Transport of a fluorescent cAMP analog in teleost proximal tubules

Abstract

Previous studies have shown 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 (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (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, in membrane vesicles from Spodoptera frugiperda (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.

Aspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4–Mediated Transport

“…To investigate whether aspirin is a substrate and can be transported by MRP4, we used DGP, obtained from HV human platelets, because MRP4 is predominantly Aspirin and MRP4 in Platelets localized in dense granule membranes (12). To demonstrate that aspirin is a substrate for MRP4, we used Fluo-cAMP, a fluorimetric analog of cAMP (17). In DGP both aspirin and SA reduced the intake of Fluo-cAMP, suggesting that both aspirin and SA interact with MRP4 solely and specifically (Fig.…”

“…Fluo-cyclic adenosine monophosphate MRP4-mediated transport. The MRP4-dependent 8-Fluocyclic adenosine monophosphate (cAMP) uptake in platelet granules was analyzed as previously described (17). Briefly DGP was incubated with 8-Fluo-cAMP, aspirin, and SA (all 20 mol/l).…”

Characterization of efflux transport proteins of the human choroid plexus papilloma cell line HIBCPP, a functional in vitro model of the blood-cerebrospinal fluid barrier

“…Fluo-cAMP was used to study ABCC4 functionality as this substance was shown to be a substrate for human ABCC4 (24). Again, PSC 833 and MK 571 were used as inhibitors in the experiment.…”

“…Substrates of ABCC1 include fluorescein methotrexate (22) and sulforhodamine 101 (23), substrates of ABCC4 are cyclic nucleotides cAMP and cGMP, adefovir and topotecan (19). Moreover, fluo-cAMP (a fluorescein-linked cAMP analogue) transport is mediated by this transporter (24). Both, ABCC1 and ABCC4 can be inhibited with MK 571.…”

Sphingolipid Signaling Reduces Basal P-Glycoprotein Activity in Renal Proximal Tubule

“…One reason for this is that in marine teleost proximal tubules the tubular lumen remains patent and all the steps in the excretory transport of fluorescent substrates can be visualized for several hours in the living tubules. Indeed, studies with teleost renal tubules have identified fluorescent substrates for specific ABC transporters, e.g., P-glycoprotein and Mrp4 (Abcc4), that have translated to mammalian systems (Schramm et al, 1995;Miller et al, 2000;Reichel et al, 2007Reichel et al, , 2010. Moreover, with regard to P-glycoprotein, there are clear molecular-level similarities between the killifish and human transporters.…”