Decreased renal organic anion secretion and plasma accumulation of endogenous organic anions in OAT1 knock-out mice

The "classical" organic anion secretory pathway of the renal proximal tubule is critical for the renal excretion of the prototypic organic anion, para-aminohippurate, as well as of a large number of commonly prescribed drugs among other significant substrates. Organic anion transporter 1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), has physiological properties consistent with a role in this pathway. However, several other transporters (e.g. OAT2, OAT3, and MRP1) have also been proposed as important PAH transporters on the basis of in vitro studies; therefore, the relative contribution of OAT1 has remained unclear. We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in the context of these other potentially functionally redundant transporters. We find that the knock-out mice manifest a profound loss of organic anion transport (e.g. para-aminohippurate) both ex vivo (in isolated renal slices) as well as in vivo (as indicated by loss of renal secretion). In the case of the organic anion, furosemide, loss of renal secretion in the knock-out results in impaired diuretic responsiveness to this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway. In addition, we have determined the levels of approximately 60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1.     

Regulation of mOAT-mediated organic anion transport by okadaic acid and protein kinase C in LLC-PK(1) cells

Organic anion transporters in the kidney proximal tubule play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites, thereby preventing their potentially toxic effects within the body. We have previously cloned a cDNA encoding an organic anion transporter from mouse kidney (mOAT) (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478; Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524). In the present study, we assessed the potential for regulation of this transporter by heterologous expression of mOAT in the pig proximal tubule-like cell line, LLC-PK(1). We report here that both protein phosphatase (PP1/PP2A) inhibitor, okadaic acid, and protein kinase C (PKC) activators down-regulate mOAT-mediated transport of para-aminohippuric acid (PAH), a prototypic organic anion, in a time- and concentrationdependent manner. However their mechanisms of action for this down-regulation are distinct. Okadaic acid modulated PAH transport, at least in part, through phosphorylation/dephosphorylation of mOAT; phosphoamino acid analysis indicated this phosphorylation occurs on serine. In contrast, PKC activation induced a decrease in the maximum transport velocity (V(max)) of PAH transport without direct phosphorylation of the transporter protein. Together these results provide the first demonstration that regulation of organic anion transport by mOAT is likely to be tightly controlled directly and indirectly by phosphatase PP1/PP2A and PKC. Our results also suggest that kinases other than PKC are involved in this process.     

Interaction of Organic Cations with Organic Anion Transporters

Studies of the organic anion transporters (Oats) have focused mainly on their interactions with organic anionic substrates. However, as suggested when Oat1 was originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478), since the Oats share close homology with organic cation transporters (Octs), it is possible that Oats interact with cations as well. We now show that mouse Oat1 (mOat1) and mOat3 and, to a lesser degree, mOat6 bind a number of “prototypical” Oct substrates, including 1-methyl-4-phenylpyridinium. In addition to oocyte expression assays, we have tested binding of organic cations to Oat1 and Oat3 in ex vivo assays by analyzing interactions in kidney organ cultures deficient in Oat1 and Oat3. We also demonstrate that mOat3 transports organic cations such as 1-methyl-4-phenylpyridinium and cimetidine. A pharmacophore based on the binding affinities of the tested organic cations for Oat3 was generated. Using this pharmacophore, we screened a chemical library and were able to identify novel cationic compounds that bound to Oat1 and Oat3. These compounds bound Oat3 with an affinity higher than the highest affinity compounds in the original set of prototypical Oct substrates. Thus, whereas Oat1, Oat3, and Oat6 appear to function largely in organic anion transport, they also bind and transport some organic cations. These findings could be of clinical significance, since drugs and metabolites that under normal physiological conditions do not bind to the Oats may undergo changes in charge and become Oat substrates during pathologic conditions wherein significant variations in body fluid pH occur.     

Analysis of three-dimensional systems for developing and mature kidneys clarifies the role of OAT1 and OAT3 in antiviral handling

The organic anion transporters OAT1 (SLC22A6, originally identified by us as NKT) and OAT3 (SLC22A8) are critical for handling many toxins, metabolites, and drugs, including antivirals (Truong, D. M., Kaler, G., Khandelwal, A., Swaan, P. W., and Nigam, S. K. (2008) J. Biol. Chem. 283, 8654-8663). Although microinjected Xenopus oocytes and/or transfected cells indicate overlapping specificities, the individual contributions of these transporters in the three-dimensional context of the tissues in which they normally function remain unclear. Here, handling of HIV antivirals (stavudine, tenofovir, lamivudine, acyclovir, and zidovudine) was analyzed with three-dimensional ex vivo functional assays using knock-out tissue. To investigate the contribution of OAT1 and OAT3 in various nephron segments, the OAT-selective fluorescent tracer substrates 5-carboxyfluorescein and 6-carboxyfluorescein were used. Although OAT1 function (uptake in oat3(-/-) tissue) was confined to portions of the cortex, consistent with a proximal tubular localization, OAT3 function (uptake in oat1(-/-) tissue) was apparent throughout the cortex, indicating localization in the distal as well as proximal nephron. This functional localization indicates a complex three-dimensional context, which needs to be considered for metabolites, toxins, and drugs (e.g. antivirals) handled by both transporters. These results also raise the possibility of functional differences in the relative importance of OAT1 and OAT3 in antiviral handling in developing and mature tissue. Because the HIV antivirals are used in pregnant women, the results may also help in understanding how these drugs are handled by developing organs.     

P-glycoprotein retains drug-stimulated ATPase activity upon covalent linkage of the two nucleotide binding domains at their C-terminal ends

P-glycoprotein (Pgp), a member of the ABC transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anti-cancer chemotherapy. We have recently obtained EM projection images of lipid-bound Pgp without nucleotide and transport substrate that showed the two halves of the transporter separated by a central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125-40131). Addition of nucleotide and/or substrate lead to a close association of the two halves of the transporter, thereby closing the central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2008) J. Biol. Chem. 283, 5769-5779). Here, we used cysteine-mediated disulfide cross-linking to further delineate the structural rearrangements of the two nucleotide binding domains (NBD1 and NBD2) that take place during catalysis. Cysteines introduced at or near the C-terminal ends of NBD1 and NBD2 allowed for spontaneous disulfide cross-linking under nonreducing conditions. For mutant A627C/S1276C, disulfide formation was with high efficiency and cross-linked Pgp retained 30-68% drug-stimulated ATPase activity compared with reduced or cysteine-less Pgp. Two other cysteine pairs (K615C/S1276C and A627C/K1260C) also formed a disulfide but to a lesser extent, and the cross-linked form of these two mutants had lower drug-stimulated ATPase activity. The data suggest that the C-terminal ends of the two NBDs of Pgp are not required to undergo significant motion with respect to one another during the catalytic cycle.     

Nucleobase transport by human equilibrative nucleoside transporter 1 (hENT1)

The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter.     

Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.     

Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated apoptosis in response to noscapine

We have previously discovered the opium alkaloid noscapine as a microtubule interacting agent that binds to tubulin, alters the dynamics of microtubule assembly, and arrests mammalian cells at mitosis (Ye, K., Ke, Y., Keshava, N., Shanks, J., Kapp, J. A., Tekmal, R. R., Petros, J., and Joshi, H. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1601-1606; Ye, K., Zhou, J., Landen, J. W., Bradbury, E. M., and Joshi, H. C. (2001) J. Biol. Chem. 276, 46697-46700; Zhou, J., Panda, D., Landen, J. W., Wilson, L., and Joshi, H. C. (2002) J. Biol. Chem. 277, 17200-17208). Here we show that noscapine does not compete with paclitaxel for tubulin binding and can efficiently inhibit the proliferation of both paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells (i.e. the parental cell line 1A9 and two derivative cell lines, 1A9PTX10 and 1A9PTX22, which harbor beta-tubulin mutations that impair paclitaxel-tubulin interaction (Giannakakou, P., Sackett, D. L., Kang, Y. K., Zhan, Z., Buters, J. T., Fojo, T., and Poruchynsky, M. S. (1997) J. Biol. Chem. 272, 17118-17125). Strikingly, these cells undergo apoptotic death upon noscapine treatment, accompanied by activation of the c-Jun NH(2)-terminal kinases (JNK). Furthermore, inhibition of JNK activity by treatment with antisense oligonucleotide or transfection with dominant-negative JNK blocks noscapine-induced apoptosis. These findings thus indicate a great potential for noscapine in the treatment of paclitaxel-resistant human cancers. In addition, our results suggest that the JNK pathway plays an essential role in microtubule inhibitor-induced apoptosis.     

Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. II. Functional alterations of renal cortical mitochondria isolated after mercuric chloride treatment

The mitochondrial functional defects occurring in the early stages of nephrotoxic renal injury secondary to mercuric chloride have been characterized. No loss of cellular integrity or major mitochondrial structural alterations occurred within the first 3 h after a subcutaneous injection of 5 mg/kg of HgCl2. At 3 h, levels of Hg2+ in renal cortex and isolated renal cortical mitochondria were 1.87 and 0.72 nmol/mg of protein, respectively. Much evidence suggested that this Hg2+ had reached the mitochondria in situ and not during the isolation process. Mitochondria isolated beginning 1 h after treatment with HgCl2 showed depressed ADP uptake. At 2 h, inhibitions of State 3 and 2,4-dinitrophenol uncoupled respiration were detected. Inhibition of 2,4-dinitrophenol-activated mitochondrial ATPase activity was present when measured on mitochondria isolated at 3 h. These effects were not reversed by 2 mM dithioerythritol, 50 mg/ml of albumin or 5 mM MgCl2. Analysis of the data in the context of information available on the in vitro effects of HgCl2 (Weinberg, J. M., Harding, P. G., and Humes, H. D. (1982) J. Biol. Chem. 257, 60-67) indicated that the mitochondrial functional effects could not be attributed to interaction of the mitochondria with Hg2+ during their isolation. These studies implicate compromised mitochondrial bioenergetic function as one of the earliest intracellular effects of Hg2+ in the production of nephrotoxicity but suggest that the intracellular process involves events in addition to those seen with direct exposure of mitochondria to Hg2+ in vitro.     

Stimulation of mitogen-activated protein kinase by G protein-coupled alpha(2)-adrenergic receptors does not require agonist-elicited endocytosis.

Agonist-elicited receptor sequestration is strikingly different for the alpha(2A)- versus alpha(2B)-adrenergic receptor (alpha(2)-AR) subtypes; the alpha(2B)-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the alpha(2A)-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648-31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999) J. Neurosci. 19, 56-63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K(+)-depleted medium eliminated alpha(2B)-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via G(i)-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.     

Localization of type I iodothyronine 5'-deiodinase to the basolateral plasma membrane in renal cortical epithelial cells

Type I iodothyronine 5'-deiodinase is an integral membrane protein catalyzing the phenolic ring deiodination of thyroxine. We recently showed that the substrate binding subunit of this approximately 50-kDa protein is selectively labeled with N-bromoacetyl-L-thyroxine, allowing ready identification of the type I enzyme without the need to maintain catalytic activity. In this study, we used both affinity labeling and catalytic activity to determine the regional distribution of this enzyme in rat kidney and to localize the enzyme to specific plasma membrane domain(s) of renal epithelial cells. The type I enzyme was present exclusively in tubular epithelial cells of the outer renal cortex and co-purified with basolateral plasma membranes; the renal medulla lacked activity. LLC-PK1 cells, derived from the proximal convoluted tubule, have abundant type I 5'-deiodinating activity. We used this homogenous cell line to verify that the type I enzyme was localized to the cytosolic surface of the basolateral membrane. Digitonin permeabilization increased affinity labeling of the enzyme 4-fold, and approximately 75% of the affinity label was incorporated into the 27-kDa substrate binding subunit. Affinity labeling of the type I enzyme in LLC-PK1 cells mimicked the affinity labeling of the substrate binding subunit of type I 5'-deiodinase in rat kidney (K?hrle, J., Rasmussen, U. B., Ekenbarger, D. M., Alex, S., Rokos, H., Hesch, R. D., and Leonard, J. L. (1990) J. Biol. Chem. 265, 6155-6163). Subcellular fractionation of LLC-PK1 cell homogenates showed that both affinity labeled and catalytically active type I enzyme were present on the cytosolic surface of the basolateral region of the renal cell membrane.     

Peptide:N-glycosidase activity found in the early embryos of Oryzias latipes (Medaka fish). The first demonstration of the occurrence of peptide:N-glycosidase in animal cells and its implication for the presence of a de-N-glycosylation system in living organisms.

The recent discovery of free oligosaccharides typical for the complex type of glycan chains terminating with a free di-N-acetylchitobiosyl structure in certain fish eggs and early embryos (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Seko, A., Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y. (1989) J. Biol. Chem. 264, 15922-15929; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) led us to find an enzyme responsible for detachment of N-linked glycan chains from glycoproteins by hydrolyzing the beta-aspartyl-glucosylamine linkage in Oryzias latipes embryos. The enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase or peptide:N-glycosidase (PNGase), was partially (2090-fold) purified, and the reaction site at which this enzyme acts was specified by analysis and identification of the reaction products. This is the first demonstration showing PNGase in animal sources, although the presence of PNGases was reported in a variety of plant extracts and bacteria. Thus, the commonality of this type of enzyme is now demonstrated, and the possible physiological role of PNGase in de-N-glycosylation as a basic biologic process is proposed.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Membrane binding characteristics of two forms of transforming growth factor-beta

Cartilage-inducing factors A and B (CIF-A and CIF-B) from bovine bone have recently been identified as transforming growth factor-beta (TGF-beta) (Seyedin, S.M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem., 261, 5693-5695) and a unique protein homologous to TGF-beta (Seyedin S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., and Graycar, J. (1987) J. Biol. Chem., 262, 1946-1949), respectively. Although the biological activities of TGF-beta and CIF-B are similar, the divergence of CIF-B from the highly conserved amino acid sequence of TGF-beta prompted an investigation of its receptor binding properties. Three classes of cell surface binding components were identified. Class A has exclusive affinity for TGF-beta; class B has greater affinity for CIF-B; and class C has equal affinity for both proteins. A high molecular weight component, the predominant binding species, was further characterized and shown to consist of two components that are either class B or class C. The differential binding properties of TGF-beta and CIF-B to cell surface components suggest that there are biological activities unique to each of the proteins.     

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.     

Fish egg glycophosphoproteins have species-specific N-linked glycan units previously found in a storage pool of free glycan chains.

Recent findings (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) of a relatively large quantity of complex-type free sialo-oligosaccharides in the unfertilized eggs of freshwater fish, Plecoglossus altivelis and Tribolodon hakonensis, prompted us to search for their progenitor glycoproteins. First we demonstrated a third occurrence of free sialoglycans in the unfertilized eggs of Medaka fish (Oryzias latipes). Next, in all three species studied, a uniformly high level of glycophosphoproteins (GPP) was identified and found to possess N-linked glycan units. The carbohydrate structures of the GPP were determined to be identical with those of the free glycans isolated from the unfertilized eggs of the respective fish species. Thus, the most likely candidate for the progenitor of free sialoglycans appeared to be the oocyte GPPs. This implies that the liberation of the free glycans by a putative peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase may represent a necessary biochemical event during vitellogenesis or oogenesis. The present results may provide insight into a new concept of a "protein N-glycosylation/de-N-glycosylation system" recently proposed by us (Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114).     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.