∼50%, and the combination of the two eliminated mediated OTA uptake. The weak base CIM was transported by OAT3 ( Ktof 80 μM) and OCT2 ( Ktof 2 μM); OCT1 had a comparatively low affinity for CIM, and CIM uptake by OAT1 was equivocal. Intact RPT accumulated CIM, with TEA and ES reducing CIM uptake by 20 and 75%, respectively, suggesting that OAT3 plays a quantitatively more significant role in CIM uptake in the early proximal tubule than OCT1/2. In single S2 segments of RPT, ES and TEA each blocked ∼50% of CIM uptake. Thus the fractional contribution of different OE transporters to renal secretion is influenced by their affinity for substrate and relative expression level in RPT.

Functional mapping of rbOCT1 and rbOCT2 activity in the S2 segment of rabbit proximal tubule

2003 ◽  
Vol 285 (6) ◽  
pp. F1149-F1159 ◽  
Author(s):  
Santi Kaewmokul ◽  
Varanuj Chatsudthipong ◽  
Kristen K. Evans ◽  
William H. Dantzler ◽  
Stephen H. Wright
Keyword(s):  
Proximal Tubule ◽  
Organic Cation ◽  
Dominant Role ◽  
Renal Proximal Tubule ◽  
Functional Mapping ◽  
Substrate Transport ◽  
Low Level ◽  
High Affinity ◽  
Relative Contribution

A strategy was developed to determine the distribution of activity mediated by the organic cation (OC) transporters OCT1 and OCT2 in rabbit renal proximal tubule (RPT). Both transporters displayed similar affinities for tetraethylammonium (TEA; in CHO-K1 cells, TEA concentrations that resulted in half-maximal transport were 19.9 and 34.5 μM for OCT1 and OCT2, respectively). Similarly, some OCs showed little capacity to discriminate between the two processes (IC50 values for ephedrine of 13.6 and 24.2 μM for OCT1 and OCT2, respectively). However, OCT2 had a higher affinity for cimetidine and [2-(4-nitro-2,1,3-benzoxadiazol-7-yl) aminoethyl]trimethylammonium (NBD-TMA; 1.3 and 1.4 μM, respectively) than did OCT1 (97.3 and 108 μM, respectively). Conversely, OCT1 had a higher affinity for tyramine and pindolol than did OCT2 (21.2 and 2.4 vs. 361 and 50 μM, respectively). We designated these as “discriminatory inhibitors” and used them to determine the relative contribution of OCT1 and OCT2 for TEA transport in single S2 segments of rabbit RPT. Cimetidine and NBD-TMA were high-affinity inhibitors of TEA transport in S2 segments (median IC50 values of 12.3 and 1.4 μM, respectively); in comparison, tyramine and pindolol were low-affinity inhibitors (265 and 69.3 μM, respectively). These IC50 values were sufficiently close to those for OCT2 to support the conclusion that TEA transport in the S2 segment of rabbit RPT is dominated by OCT2. However, the profile of inhibition of tyramine (an OCT1-selective substrate) transport in single S2 segments indicated that, despite a comparatively low level of expression, OCT1 can play a dominant role in the uptake of selected OC substrates.

Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+gradient

2003 ◽  
Vol 284 (4) ◽  
pp. F763-F769 ◽  
Author(s):  
Douglas H. Sweet ◽  
Lauretta M. S. Chan ◽  
Ramsey Walden ◽  
Xiao-Ping Yang ◽  
David S. Miller ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Organic Anion ◽  
Renal Proximal Tubule ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
Renal Proximal Tubule Cells ◽  
Rat Renal Cortical Slices ◽  
Cortical Slices

Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na+ gradient through Na+-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na+-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li+ or methylsuccinate (MS) or when Na+ was absent. All these treatments inhibit the Na+-dicarboxylate cotransporter, but not rat Oat3. Li+, MS, and Na+ removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na+ sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li+, MS, taurocholate, and removal of Na+. Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na+ gradient.

scholarly journals Ochratoxin A Secretion in Primary Cultures of Rabbit Renal Proximal Tubule Cells

1999 ◽  
Vol 10 (1) ◽  
pp. 13-20
Author(s):  
CARLOTTA E. GROVES ◽  
GRAZYNA NOWAK ◽  
MARK MORALES
Keyword(s):  
Proximal Tubule ◽  
Kinetic Analysis ◽  
Ochratoxin A ◽  
Organic Anion ◽  
Primary Cultures ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
High Affinity ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Abstract. Primary cultures of rabbit renal proximal tubule cells grown under improved culture conditions were used to study the transepithelial transport of the nephrotoxic mycotoxin ochratoxin A. The basal-to-apical transepithelial flux, i.e., secretion, of this fluorescence organic acid was measured in primary cultures of rabbit renal proximal tubule cells. The basal-to-apical flux of ochratoxin A increased with time and reached a steady state after 12 h. On the other hand, the apical-to-basal flux, i.e., reabsorption, of ochratoxin A was minimal over time. The secretory flux of ochratoxin A was as much as eightfold greater than the reabsorptive flux, indicating that net secretion is the primary mechanism for ochratoxin A clearance by the proximal tubule. The kinetic analysis of ochratoxin A flux revealed secretion to be a saturable and very high-affinity process with an apparent K50 of 0.33 ± 0.21 mM. A saturating concentration of the prototypical organic anion substrate para-aminohippurate (PAH) reduced ochratoxin A secretion by approximately 75%. The kinetic analysis of PAH inhibition of ochratoxin A secretion revealed an IC50 of 195 mM, which is similar to the IC50 for PAH inhibition of peritubular ochratoxin A uptake in tubule suspensions and the Km values for peritubular PAH uptake. The organic anions probenecid, octanoate, and α-ketoglutarate reduced ochratoxin A excretion to the same degree as PAH, whereas the amino acid phenylalanine had a minimal effect on ochratoxin A secretion. Thus, collectively, these observations indicate that the secretion of ochratoxin A in primary cultures of rabbit renal proximal tubules is limited to the organic anion secretory pathway. The high affinity measured for the basal-to-apical flux of ochratoxin A suggests that at concentrations typical of naturally occurring exposures, transepithelial secretion by the organic anion transport pathway represents a significant avenue for excretion of this mycotoxin by the renal proximal tubule.

scholarly journals Metabolism of Dehydroepiandrosterone Sulfate and Estrone-Sulfate by Human Platelets

2012 ◽  
pp. 381-388 ◽  
Author(s):  
A. GARRIDO ◽  
Y. MUÑOZ ◽  
W. SIERRALTA ◽  
L. VALLADARES
Keyword(s):  
Organic Anion ◽  
Dehydroepiandrosterone Sulfate ◽  
Human Platelets ◽  
Steroid Sulfatase ◽  
Organic Anion Transporting Polypeptide ◽  
Estrone Sulfate ◽  
High Affinity ◽  
Sulfatase Activity ◽  
17Β Estradiol ◽  
Estrone Sulphate

The aim of the present research was to study the uptake of DHEAS, and to establish the intracrine capacity of human platelets to produce sex steroid hormones. The DHEAS transport was evaluated through the uptake of [3H]-DHEAS in the presence or absence of different substrates through the organic anion transporting polypeptide (OATP) family. The activity of sulfatase enzyme was evaluated, and the metabolism of DHEAS was measured by the conversion of [3H]-DHEAS to [3H]-androstenedione, [3H]-testosterone, [3H]-estrone and [3H]-17β-estradiol. Results indicated the existence in the plasma membrane of an OATP with high affinity for DHEAS and estrone sulphate (E1S). The platelets showed the capacity to convert DHEAS to active DHEA by the steroid-sulfatase activity. The cells resulted to be a potential site for androgens production, since they have the capacity to produce androstenedione and testosterone; in addition, they reduced [3H]-estrone to [3H]-17β-estradiol. This is the first demonstration that human platelets are able to import DHEAS and E1S using the OATP family and to convert DHEAS to active DHEA, and to transform E1S to 17β-estradiol.

scholarly journals Growth, lifetime, directional movement and myosin-dependent motility of mutant keratin granules in cultured cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. M. Lehmann ◽  
R. E. Leube ◽  
R. Windoffer
Keyword(s):  
Digital Image Analysis ◽  
Cultured Cells ◽  
Epidermolysis Bullosa Simplex ◽  
Continuous Growth ◽  
Relative Contribution ◽  
Complete Life Cycle ◽  
Directional Movement ◽  
Multiple Characteristics ◽  
Cell Centre

AbstractIntermediate filament polypeptides (IFPs) are prominent components of cytoplasmic aggregates, which are pathognomonic for multiple diseases. Recent observations in cultured cells suggest that they are dynamic and subject to regulated turnover. The emerging concept is that multiple factors contribute to motility and turnover of IFP-containing aggregates. To understand their relative contribution, quantitative tools are needed. The current study addresses this need using epithelial cells producing mutant keratin IFPs that have been identified as the cause of the hereditary blister-forming skin disease epidermolysis bullosa simplex. Digital image analysis of individual granules allowed mapping of their complete life cycle, with information on multiple characteristics at any given time-point. The deduced signet features revealed rapid granule fusion and directed transport from the periphery towards the cell centre, and a limited, ~ 30 min lifetime with a slow, continuous growth phase followed by fast disassembly. As paradigmatic proof-of-principle, we demonstrate that inhibition of myosin II selectively reduces granule movement, linking keratin granule motility to retrograde cortical acto-myosin flow. The newly developed methods and established parameters will help in the characterization of known and the identification of novel regulators of IFP-containing aggregates.

scholarly journals Identification of a Novel Human Organic Anion Transporting Polypeptide as a High Affinity Thyroxine Transporter

2002 ◽  
Vol 16 (10) ◽  
pp. 2283-2296 ◽  
Author(s):  
F. Pizzagalli ◽  
B. Hagenbuch ◽  
B. Stieger ◽  
U. Klenk ◽  
G. Folkers ◽  
...  
Keyword(s):  
Organic Anion ◽  
Organic Anion Transporting Polypeptide ◽  
High Affinity

Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

2007 ◽  
Vol 293 (1) ◽  
pp. G271-G278 ◽  
Author(s):  
Chitrawina Mahagita ◽  
Steven M. Grassl ◽  
Pawinee Piyachaturawat ◽  
Nazzareno Ballatori
Keyword(s):  
Bile Acid ◽  
Transport Mechanism ◽  
Organic Anion ◽  
Large Family ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Facilitated Diffusion ◽  
Transport Activity ◽  
Estrone Sulfate ◽  
Anion Transporter

Organic anion transporting polypeptides (OATP/ SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 ( Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 ( SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C ( SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.

scholarly journals Organic anion transporting polypeptide�1A2 mediates fentanyl uptake in cultured cells

2019 ◽  
Author(s):  
Xing‑Chen Huo ◽  
Hui‑Wen Yang ◽  
Li‑Hua Huang ◽  
Fan Zhang ◽  
Zi‑Ye Pan ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Organic Anion ◽  
Organic Anion Transporting Polypeptide

Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene

1993 ◽  
Vol 104 (3) ◽  
pp. 695-704 ◽  
Author(s):  
N. Cartier ◽  
R. Lacave ◽  
V. Vallet ◽  
J. Hagege ◽  
R. Hellio ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Proximal Tubule ◽  
Pyruvate Kinase ◽  
Cell Lines ◽  
Cultured Cells ◽  
Simian Virus ◽  
T Antigen ◽  
Regulatory Sequence ◽  
Proximal Tubule Cell ◽  
Proximal Tubule Cells

Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5′ regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.

Renal organic anion secretion: evidence for dopaminergic and adrenergic regulation

1996 ◽  
Vol 271 (5) ◽  
pp. R1372-R1379 ◽  
Author(s):  
P. A. Halpin ◽  
J. L. Renfro
Keyword(s):  
Proximal Tubule ◽  
Organic Anion ◽  
Primary Cultures ◽  
Regulatory Control ◽  
Dichlorophenoxyacetic Acid ◽  
Adrenergic Stimulation ◽  
Anion Secretion ◽  
Ussing Chambers ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Organic Anion Secretion

To examine possible regulatory control of renal proximal tubule organic anion secretion, winter flounder (Pleuronectes americanus) proximal tubule primary cultures were mounted in Ussing chambers. Unidirectional fluxes of [2,4-(14)C]dichlorophenoxyacetic acid were determined under short-circuited conditions. Phorbol 12-myristate 13-acetate (1 microM) caused a significant (P < 0.01) inhibition of net 2,4-dichlorophenoxyacetic acid secretion. Preincubation with staurosporine (1 microM) blocked the phorbol 12-myristate 13-acetate-induced decrease in secretion. Neither forskolin (10 microM) nor W-7 (20 microM) had any effect on net transport. Elevation of intracellular calcium activity with either A-23187 or thapsigargin produced a slight, transient decrease in transport. Addition of dopamine (1 microM) to the peritubular side, but not the luminal side, caused a significant (P < 0.01) decrease in net secretion. Both the alpha-adrenergic agonist oxymetazoline (10 microM) and depletion of intracellular Na+ transiently, but significantly (P < 0.05), increased net transport. The data indicate that renal organic anion excretion may be regulated through dopaminergic inhibition and alpha-adrenergic stimulation of net transepithelial secretion.

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