Submitted on January 17, 2007
Accepted on September 5, 2007
Transport of a fluorescent cAMP analog in teleost proximal tubules
Valeska Reichel1, Rosalinde Masereeuw2, Jeroen J.M.W. van den Heuvel2, David S Miller3, and Gert Fricker1*
1 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany
2 Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands
3 Laboratory of Pharmacology and Chemistry, NIH/NIEHS, Research Triangle Park, North Carolina, United States
* To whom correspondence should be addressed. E-mail: gert.fricker{at}uni-hd.de.
Previous studies showed that killifish (Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady state luminal accumulation of fluo-cAMP was concentrative, specific and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (or-ganic anion transporter (Oat) inhibitor) or PSC833 (p-glycoprotein inhibitor), but cell to lu-men transport was reduced in a concentration-dependent manner by MK571, LTC4, AZT, cAMP and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate, FL-MTX, neither cAMP, adefovir nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were ex-posed to endothelin-1, Na nitroprusside (an NO generator) or phorbol ester (protein kinase C, PKC, activator), all of which signal substantial reductions in cell to lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin and this reduction was blocked by the PKA inhibitor, H-89. Finally, Finally, in membrane vesicles from Sf9 cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell to lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.
