The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. ؉ uptake by 29 ؎ 3% (n ‫؍‬ 10). hANP (10 nM) or 8-bromo-cGMP (100 M) also decreased ASP ؉ uptake by 17 ؎ 3 (n ‫؍‬ 9) or 32 ؎ 5% (n ‫؍‬ 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP-and the cGMP-dependent protein kinases.The proximal tubule is the site of secretion and reabsorption of endogenous metabolites and xenobiotics in the kidney. Many of these substances are organic cations. As several drugs are among these organic cations, specific knowledge about properties of organic cation transport in the human proximal tubule is of great importance. The first organic cation transporter was cloned from rat (rOCT1) in 1994 (1). The first human organic cation transporters (hOCT1 and hOCT2) were cloned 3 years later (2, 3), and three other members of this family (hOCTN1, hOCTN2, and hOCT3, originally named EMT) followed (4 -6). Previous functional studies (7, 8) on organic cation transport in the proximal tubule of the rat showed the differences between transport across the luminal or basolateral membrane. After cloning of the transporters data from functional investigations of these transporters together with those obtained by microperfusion experiments and transport studies with membrane vesicles (8 -12) led to a model of organic cation transport in the proximal tubule. OCTN1 was shown to be a H ϩ /organic cation exchanger (4, 13), whereas OCT1 has been characterized as a functionally different potential driven uniporter. Immunohistochemistry localized rOCT1 and rOCT2 to the basolateral membrane of proximal tubules (14 -16). The OCTN1 is most likely located in the luminal membrane of proximal tubules (17, 18). Thus, organic cation transporters expressed in the luminal and basolateral membranes of the proximal tubule are molecularly and functionally very different members of this protein family.After cloning of the first transporters, studies were performed to gain information on properties of these organic cation transporters expressed in Xenopus laevis oocytes or cell lines (19 -21). From these studies we have information on their electrogeneity, substrate specificities, and inhibitors with K m and K i values for the distinct cation transporters from rat and man. The homologous transporters of these two species differ in K m and K i values for the investigated cations (19 -24). Properties found for the cloned rOCT1 and rOCT2 do not...

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]- N-methylpyridinium (ASP+). ASP+ uptake was electrogenic and inhibited by TPA+ (EC50 = 2.7 μM), tetraethylammonium (EC50 = 35 μM), cimetidine (EC50 = 36 μM), or quinine (EC50 = 6.7 μM). Stimulation with carbachol or ATP decreased initial uptake by 44 ± 3 ( n = 14) and 34 ± 4% ( n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 ± 4% ( n = 40). Inhibition of calmodulin (CaM), Ca2+/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 ± 2 ( n = 15), 40 ± 4 ( n = 30), and 31 ± 4% ( n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC50 value for the inhibition of ASP+ uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.

The reticulocyte haemglobin content is superior to the percentage of hypochromic red cells in detecting iron deficiency in haemodialysis patients.

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.