Expression, distribution and functions of organic anion transporters in the rat epididymis

The nature of my work is to study the way by which organic substances cross the blood-epididymis barrier. My work is inspired by the anatomical and functional analogies between the epididymis and the kidney tubule. In the latter, elimination of toxic metabolites and foreign substances are known to occur through arrays of membrane proteins uniquely adapted to transport cationic and anionic organic substances across membranes. My work is to unravel the role of organic anion transporters in the epididymis using a combination of molecular biology and cellular physiology techniques carried out on cultured epithelia as well as on freshly isolated cells from the rat epididymis.;Using RT-PCR and Western Blot analysis, I identified the expression of organic anion transporter genes, OAT1, OAT2 and OAT3 mRNA in the rat epididymis. Immunohistochemistry revealed that OAT1 protein is present in the basal side of the principal cells of both the corpus and cauda epididymidis. OAT3 is seen in the lamina propria and blood vessels but not on the principal or other epithelial cells. Immunohistochemistry, however, failed to detect OAT2 protein. OAT1 was cloned from the rat cauda epididymidis using standard cloning techniques and verified by gene sequencing. Functional studies of the cloned protein expressed in human embryonic kidney (HEK) cells revealed similarities between the rat epididymal OAT1 and kidney OAT1 in that both transporters take up para-aminohippurate (PAH) into cells with similar kinetics and efficacy. Rat epididymal OAT1 was able to take up the anti-fertility drug, lonidamine.;The functions of OAT1 in the rat epididymis were studied by measuring the secretion (basal-to-apical) of PAH by epididymal epithelia in culture. The transport exhibited non-genomic up-regulation and down-regulation by PKA and PKC, respectively. These short-term regulations are not unlike those reported for the kidney OAT1 and are thought mediated through agonists-stimulated MAPK, PLA2 or COX-1 pathway. Since COX-1 has previously been shown to be exclusively present in the basal cells, it is conceivable that organic anion transport by the principal cells is regulated by the basal cells in the manner that principal cell Cl- secretion is regulated by the basal cells.;Another family of organic anion transporters, the multi-drug resistant-associated-protein (MRP) is another subject of my study. Reverse transcription PCR and immunohistochernistry showed that MRP1 is expressed by the basal cells. MRP1 was cloned from the rat epididymis and expressed in HEK cells with a view to delineate its functions. HEK cells expressed with MRP1 showed little accumulation of calcein-AM compared to cells not expressed with the protein, or cells expressed with the protein but treated with MK571, an inhibitor of MRP1. This ability of MRP1 to eliminate calcein-AM from the cells was competitively reduced by verapamil, omeprazole, lonidamine or methotrexate, all are pharmacological agents commonly used for diverse clinical conditions. These results indicate that MRP1 is a multi-specific drug transporter. Freshly isolated basal cells also excluded verapamil and this effect was blocked by MK571.;In summary, my work has focused on the expression, distribution and functions of two major organic anion transporters, OAT1 and MRP1 thought to be the molecular entities involved in the transport of organic substances across the epididymal tubule. This membrane- and cell-specific placement of the two organic anion transporters that have different substrate specificity and mediate counter flows of substrates offer a mechanism whereby organic substance entry into the epididymis can be 'filtered'. Having low substrate specificity, OAT1 indiscriminately brings into the cells a diverse spectrum of organic anions, some of which might be useful organic substances that nourish spermatozoa, while others could be sperm toxicants. By virtue of the drug-specific MRP1, basal cells expel some of these toxicants from the epididymis. In this way, basal cells play a protective role. They prevent access of foreign substances to spermatozoa stored in the epididymis. This hypothesis is speculative albeit an attractive one. Further work is required to lay the experimental groundwork for this hypothesis.