| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universitaetsklinikum Muenster, Muenster, Germany
2 Institut fuer Anatomie und Zellbiologie, Universitaet Wuerzburg, Wuerzburg, Germany
3 Institute of Biotechnology, Vilnius, Lithuania
* To whom correspondence should be addressed. E-mail: gciari{at}uni-muenster.de.
Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT-regulation is substrate-specific, suitable fluorescent organic cations were selected by comparing their uptake in human embryonic kidney (HEK293) cells (WT) and in HEK293 cells stably transfected with hOCT2 (hOCT2). N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. Km-values of hOCT2-mediated uptake of 95 µM (amiloride) and 24 µM (ASP) were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured (IC50 in µM for amiloride and ASP, respectively: tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, tetrapentylammonium (TPA) 7 and 2). Amiloride and ASP-uptakes were significantly reduced by inhibition of Ca2+/calmodulin-complex (-55±5%, n=10 and -63±2%, n=15, for amiloride and ASP, respectively), stimulation of PKC (-54±5%, n=14 and -31±6%, n=26) and of PKA (-16±5%, n=16 and -18±4%, n=40) and increased by inhibition of PI3K (+28±6%, n=8 and +55±17%, n=16). Inhibition of Ca2+/calmodulin-complex resulted in a significant decrease of Vmax (160 to 99 photons/sec) that can be in part explained by a reduction of the membrane-associated hOCT2 (-22±6%, n=9) as determined by FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of Ca2+/calmodulin-complex causes changes in transport capacity via hOCT2-trafficking.
This article has been cited by other articles:
|
|
 |
|
  W.-K. Lee, M. Reichold, B. Edemir, G. Ciarimboli, R. Warth, H. Koepsell, and F. Thevenod Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1504 - F1513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Pabla, R. F. Murphy, K. Liu, and Z. Dong The copper transporter Ctr1 contributes to cisplatin uptake by renal tubular cells during cisplatin nephrotoxicity Am J Physiol Renal Physiol, March 1, 2009; 296(3): F505 - F511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sturm, V. Gorboulev, D. Gorbunov, T. Keller, C. Volk, B. M. Schmitt, P. Schlachtbauer, G. Ciarimboli, and H. Koepsell Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity Am J Physiol Renal Physiol, September 1, 2007; 293(3): F767 - F779. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Horvath, N. Schmid, M. A. Fragoso, A. Schmid, G. E. Conner, M. Salathe, and A. Wanner Epithelial Organic Cation Transporters Ensure pH-Dependent Drug Absorption in the Airway Am. J. Respir. Cell Mol. Biol., January 1, 2007; 36(1): 53 - 60. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
| Visit Other APS Journals Online |
