The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine.

Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Tubular reabsorption of sulfate is achieved by the sodium-dependent sulfate transporter, NaSi-1, located at the apical membrane, and the sulfate-anion exchanger, sat-1, located at the basolateral membrane. To delineate the physiological role of rat sat-1, [(35)S]sulfate and [(14)C]oxalate uptake into sat-1-expressing oocytes was determined under various experimental conditions. Influx of [(35)S]sulfate was inhibited by bicarbonate, thiosulfate, sulfite, and oxalate, but not by sulfamate and sulfide, in a competitive manner with K(i) values of 2.7 +/- 1.3 mM, 101.7 +/- 9.7 microM, 53.8 +/- 10.9 microM, and 63.5 +/- 38.7 microM, respectively. Vice versa, [(14)C]oxalate uptake was inhibited by sulfate with a K(i) of 85.9 +/- 9.5 microM. The competitive type of inhibition indicates that these compounds are most likely substrates of sat-1. Physiological plasma bicarbonate concentrations (25 mM) reduced sulfate and oxalate uptake by more than 75%. Simultaneous application of sulfate, bicarbonate, and oxalate abolished sulfate as well as oxalate uptake. These data and electrophysiological studies using a two-electrode voltage-clamp device provide evidence that sat-1 preferentially works as an electroneutral sulfate-bicarbonate or oxalate-bicarbonate exchanger. In kidney proximal tubule cells, sat-1 likely completes sulfate reabsorption from the ultrafiltrate across the basolateral membrane in exchange for bicarbonate. In hepatocytes, oxalate extrusion is most probably mediated either by an exchange for sulfate or bicarbonate.

Organic anions are taken up from the blood into proximal tubule cells by organic anion transporters 1 and 3 (OAT1 and OAT3) in exchange for dicarboxylates. The released dicarboxylates are recycled by the sodium dicarboxylate cotransporter 3 (NaDC3). In this study, we tested the substrate specificities of human NaDC3, OAT1, and OAT3 to identify those dicarboxylates for which the three cooperating transporters have common high affinities. All transporters were stably expressed in HEK293 cells, and extracellularly added dicarboxylates were used as inhibitors of3 H]estrone-3-sulfate (OAT3) uptake. Human NaDC3 was stably expressed as proven by immunochemical methods and by sodiumdependent uptake of succinate (K 0.5 for sodium activation, 44.6 mM; Hill coefficient, 2.1; K m for succinate, 18 M). NaDC3 was best inhibited by succinate (IC 50 25.5 M) and less by ␣-ketoglutarate (IC 50 69.2 M) and fumarate (IC50 95.2 M). Dicarboxylates with longer carbon backbones (adipate, pimelate, suberate) had low or no affinity for NaDC3. OAT1 exhibited the highest affinity for glutarate, ␣-ketoglutarate, and adipate (IC50 between 3.3 and 6.2 M), followed by pimelate (18.6 M) and suberate (19.3 M). The affinity of OAT1 to succinate and fumarate was low. OAT3 showed the same dicarboxylate selectivity with ϳ13-fold higher IC50 values compared with OAT1. The data 1) reveal ␣-ketoglutarate as a common high-affinity substrate of NaDC3, OAT1, and OAT3 and 2) suggest potentially similar molecular structures of the binding sites in OAT1 and OAT3 for dicarboxylates.EFFICIENT EXCRETION OF ANIONIC endogenous waste products and exogenous compounds including drugs and toxins is an important task of the kidneys. Many organic anions (OA) undergo active trans-cellular secretion in renal proximal tubules, involving OA uptake across the basolateral membrane and OA release across the luminal membrane of proximal tubule cells. The uptake of OA from blood across the basolateral membrane into tubule cells is remarkable in several ways. First, OA uptake is considered the rate-limiting step in secretion; second, the uptake occurs against an opposing intracellular negative membrane potential difference; third, a multitude of chemically different OA is accepted as substrates for secretion (8,30,36). The mechanism by which OA overcome the opposing driving force during basolateral uptake was clarified in studies with basolateral membrane vesicles isolated from rat kidneys. Shimada et al. (29) and Pritchard (25) suggested the cooperation of two transporters: a sodium-driven dicarboxylate cotransporter and an OA/dicarboxylate exchanger.As regards the molecular identity of these cooperating transporters, the sodium-dicarboxylate cotransporter 3 (NaDC3, SLC13A3) takes up dicarboxylates into the cells and thus provides substrates for exchange against extracellular OA through the organic anion transporters 1 and 3 (OAT1, SLC22A6; and OAT3, SLC22A8). NaDC3 was cloned from human, rat, mouse, and winter flounder and found to be expressed in kidneys, liver, placenta, and...

a]27D -61°(c 0.5, CHC13) [lit.39 mp 158-159 °C; [a]26D -67.8°(c 0.5, CHC13)]. 17/S-(Salicylideneamino)-5-androsten-3d-ol was sublimed at 160 °C (0.02 mm); mp 224-226 °C.