Molecular identification of a novel carnitine transporter specific to human testis. Insights into the mechanism of carnitine recognition.

l-Carnitine is an essential component of mitochondrial fatty acid beta-oxidation and plays a pivotal role in the maturation of spermatozoa within the male reproductive tract. Epididymal plasma contains the highest levels of l-carnitine found in the human body, and initiation of sperm motility occurs in parallel to l-carnitine increase in the epididymal lumen. Using a specific carrier, epididymal epithelium secretes l-carnitine into the lumen by an active transport mechanism; however, the structure-activity relationship comprising the carnitine-permeation pathway is poorly understood. We discovered a novel carnitine transporter (CT2) specifically located in human testis. Analyzing the primary structure of CT2 revealed that it is phylogenetically located between the organic cation transporter (OCT/OCTN) and anion transporter (OAT) families. Hence, CT2 represents a novel transporter family. When expressed in Xenopus oocytes, CT2 mediates the high affinity transport of l-carnitine but does not accept mainstream OCT/OCTN cationic or OAT anionic substrates. We synthesized and tested various carnitine-related compounds and investigated the physicochemical properties of substrate recognition by semi-empirical computational chemistry. The data suggest that the quaternary ammonium cation bulkiness and relative hydrophobicity be the most important factors that trigger CT2-substrate interactions. Immunohistochemistry showed that the CT2 protein is located in the luminal membrane of epididymal epithelium and within the Sertoli cells of the testis. The identification of CT2 represents an interesting evolutionary link between OCT/OCTNs and OATs, as well as provides us with an important insight into the maturation of human spermatozoa.

Biosynthesis of S-(2-chloro-1,1,2-trifluoroethyl)glutathione in rat and human hepatocytes and in Hep G2 cells.

Chlorotrifluoroethene is nephrotoxic in rats, and glutathione S-transferase-catalyzed S-(2-chloro-1,1,2-trifluorethyl)glutathione (CTFG) formation is the initial step in its bioactivation. CTFG biosynthesis and the activities of cytosolic and microsomal glutathione S-transferases were measured in rat and human hepatocytes and in human hepatoma-derived Hep G2 cells. Hepatocytes of > or = 88% viability were obtained from rat or human liver slices by collagenase or collagenase+dispase digestion, respectively. Hep G2 cells were grown in modified Earle's medium supplemented with 10% (v/v) fetal calf serum. Cells and subcellular fractions were exposed to chlorotrifluoroethene, and CTFG formation was quantified by HPLC. Both human liver and Hep G2 cell subcellular fractions catalyzed CTFG formation, and human and rat microsomal fractions exhibited higher specific activities than cytosolic fractions with chlorotrifluoroethene as the substrate. Time-dependent formation of CTFG was observed in all cell preparations. The presence of microsomal glutathione S-transferase was demonstrated by Western blotting with antimicrosomal glutathione S-transferase antibodies in rat and human liver tissue and in Hep G2 cells. Cytosolic and microsomal glutathione S-transferase activities were lower in Hep G2 cells than in rat and human liver tissues. These results demonstrate that human hepatocytes and Hep G2 cells are competent to synthesize CTFG and that Hep G2 cells may provide a useful model for studying human liver-catalyzed glutathione S-conjugate formation.