Expression of organic anion transporting polypeptide E (OATP-E) in human placenta.

PURPOSE: To examine the expression of multifunctional Na(+)-independent organic anion transporting polypeptide, termed OATP-E, involved in the transport of thyroid hormone in human placenta. METHODS: Western blot analysis was performed using a specific antibody against OATP-E in human placenta to confirm the expression of OATP-E at the protein level. Immunohistochemistry was also performed using the specific antibody against OATP-E on frozen sections of human placenta. RESULTS: By Western blot analysis, a single band for OATP-E was observed in human placenta. Immunohistochemistry revealed that OATP-E was predominantly expressed at the apical surface of the syncytiotrophoblasts in placenta. CONCLUSION: These results reveal that OATP-E is expressed in human placenta, suggesting a functional role for the transplacental transfer of thyroid hormone.

An inwardly rectifying K(+) channel, Kir4.1, expressed in astrocytes surrounds synapses and blood vessels in brain.

Glial cells express inwardly rectifying K(+) (Kir) channels, which play a critical role in the buffering of extracellular K(+). Kir4.1 is the only Kir channel so far shown to be expressed in brain glial cells. We examined the distribution of Kir4.1 in rat brain with a specific antibody. The Kir4.1 immunostaining distributed broadly but not diffusely in the brain. It was strong in some regions such as the glomerular layer of the olfactory bulb, the Bergmann glia in the cerebellum, the ependyma, and pia mater, while little activity was detected in white matter of the corpus callosum or cerebellar peduncle. In the olfactory bulb, Kir4.1 immunoreactivity was detected in a scattered manner in about one-half of the glial fibrillary acidic protein-positive astrocytes. Immunoelectron microscopic examination revealed that Kir4.1 channels were enriched on the processes of astrocytes wrapping synapses and blood vessels. These data suggest that Kir4.1 is expressed in a limited population of brain astrocytes and may play a specific role in the glial K(+)-buffering action.

LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers.

BACKGROUND & AIMS: One approach to the development of targeted cancer chemotherapy exploits increased uptake of the agent into neoplastic cells. In this scenario, higher concentrations of the agent in cancer cells are responsible for differential killing, whereas the low concentration in normal human cells decreases side effects. The aim of this study was to isolate an organic anion transporter that is weak in normal cells, but abundantly expressed in cancer cells, to deliver the anticancer drugs to the cells. METHODS: A human liver complementary DNA (cDNA) library was screened with liver-specific transporter (LST)-1 cDNA as a probe. Northern blot analyses were performed using the isolated cDNA (termed LST-2). An LST-2-specific antibody was raised, and immunohistochemical analyses including immunoelectron microscopy were performed. Xenopus oocyte expression system was used for functional analysis. We also established a permanent cell line that consistently expresses LST-2 to examine the relationship between methotrexate uptake and sensitivity. RESULTS: The isolated cDNA, LST-2, has 79.7% of overall homology with human LST-1. LST-2 exclusively expressed in the liver under normal conditions and its immunoreactivity was highest at the basolateral membrane of the hepatocytes around the central vein. Although its weak expression in the liver, LST-2 is abundantly expressed in the gastric, colon, and pancreatic cancers. On the other hand, the LST-1 was only detected in a hepatic cell line. LST-2 transports methotrexate in a saturable and dose-dependent manner. Furthermore, introduction of the LST-2 gene into mammalian cells potentiates sensitivity to methotrexate. CONCLUSIONS: LST-2 is one of the prime candidate molecules for determining methotrexate sensitivity and may be a good target to deliver anticancer drugs to the gastrointestinal cancers.

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3',5-triiodo-L-thyronine (K(m), 0.9 microM), thyronine, and rT(3) in a Na(+)-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The rat counterpart, oatp-E, also transported 3,3',5-triiodo-L-thyronine. In addition, in this study we revealed that human OATP, which is exclusively expressed in the brain, transported 3,3',5-triiodo-L-thyronine (K(m), 6.5 microM), T(4) (K(m), 8.0 microM), and rT(3). These data suggest that in humans, several different molecules are involved in transporting thyroid hormone: OATP in the brain, LST-1 in the liver, and OATP-E in peripheral tissues.

Functional Kir7.1 channels localized at the root of apical processes in rat retinal pigment epithelium.

1. The inwardly rectifying K+ channel current (IK(IR)) recorded from isolated retinal pigmented epithelial (RPE) cells showed poor dependence on external K+ ([K+]o) and low sensitivity to block by Ba2+. We examined the molecular identity and specific subcellular localization of the KIR channel in RPE cells. 2. The Kir7.1 channel current heterologously expressed in HEK293T cells (human embryonic kidney cell line) showed identical properties to those of the RPE IK(IR), i.e. poor dependence on [K+]o and low sensitivity to Ba2+ block. 3. Expression of Kir7.1 mRNA and protein was detected in RPE cells by RT-PCR and immunoblot techniques, respectively. 4. Immunohistochemical studies including electron microscopy revealed that the Kir7.1 channel was localized specifically at the proximal roots of the apical processes of RPE cells, where Na+,K+-ATPase immunoreactivity was also detected. 5. The middle-distal portions of apical processes of RPE cells in the intact tissue exhibited immunoreactivity of Kir4.1, a common KIR channel. In the isolated RPE cells, however, Kir4.1 immunoreactivity was largely lost, while Kir7.1 immunoreactivity remained. 6. These data indicate that the only IK(IR) recorded in isolated RPE cells is derived from the functional Kir7.1 channel localized at the root of apical processes. Co-localization with Na+,K+-ATPase suggests that the Kir7.1 channel may provide the pathway for recycling of K+ to maintain pump activity and thus is essential for K+ handling in RPE cells.

C-terminal tails of sulfonylurea receptors control ADP-induced activation and diazoxide modulation of ATP-sensitive K(+) channels.

The ATP-sensitive K(+) (K(ATP)) channels are composed of the pore-forming K(+) channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle-type K(ATP) channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K(ATP) channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K(ATP) channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K(ATP) channels containing different SURs.

Molecular identification of a rat novel organic anion transporter moat1, which transports prostaglandin D(2), leukotriene C(4), and taurocholate.

We have isolated a rat novel multispecific organic anion transporter, moat1. The isolated clones were originated by alternative splicing of the moat1 mRNA. The nucleotide sequences predict a protein of 682 amino acids with moderate sequence similarity to LST-1, the oatp family, and the prostaglandin transporter. Northern blot analysis of rat moat1 identified a predominant transcript of 4.4 kilonucleotides in all tissues. Northern blot and in situ hybridization analyses of rat brain further indicated that moat1 mRNA is widely distributed in neuronal cells of the central nervous system, especially in the hippocampus and cerebellum. moat1 transports prostaglandin D(2) (K(m); 35.5 nM), leukotriene C(4) (K(m); 3.2 microM) and taurocholate (K(m); 17.6 microM) in a sodium-independent manner. moat1 also transports prostaglandin E(1), E(2), thromboxane B(2), and iloprost but not dehydroepiandrosterone sulfate and digoxin, of which the substrate specificity is similar, but definitively different from those of any other organic anion transporters.

In vivo formation of a proton-sensitive K+ channel by heteromeric subunit assembly of Kir5.1 with Kir4.1.

Kir5.1 is an inwardly rectifying K+ channel (Kir) subunit, whose physiological function is unknown. Human embryonic kidney HEK293T cells co-transfected with rat Kir5.1 and Kir4.1 cDNA expressed a functional K+ channel, whose properties were significantly different from those of the homomeric Kir4.1 channel. Formation of a Kir4. 1/Kir5.1 assembly in HEK293T was confirmed biochemically. We found that heteromeric Kir4.1/Kir5.1 channel activity was affected by internal pH levels between 6.0 and 8.0, when the homomeric Kir4.1 channel activity was relatively stable. Changing external pH in this range had no effect on either Kir channel. Western blot analysis using specific antibodies revealed that Kir4.1 and Kir5.1 proteins were expressed in kidney and brain, but co-immunoprecipitated only from kidney. These results indicate that the co-assembly of Kir5.1 with Kir4.1 occurs in vivo, at least in kidney. The heteromeric Kir4. 1/Kir5.1 channel may therefore sense intracellular pH in renal epithelium and be involved in the regulation of acid-base homeostasis.

Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevis oocyte.

Recent studies showed that coexpression of Kir6.1 or Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B) reconstituted an inwardly rectifying, ATP-sensitive K(+) channel that was inhibited by glibenclamide (2, 15-17). Here we report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B) from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp open reading frame that encodes a 1,545-amino acid polypeptide, showing 67% shared identity with SUR1 (a pancreatic beta-cell isoform) and 98% with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle isoform). Consistent with the predicted structures of other members of the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B contains 17 putative membrane-spanning segments. Also, predicted Walker A and B consensus binding motifs, present in other ABC members, are conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the rSUR2B transcript was investigated using RT-PCR and Southern blot of microdissected tubules. The rSUR2B transcript was detected in proximal tubule, cortical thick ascending limb, distal collecting tubule, cortical collecting duct, and outer medullary collecting duct, but not medullary thick ascending limb. This distal distribution overlaps with that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in Xenopus laevis oocytes resulted in whole cell Ba(2+)-sensitive K(+) currents that were inhibited by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In contrast, rSUR2B did not confer significant glibenclamide sensitivity to oocytes coinjected with ROMK1 or ROMK3. The interaction between ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in vitro translated rSUR2B and ROMK2. In agreement with the functional data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but not ROMK1 and ROMK3, can interact with rSUR2B to confer a sulfonylurea-sensitive K(+) channel, implicating SUR proteins in forming and regulating renal ATP-sensitive K(+) channels. The ROMK isoform specificity of glibenclamide effects suggests that the NH(2) terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions.

Anchoring proteins confer G protein sensitivity to an inward-rectifier K(+) channel through the GK domain.

Anchoring proteins cluster receptors and ion channels at postsynaptic membranes in the brain. They also act as scaffolds for intracellular signaling molecules including synGAP and NO synthase. Here we report a new function for intracellular anchoring proteins: the regulation of synaptic ion channel function. A neuronal G protein-gated inwardly rectifying K(+) channel, Kir3.2c, can not be activated either by M(2)-muscarinic receptor stimulation or by G(betagamma) overexpression. When coexpressed with SAP97, a member of the PSD/SAP anchoring protein family, the channel became sensitive to G protein stimulation. Although the C-terminus of Kir3. 2c bound to the second PDZ domain of SAP97, functional analyses revealed that the guanylate kinase (GK) domain of SAP97 is crucial for sensitization of the Kir3.2c channel to G protein stimulation. Furthermore, SAPAP1/GKAP, which binds specifically to the GK domain of membrane-associated guanylate kinases, prevented the SAP97-induced sensitization. The function of a synaptic ion channel can therefore be controlled by a network of various intracellular proteins.

Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis.

1. One of the features of weaver mutant mice is male infertility, which suggests that Kir3.2, a G-protein-gated inwardly rectifying K+ channel subunit, may be involved in spermatogenesis. Therefore, we have characterized the Kir3.2 isoform in mouse testis using immunological, molecular biological and electrophysiological techniques. 2. Testicular membrane contained a protein that was recognized by the antibody specific to the C-terminus of Kir3.2c (aG2C-3). Its molecular mass was approximately 45 kDa, which was smaller than that of Kir3.2c ( approximately 48 kDa). The immunoprecipitant obtained from testis with aG2C-3 contained a single band of the 45 kDa protein, which could not be detected by the antibody to the N-terminus common to the known Kir3.2 isoforms (aG2N-2). 3. A novel alternative splicing variant of Kir3.2, designated Kir3.2d, was isolated from a mouse testis cDNA library. The cDNA had an open reading frame encoding 407 amino acids, whose molecular mass was calculated to be approximately 45 kDa. Kir3.2d was 18 amino acids shorter than Kir3.2c at its N-terminal end, which was the only difference between the two clones. The 18 amino acid region possesses the epitope for aG2N-2. 4. In heterologous expression systems of both Xenopus oocytes and mammalian cells (HEK 293T), Kir3.2d either alone or with Kir3.1 exhibited G-protein-gated inwardly rectifying K+ channel activity. 5. Prominent Kir3.2d immunoreactivity in the testis was detected exclusively in the acrosomal vesicles of spermatids, while Kir3.1 immunoreactivity was diffuse in the spermatogonia and spermatocytes. These results indicate the possibility that the testicular variant of Kir3.2, Kir3. 2d, may assemble to form a homomultimeric G-protein-gated K+ channel and be involved in the development of the acrosome during spermiogenesis.

Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2.

Two complementary DNAs for the organic anion transporter subtypes oatp2 and oatp3, which transport thyroid hormones as well as taurocholate, were isolated from a rat retina cDNA library. The sequence of oatp2 is identical to that recently reported (Noé, B., Hagenbuch, B., Stieger, B., and Meier, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10346-10350), whereas the sequence of oatp3 is novel. oatp3 consists of 670 amino acid residues and exhibits a structural architecture common to the organic anion transporter family, possessing the 12 putative membrane-spanning segments. Oocytes injected with oatp2 and oatp3 cRNAs showed taurocholate uptake in a saturable manner. The oatp2 and oatp3 cRNA-injected oocytes also showed significant uptake of both thyroxine and triiodothyronine. Northern blot and in situ analyses showed that the oatp2 mRNA was widely expressed in neuronal cells of the central nervous system, especially in the hippocampus, cerebellum, and choroid plexus as well as in the retina and liver. The oatp3 mRNA was highly expressed in the kidney and moderately abundant in the retina. This suggests that oatp2 and oatp3 are multifunctional transporters involved in the transport of thyroid hormones in the brain, retina, liver, and kidney.

Myeloperoxidase-antineutrophil cytoplasmic antibody-positive crescentic glomerulonephritis complicating the course of Graves' disease: report of three adult cases.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis has been recently recognized in Graves' disease patients treated with propylthiouracil. We have experienced three adult cases of Graves' disease with main features being renal derangements. All three patients, who were between the ages of 22 and 82 years, had been treated with propylthiouracil for 2 to 5 years after a diagnosis of Graves' disease. After several weeks of upper respiratory tract infection or flu-like symptoms, they abruptly began to manifest proteinuria and hematuria concomitant with severe anemia. Their serum creatinine increased from normal levels to 1.2 to 3.6 mg/dL. Renal biopsy revealed crescentic glomerulonephritis without deposition of immune complexes (ie, pauci-immune type). Crescent formations were observed in 40% to 60% of the glomeruli in all three cases. The serum from the patients revealed positive perinuclear-ANCA and negative cytoplasmic-ANCA (C-ANCA) pattern, and myeloperoxidase (MPO)-ANCA titers were 120 to 502 ELISA Units/mL (normal, < 10 ELISA Units/mL). A withdrawal of propylthiouracil with or without immunosuppressive therapy ameliorated their renal derangements. Graves' disease patients should be placed under vigilant observation by monitoring their urinalysis and serum creatinine, especially when being treated with antithyroid drugs and when suffering from flu-like symptoms.

Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T cells.

The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-gamma 1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.