Role of MAPK and PKA in regulation of rbOCT2-mediated renal organic cation transport

2007 ◽  
Vol 293 (1) ◽  
pp. F21-F27 ◽  
Author(s):  
Sunhapas Soodvilai ◽  
Atip Chatsudthipong ◽  
Varanuj Chatsudthipong
Keyword(s):  
Organic Cation ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Inhibitory Effect ◽  
Cell System ◽  
Proximal Tubules ◽  
Common Pathway ◽  
Renal Proximal Tubules ◽  
Organic Cation Transporter 2 ◽  
Heterologous Cell

The effects of protein kinases MAPK and PKA on the regulation of organic cation transporter 2 (OCT2) were investigated both in a heterologous cell system [Chinese hamster ovary (CHO-K1) cells stably transfected with rabbit (rb)OCT2] and in native intact rabbit renal proximal S2 segments. Inhibition of MEK (by U-0126) or PKA (by H-89) reduced transport activity of rbOCT2 in CHO-K1 cells. The inhibitory effect of U-0126 combined with H-89 produced no additive effect, indicating that the action of PKA and MAPK in the regulation of rbOCT2 is in a common pathway. Activation of PKA by forskolin stimulated rbOCT2 activity, and this stimulatory effect was eliminated by H-89, indicating that the stimulation required PKA activation. In S2 segments of rabbit renal proximal tubules, activation of MAPK (by EGF) and PKA (by forskolin) stimulated activity of rbOCT2, and this activation was abolished by U-0126 and H-89, respectively. This is the first study to show that MAPK and PKA are involved, apparently in a common pathway, in the regulation of OCT2 activity in both a heterologous cell system and intact renal proximal tubules.

Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules

2016 ◽  
Vol 31 (4) ◽  
pp. 1421-1433 ◽  
Author(s):  
Ulf Schulze ◽  
Sabine Brast ◽  
Alexander Grabner ◽  
Christian Albiker ◽  
Beatrice Snieder ◽  
...  
Keyword(s):  
Organic Cation ◽  
Organic Cation Transporter ◽  
Proximal Tubules ◽  
Renal Proximal Tubules ◽  
Organic Cation Transporter 2 ◽  
Tetraspanin Cd63

Basolateral localization of organic cation transporter 2 in intact renal proximal tubules

2000 ◽  
Vol 279 (5) ◽  
pp. F826-F834 ◽  
Author(s):  
Douglas H. Sweet ◽  
David S. Miller ◽  
John B. Pritchard
Keyword(s):  
Plasma Membrane ◽  
Organic Cation ◽  
Apical Membrane ◽  
Organic Cation Transporter ◽  
Proximal Tubules ◽  
Renal Cells ◽  
Renal Proximal Tubules ◽  
Lateral Membrane ◽  
Organic Cation Transporter 2 ◽  
Specific Potential

The localization of organic cation transporter 2 (OCT2) within renal cells is the subject of considerable controversy, resulting in marked uncertainty as to its function. To resolve this issue, we made an OCT2/green fluorescent protein (GFP) fusion construct (rOCT2-GFP) and determined its localization within Xenopus laevis oocytes and renal cells using confocal microscopy. Oocytes expressing rOCT2-GFP exhibited plasma membrane fluorescence as well as greatly increased specific, potential-driven uptake of [14C]tetraethylammonium (TEA). Polarized monolayers of renal epithelial cell lines [LLC-PK1 and Madin-Darby canine kidney (MDCK)] transiently transfected with pEGFP-C3, which codes for a cytoplasmic GFP, showed a diffuse, evenly distributed cytoplasmic signal with no plasma membrane fluorescence. In contrast, cells transiently transfected with pEGFP-C3/rOCT2 (the vector coding for rOCT2-GFP) showed predominantly plasma membrane fluorescence, which was most prominent in the lateral membrane. MDCK cells stably expressing rOCT2-GFP (MDCK/rOCT2-GFP) maintained in long-term culture showed a greatly increased basal and lateral membrane fluorescence. When grown on porous supports, MDCK/rOCT2-GFP monolayers showed specific, potential-driven TEA uptake from the basal side. Finally, expression and distribution of rOCT2-GFP were investigated in isolated killifish ( Fundulus heteroclitus) renal proximal tubules. On expression of rOCT2-GFP, transfected tubules showed marked basal and lateral membrane fluorescence, with no detectable signal at the apical membrane. In contrast, tubules expressing a luminal sodium-dicarboxylate cotransporter (rbNaDC-1)-GFP construct showed apical membrane fluorescence, and tubules expressing cytoplasmic GFP had a diffuse cytoplasmic fluorescence. These results indicate that rOCT2 is basolateral in renal proximal tubule cells.

Daunomycin secretion by killfish renal proximal tubules

1995 ◽  
Vol 269 (2) ◽  
pp. R370-R379 ◽  
Author(s):  
D. S. Miller
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Organic Cation Transporter ◽  
Proximal Tubules ◽  
Epifluorescence Microscopy ◽  
Buffer Solution ◽  
Simple Diffusion ◽  
Renal Proximal Tubules ◽  
Cellular Accumulation ◽  
Tubular Lumen

Epifluorescence microscopy and video-image analysis were used to measure the uptake of the fluorescent anthracycline daunomycin by intact killifish renal proximal tubules. When tubules were incubated in medium containing 2-5 microM daunomycin, the drug accumulated in the cells and the tubular lumen. At steady state, luminal fluorescence was two to three times greater than cellular fluorescence. Luminal accumulation of daunomycin was reduced when tubules were exposed to the multidrug-resistance (MDR) transporter modifiers verapamil and cyclosporin A (CSA), but not tetraethylammonium (TEA), a model substrate for the renal organic cation transport system. NaCN and vanadate reduced luminal drug accumulation. In contrast, cellular daunomycin accumulation was not affected by verapamil, CSA, TEA, or vanadate and was only slightly reduced by NaCN. When the pH of the buffer solution was decreased from 8.25 to 7.25, luminal, but not cellular, accumulation of daunomycin was again reduced by CSA; however, TEA now reduced cellular and luminal accumulation. These findings are consistent with daunomycin being actively secreted in killifish proximal tubule by two mechanisms. At pH 8.25, daunomycin crossed the basolateral membrane by simple diffusion and was secreted into the tubular lumen by the MDR transporter. At pH 7.25, daunomycin was transported across the basolateral membrane by simple diffusion and carrier-mediated uptake on the organic cation transporter and was secreted into the lumen by the MDR transporter and the organic cation/H+ exchanger.

Peritubular organic cation transport in isolated rabbit proximal tubules

1994 ◽  
Vol 266 (3) ◽  
pp. F450-F458 ◽  
Author(s):  
C. E. Groves ◽  
K. K. Evans ◽  
W. H. Dantzler ◽  
S. H. Wright
Keyword(s):  
Organic Cation ◽  
High Capacity ◽  
Organic Cation Transporter ◽  
Cation Transport ◽  
Proximal Tubules ◽  
Organic Cation Transport ◽  
Inhibitory Potency ◽  
Renal Proximal Tubules ◽  
Michaelis Constants ◽  
Maximal Capacity

The physiological characteristics of peritubular organic cation transport were examined by measuring the transport of the organic cation tetraethylammonium (TEA) in rabbit renal proximal tubule suspensions and isolated nonperfused rabbit renal proximal tubules. Peritubular organic cation transport in both single S2 segments and suspensions of isolated renal proximal tubules was found to be a high-capacity, high-affinity, carrier-mediated process. For tubule suspensions, the maximal capacity of the carrier for TEA (Jmax) and the concentration of TEA at 1/2 Jmax (Kt) (1.49 +/- 0.21 nmol.min-1.mg dry wt-1 and 131 +/- 16 microM, respectively), did not differ significantly from those measured in single S2 segments (Jmax, 1.16 +/- 0.075 nmol.min-1.mg dry wt-1; Kt, 108 +/- 10 microM). In addition, the pattern of inhibition of peritubular TEA transport by long-chain n-tetraalkylammonium compounds (n = 1-5) was both qualitatively and quantitatively similar in single S2 segments and tubule suspensions, exhibiting an increase in inhibitory potency with increasing alkyl chain length. For example, in tubule suspensions, apparent Michaelis constants for inhibition of TEA uptake ranged from 1.3 mM for tetramethylammonium (TMA) to 0.8 microM for tetrapentylammonium (TPeA). To determine whether these compounds were substrates for the peritubular organic cation transporter, their effect on the efflux of [14C]TEA from tubule suspensions was examined. A concentration of 0.5 mM of the short-chain tetraalkyls TMA or TEA increased the efflux of [14C]TEA (i.e., trans-stimulated) from tubules in suspension. The longer-chain tetraalkyls tetrapropylammonium, tetrabutylammonium, and TPeA all decreased the efflux of [14C]TEA from tubules in suspension; TPeA completely blocked efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Fluorescent organic cations for human OCT2 transporters screening: uptake in CHO cells stably expressing hOCT2

ADMET & DMPK ◽  
2017 ◽  
Vol 5 (2) ◽  
pp. 135 ◽  
Author(s):  
Malachy C. Ugwu ◽  
Ryan Pelis ◽  
Charles O. Esimone ◽  
Remigius U. Agu
Keyword(s):  
Cho Cells ◽  
Organic Cation ◽  
Rhodamine 6G ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Rhodamine 123 ◽  
Intracellular Uptake ◽  
Wild Type ◽  
Transfected Cells ◽  
Organic Cation Transporter 2

<p class="ADMETabstracttext">The aim of this study was to assess the suitability of amiloride, rhodamine 6G and rhodamine 123 as non-radioactive substrates for characterizing hOCT2 using CHO cells. The uptake characteristics of these compounds were compared in wild-type (WT) and human organic cation transporter 2 (hOCT2)-stably transfected Chinese Hamster Ovary (CHO) cells. All the compounds were accumulated by the CHO-hOCT2 cells. Intracellular uptake of the compounds was higher in CHO cells stably-expressing hOCT2 compared to the WT. The uptake was concentration–dependent and saturable (except for rhodamine 123). The affinities of the compounds for the hOCT2 (in descending order) were: amiloride (K<sub>m</sub> = 72.63 ± 12.02 µM) &gt; rhodamine 6 G (K<sub>m</sub> = 82.47 ± 29.15 µM). Uptake of amiloride in transfected cells was pH -dependent and significantly inhibited by hOCT2 inhibitors (quinine, verapamil and quinidine). Based on our kinetic data and other considerations, we recommend the use of amiloride for characterizing hOCT2 transporters.</p>

Functional characterization of ORCTL2 - an organic cation transporter expressed in the renal proximal tubules

FEBS Letters ◽  
1998 ◽  
Vol 433 (3) ◽  
pp. 245-250 ◽  
Author(s):  
Mark Reece ◽  
Dirk Prawitt ◽  
John Landers ◽  
Christina Kast ◽  
Philippe Gros ◽  
...  
Keyword(s):  
Organic Cation ◽  
Functional Characterization ◽  
Organic Cation Transporter ◽  
Proximal Tubules ◽  
Renal Proximal Tubules

Specificity of basolateral organic cation transport in snake renal proximal tubules

1996 ◽  
Vol 270 (5) ◽  
pp. R1025-R1030
Author(s):  
Y. K. Kim ◽  
W. H. Dantzler
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Cation Transport ◽  
Inhibitory Effect ◽  
Proximal Tubules ◽  
Organic Cation Transport ◽  
Renal Proximal Tubules ◽  
Basolateral Side ◽  
Michaelis Constants ◽  
Kinetics Of

We examined the specificity of basolateral organic cation transport in isolated snake (Thamnophis spp.) renal proximal tubules by determining the inhibitory effect of a series of n-tetraalkylammonium (n-TAA) compounds (n = 1-5) on the basolateral uptake of [3H]tetraethylammonium (TEA). The inhibitory potency increased with increasing alkyl chain length, with the apparent Michaelis constants for inhibition of TEA uptake ranging from 3.3 mM for tetramethylammonium (TMA) to 1.0 microM for tetrapentylammonium (TPeA). Thus the apparent affinity of the carrier for n-TAA compounds increases with their increasing hydrophobicity. Because previous data suggested that TEA transport across the basolateral membrane may be asymmetrical and that the exit step may be regulated differently from the entry step, we examined the kinetics of [3H]TEA efflux across the basolateral membrane, Efflux, like entry, occurred by a saturable process that could be described adequately by Michaelis-Menten kinetics. However, the concentration of TEA at one-half Jmax (Kt) for efflux (approximately 110 microM) was about six times the Kt for uptake (approximately 18 muM), indicating that the affinity of the carrier for TEA is greater in the uptake direction than in the efflux direction or that there are separate carriers with different affinities for uptake and efflux. In either case, this difference would favor movement of TEA taken up at the basolateral side across the cells and into the lumen over movement back into the peritubular fluid.

scholarly journals Kinetic basis of metformin-MPP interactions with organic cation transporter OCT2

2019 ◽  
Vol 317 (3) ◽  
pp. F720-F734 ◽  
Author(s):  
Philip J. Sandoval ◽  
Mark Morales ◽  
Timothy W. Secomb ◽  
Stephen H. Wright
Keyword(s):  
Organic Cation ◽  
Competitive Inhibition ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Inhibitory Interaction ◽  
Ligand Interaction ◽  
Ovary Cells ◽  
Multiple Substrates ◽  
Organic Cation Transporter 2

Organic cation transporter 2 (OCT2) clears the blood of cationic drugs. Efforts to understand OCT2 selectivity as a means to predict the potential of new molecular entities (NMEs) to produce unwanted drug-drug interactions typically assess the influence of the NMEs on inhibition of transport. However, the identity of the substrate used to assess transport activity can influence the quantitative profile of inhibition. Metformin and 1-methyl-4-phenylpyridinium (MPP), in particular, display markedly different inhibitory profiles, with IC50 values for inhibition of MPP transport often being more than fivefold greater than IC50 values for the inhibition of metformin transport by the same compound, suggesting that interaction of metformin and MPP with OCT2 cannot be restricted to competition for a single binding site. Here, we determined the kinetic basis for the mutual inhibitory interaction of metformin and MPP with OCT2 expressed in Chinese hamster ovary cells. Although metformin did produce simple competitive inhibition of MPP transport, MPP was a mixed-type inhibitor of metformin transport, decreasing the maximum rate of mediated substrate transport and increasing the apparent Michaelis constant ( Ktapp) for OCT2-mediated metformin transport. Furthermore, whereas the IC50 value for metformin’s inhibition of MPP transport did not differ from the Ktapp value for metformin transport, the IC50 value for MPP’s inhibition of metformin transport was less than its Ktapp value for transport. The simplest model to account for these observations required the influence of a distinct inhibitory site for MPP that, when occupied, decreases the translocation of substrate. These observations underscore the complexity of ligand interaction with OCT2 and argue for use of multiple substrates to obtain the needed kinetic assessment of NME interactions with OCT2.

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