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Expression of human polyspecific renal organic cation transport activity in Xenopus laevis oocytes
J K Chun1, M Piquette-Miller, L Zhang
1Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Abstract:
Polyspecific organic cation transporters in the basolateral and brush border membrane of the kidney play a role in the elimination of many clinically important drugs and endogenous compounds. In this study we report the functional expression of organic cation transport activity in Xenopus laevis oocytes injected with poly(A)+RNA (mRNA) isolated from human kidney. Uptake of [14C]tetraethylammonium (TEA) was measured in mRNA-injected or water-injected oocytes, 4 days after injection. In oocytes injected with 50 ng of mRNA isolated from human renal cortex, the uptake of [14C]TEA was significantly increased in comparison with water-injected oocytes (7.2 +/- 0.6 and 3.5 +/- 0.3 pmol/oocyte/h, respectively). Injection of 20 ng of an enriched size-fraction (fraction C) of mRNA (mean size of 2.3 kb) resulted in further enhancement of [14C]TEA uptake: [14C]TEA uptake was enhanced six-to seven-fold in oocytes injected with fraction C (23.7 +/- 3.7 pmol/oocyte/h) in comparison with water-injected oocytes. The uptake of TEA in mRNA-injected oocytes was significantly inhibited by 5 mM of unlabeled TEA, cimetidine, and N1-methylnicotinamide. These data suggest that polyspecific organic cation transport activity can be successfully expressed in Xenopus laevis oocytes injected with mRNA isolated from human kidney.
Insights
Researchers expressed human kidney organic cation transporters in frog eggs using messenger RNA (mRNA). This functional expression allows for studying how these transporters eliminate drugs and other compounds from the body.
Area of Science:
- Pharmacology and Toxicology
- Molecular Biology
- Renal Physiology
Background:
- Polyspecific organic cation transporters in kidney membranes are crucial for drug and endogenous compound elimination.
- Understanding these transporters is vital for predicting drug efficacy and toxicity.
Purpose of the Study:
- To functionally express human kidney organic cation transport activity in Xenopus laevis oocytes.
- To characterize the transport of organic cations using messenger RNA (mRNA) from human kidney tissue.
Main Methods:
- Isolation of poly(A)+RNA (mRNA) from human renal cortex.
- Injection of mRNA into Xenopus laevis oocytes.
- Measurement of [14C]tetraethylammonium (TEA) uptake in injected oocytes.
- Inhibition studies using unlabeled TEA, cimetidine, and N1-methylnicotinamide.
Main Results:
- mRNA-injected oocytes showed significantly increased [14C]TEA uptake compared to controls.
- An enriched mRNA fraction (2.3 kb) further enhanced [14C]TEA uptake six-to seven-fold.
- Uptake was significantly inhibited by unlabeled TEA, cimetidine, and N1-methylnicotinamide, confirming transporter activity.
Conclusions:
- Xenopus laevis oocytes can successfully express functional polyspecific organic cation transport activity from human kidney mRNA.
- This model system provides a valuable tool for studying kidney organic cation transporters and their role in xenobiotic elimination.