Expression of a human renal sodium nucleoside cotransporter in Xenopus laevis oocytes

In this study, Xenopus laevis oocytes injected with poly(A)+ RNA (mRNA) isolated from human kidney were used to express a Na(+)-nucleoside cotransporter. Na(+)-stimulated [3H]thymidine uptake was enhanced 2-3-fold in oocytes injected with 50 ng poly(A)+ RNA and 4-5-fold in oocytes injected with 20 ng of a size-fractionated human renal cortex mRNA fragment (2-3 kb) in comparison with water-injected oocytes. Na(+)-dependent thymidine uptake in oocytes injected with the 2-3 kb mRNA fragment was inhibited significantly by thymidine and guanosine but not by formycin B, consistent with the N4 Na(+)-nucleoside cotransporter. The Km (28 microM) of Na(+)-dependent thymidine uptake in the oocytes injected with the 2-3 kb mRNA fragment was similar to the Km (27 microM) of Na(+)-dependent thymidine uptake obtained in human renal brush border membrane vesicles. These data suggest for the first time that a Na(+)-nucleoside cotransporter from human kidney can be expressed in X. laevis oocytes.     

Comparative analysis of mammalian stanniocalcin genes

The recent discovery of mammalian stanniocalcin (STC) prompted an investigation of its gene structure and expression pattern to study its function and regulation. We show that both the human and mouse genes are composed of four exons spanning about 13 kb, with 85% nucleotide sequence identity in coding regions. Remarkably high sequence conservation between species also exists in the approximately 3-kb 3'-untranslated region. Comparative analysis of the 5'-untranslated region and flanking DNA from the rat and human STC genes showed long stretches of CAG trinucleotide repeats and an additional (CA)25 dinucleotide repeat unique to the rat promoter. An analysis of STC expression in the mouse showed that ovary contained the highest level of messenger RNA, with lower, but detectable, levels in most tissues. In situ hybridization revealed strong, specific hybridization over the thecal-interstitial cells of the ovarian stroma, whereas immunohistochemical analysis indicated that STC was present not only in the stroma, but also in the corpora lutea and oocyte of the developing follicle. Consequently, STC may act as a signaling molecule between the thecal-interstitial cell compartment and the corpus luteum and oocyte, thereby regulating the activity of these structures in some way. These findings suggest that in addition to its role in mineral metabolism, STC has acquired an important function in reproduction during its evolution to mammals.     

Dopamine regulates phosphate uptake by opossum kidney cells through multiple counter-regulatory receptors

The purpose of this study was to determine the mechanisms of dopamine regulation of phosphate uptake in opossum kidney (OK) cells, a model of proximal renal tubules. Dopamine stimulated cAMP generation and inhibited radiolabeled phosphate uptake into OK cell monolayers by 14.4 +/- 1.8%. The effect of dopamine was transient, as phosphate uptake returned toward control level by 3 h despite the continued presence of dopamine. Pretreatment with pertussis toxin increased dopamine inhibition of phosphate uptake to 25 +/- 3%, increased the duration of the dopamine effect to at least 3 h, and enhanced cAMP generation. In an OK cell clone that overexpressed cAMP phosphodiesterase, dopamine did not inhibit phosphate uptake, but pharmacologic inhibition of protein kinase A activation did not prevent dopamine inhibition of phosphate uptake. A DA1 receptor agonist inhibited phosphate uptake more potently than dopamine (29.5 +/- 1.1%) or a DA2 receptor agonist (7.9 +/- 2%). However, both DA1 and DA2 receptor antagonists completely blocked dopamine inhibition of phosphate uptake. DA1, but not the DA2, antagonists blocked dopamine-stimulated cAMP generation. Treatment with alpha-adrenergic receptor antagonists potentiated dopamine inhibition of phosphate uptake to the same extent as pertussis toxin and was not additive with pertussis toxin. It is concluded that dopamine inhibits phosphate uptake through DA1 and DA2 receptor stimulation by cAMP-dependent and -independent pathways and activates a pertussis toxin-sensitive counter-regulatory pathway that attenuates this response through alpha-adrenergic receptor stimulation.     

Heavy metals mercury, cadmium, and chromium inhibit the activity of the mammalian liver and kidney sulfate transporter sat-1

Heavy metal intoxication leads to defects in cellular uptake mechanisms in the mammalian liver and kidney. We have studied the effects of several heavy metals, including mercury, lead, cadmium, and chromium (at concentrations of 1 to 1000 microM), on the activity of the mammalian sulfate transporter sat-1(2) in Xenopus oocytes. sat-1 encodes a sulfate/bicarbonate anion exchanger expressed in the rat liver and kidney. Mercury (10 microM) strongly inhibited sat-1 transport by reducing Vmax by eightfold but not its Km for inorganic sulfate (Si). Lead (up to 1 mM) was unable to significantly inhibit sat-1 transporter activity. Cadmium (500 microM) showed weak inhibition of sat-1 transport by decreasing only sat-1 Vmax. Chromium (100 microM) strongly inhibited sat-1 transport by reducing Km for Si by sevenfold, most probably by binding to the Si site, due to the strong structural similarity between the CrO2-4 and SO2-4 substrates. This study presents the first characterization of heavy metal inhibition of the hepatic and renal sulfate/bicarbonate transporter sat-1, through various mechanisms, which may lead to sulfaturia following heavy metal intoxication.     

Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine

Recently, we cloned the human cation transporter hOCT2, a member of a new family of polyspecific transporters from kidney, and demonstrated electrogenic uptake of tetraethylammonium, choline, N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. Using polymerase chain reaction amplification, cDNA sequencing, in situ hybridization, and immunohistochemistry, we now show that hOCT2 message and protein are expressed in neurons of the cerebral cortex and in various subcortical nuclei. In Xenopus laevis oocytes expressing hOCT2, electrogenic transport of norepinephrine, histamine, dopamine, serotonin, and the antiparkinsonian drugs memantine and amantadine was demonstrated by tracer influx, tracer efflux, electrical measurements, or a combination. Apparent Km values of 1.9 +/- 0.6 mM (norepinephrine), 1.3 +/- 0.3 mM (histamine), 0.39 +/- 0.16 mM (dopamine), 80 +/- 20 microM (serotonin), 34 +/- 5 microM (memantine), and 27 +/- 3 microM (amantadine) were estimated. Measurement of trans-effects in depolarized oocytes and human embryonic kidney cells expressing hOCT2 suggests that there were different rates and specificities for cation influx and efflux. The hypothesis is raised that hOCT2 plays a physiological role in the central nervous system by regulating interstitial concentrations of monoamine neurotransmitters that have evaded high affinity uptake mechanisms. We show that amantadine does not interact with the expressed human Na+/Cl- dopamine cotransporter. However, concentrations of amantadine that are effective for the treatment of Parkinson's disease may increase the interstitial concentrations of dopamine and other aminergic neurotransmitters by competitive inhibition of hOCT2.     

Physiological effects of medetomidine-zolazepam-tiletamine immobilization in black bears

Proteasomes are nonlysosomal multicatalytic proteases involved in antigen processing. Three of the 10 mammalian proteasome beta subunits (LMP2, LMP7, and LMP10) are induced by IFN-gamma. Two of these (LMP2 and LMP7) are encoded in the major histocompatibility complex of both human (chromosome 6) and mouse (chromosome 17). However, the human homologue of Lmp10, MECL1, is found on chromosome 16. Here we show that in mice, Lmp10 is a single-copy gene localized to chromosome 8, in a region of conserved synteny with human chromosome 16. Sequencing of a 129/SvJ strain genomic clone revealed that the gene has eight exons spanning 2.3 kb. Characterization of a full-length mouse cDNA clone indicates that Lmp10 encodes a protein of 273 amino acids with a calculated molecular weight of 29 kDa and an isoelectric point of 6.86. Northern analysis of Lmp2, Lmp7, and Lmp10 showed expression in heart, liver, thymus, lung, and spleen, but not in brain, kidney, skeletal muscle, or testis.     

DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate

Biosynthesis of glycosylphosphatidylinositol and N-glycan precursor is dependent upon a mannosyl donor, dolichol phosphate-mannose (DPM). The Thy-1negative class E mutant of mouse lymphoma and Lec15 mutant Chinese hamster ovary (CHO) cells are incapable of DPM synthesis. The class E mutant is defective in the DPM1 gene which encodes a mammalian homologue of Saccharomyces cerevisiae Dpm1p that is a DPM synthase, whereas Lec15 is a different mutant, indicating that mammalian DPM1 is not sufficient for DPM synthesis. Here we report expression cloning of a new gene, DPM2, which is defective in Lec15 cells. DPM2, an 84 amino acid membrane protein expressed in the endoplasmic reticulum (ER), makes a complex with DPM1 that is essential for the ER localization and stable expression of DPM1. Moreover, DPM2 enhances binding of dolichol phosphate, a substrate of DPM synthase. Mammalian DPM1 is catalytic because a fusion protein of DPM1 that was stably expressed in the ER synthesized DPM without DPM2. Therefore, biosynthesis of DPM in mammalian cells is regulated by DPM2.     

In vivo suppression of the renal Na+/Pi cotransporter by antisense oligonucleotides

A 20-mer phosphorothioate oligonucleotide (AS1) was designed to hybridize to the message for the rat kidney sodium phosphate cotransporter NaPi-2 close to the translation initiation site. Single intravenous doses of this oligonucleotide were given to rats maintained on a low phosphorus diet to increase NaPi-2 expression. At 3 days after oligonucleotide infusion, rats receiving 2.5 micromol of AS1 exhibited a reduction in renal NaPi-2 to cyclophilin mRNA ratio by 40% +/- 17%, and rats receiving 7.5 micromol of AS1 exhibited a reduction in NaPi-2 to cyclophilin mRNA ratio by 46% +/- 21%. Reversed-sequence AS1 was without effect. The higher dose of 7.5 micromol of AS1 also reduced the rate of phosphate uptake into renal brush border membrane vesicles and the expression of NaPi-2 protein detected by Western blotting in these vesicles. Reversed sequence AS1 was again without effect on these parameters. These results suggest that systemically infused oligonucleotides can exert antisense effects in the renal proximal tubule.     

Preparation of a monoclonal antibody specific for human stanniocalcin

Stanniocalcin (STC) is a glycoprotein hormone that was first identified in fish, where it regulates the calcium level in the body fluid. The cDNA which encodes human STC has recently been reported but the function has not been completely elucidated. We have prepared a monoclonal antibody against human STC using an analogous peptide of the putative antigenic domain in human STC; it was conjugated with keyhole limpet hemocyanin (KLH). The monoclonal antibody specifically stained the distal convoluted tubules in human kidney which is a putative target organ of STC. The ELISA was established using the monoclonal antibody and recombinant human STC as a standard antigen. The monoclonal antibody prepared in this study provides a useful tool for clinical studies of STC in human.     

Expression of an antigen homologous to the human CO17-1A/GA733 colon cancer antigen in animal tissues

The CO17-1A/GA733 antigen is associated with human carcinomas and some normal epithelial tissues. This antigen has shown promise as a target in approaches to passive and active immunotherapy of colorectal cancer. The relevance of animal models for studies of immunotherapy targeting this antigen in patients is dependent on the expression of the antigen on normal animal tissues. Immunohistoperoxidase staining with polyclonal rabbit antibodies to the human antigen revealed the human homologue on normal small intestine, colon and liver of mice, rats and non-human primates, whereas mouse monoclonal antibodies to the CO17-1A or GA733 epitopes on the human antigen did not detect the antigen. Polyclonal rabbit antibodies, elicited by the murine antigen homologue derived from recombinant baculovirus-infected insect cells, immunoprecipitated the antigen from mouse small intestine, colon, stomach, kidney and lung. The isolated recombinant murine protein bound polyclonal, but not monoclonal, antibodies to the human CO17-1A/GA733 antigen, and recombinant human antigen bound polyclonal antibodies elicited by the murine antigen homologue. Thus, the antigen homologue expressed by animal tissues is similar, but not identical, to the human antigen. These results have important implications for experimental active and passive immunotherapy targeting the CO17-1A/GA733 antigen.     

Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems

Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability.     

Adrenal tumors and pregnancy

The lats gene has been identified as a tumour suppressor in Drosophila melanogaster using mosaic screens. Mosaic flies carrying somatic cells that are mutant for lats develop large tumours in many organs. The human LATS1 homologue rescues embryonic lethality and inhibits tumour growth in lats mutant flies, demonstrating the functional conservation of this gene. Biochemical and genetic analyses have revealed that LATS1 functions as a negative regulator of CDC2 (ref. 3). These data suggest that mammalian LATS1 may have a role in tumorigenesis. To elucidate the function of mammalian LATS1, we have generated Lats1-/- mice. Lats1-/- animals exhibit a lack of mammary gland development, infertility and growth retardation. Accompanying these defects are hyperplastic changes in the pituitary and decreased serum hormone levels. The reproductive hormone defects of Lats1-/- mice are reminiscent of isolated LH-hypogonadotropic hypogonadism and corpus luteum insufficiency in humans. Furthermore, Lats1-/- mice develop soft-tissue sarcomas and ovarian stromal cell tumours and are highly sensitive to carcinogenic treatments. Our data demonstrate a role for Lats1 in mammalian tumorigenesis and specific endocrine dysfunction.     

hPepT1-mediated epithelial transport of bacteria-derived chemotactic peptides enhances neutrophil-epithelial interactions

Intestinal epithelial cells express hPepT1, an apical transporter responsible for the uptake of a broad array of small peptides. As these could conceivably include n-formyl peptides, we examined whether hPepT1 could transport the model n-formylated peptide fMLP and, if so, whether such cellular uptake of fMLP influenced neutrophil-epithelial interactions. fMLP uptake into oocytes was enhanced by hPepT1 expression. In addition, fMLP competitively inhibited uptake of a known hPepT1 substrate (glycylsarcosine) in hPepT1 expressing oocytes. hPepT1 peptide uptake was further examined in a polarized human intestinal epithelial cell line (Caco2-BBE) known to express this transporter. Epithelial monolayers internalized apical fMLP in a fashion that was competitively inhibited by other hPepT1 recognized solutes, but not by related solutes that were not transported by hPepT1. Fluorescence analyses of intracellular pH revealed that fMLP uptake was accompanied by cytosolic acidification, consistent with the known function of hPepT1 as a peptide H+ cotransporter. Lumenal fMLP resulted in directed movement of neutrophils across epithelial monolayers. Solutes that inhibit hPepT1-mediated fMLP transport decreased neutrophil transmigration by approximately 50%. Conversely, conditions that enhanced the rate of hPepT1-mediated fMLP uptake (cytosolic acidification) enhanced neutrophil-transepithelial migration by approximately 70%. We conclude that hPepT1 transports fMLP and uptake of these peptide influences neutrophil-epithelial interactions. These data (a) emphasize the importance of hPepT1 in mediating intestinal inflammation, (b) raise the possibility that modulating hPepT1 activity could influence states of intestinal inflammation, and (c) provide the first evidence of a link between active transepithelial transport and neutrophil-epithelial interactions.     

Role of aquaporin water channels in kidney and lung

Several aquaporin-type water channels are expressed in mammalian kidney and lung: AQP1 in lung microvessels and kidney proximal tubule, thin descending limb of Henle, and vasa recta; AQP2 in apical membrane of collecting duct epithelium; AQP3 and AQP4 in basolateral membranes of airway and collecting duct epithelium; and AQP5 in alveolar epithelium. Novel quantitative fluorescence methods demonstrated very high water permeabilities of the alveolar epithelial and endothelial barriers, and moderately high water permeability across distal airways. In the kidney, water permeability is high in proximal tubule and thin descending limb of Henle, and regulated by vasopressin in collecting duct. The author's laboratory has studied the role of aquaporins in organ physiology using transgenic knockout mice lacking specific aquaporins. AQP1 null mice are mildly growth-retarded, manifest a severe urinary concentrating defect, and have reduced water permeability between airspace and capillary compartments. AQP4 null mice appear normal grossly except for a mild defect in maximum urinary concentrating ability. AQP2-deficient humans have hereditary non-X-linked nephrogenic diabetes insipidus (NDI). In transfected mammalian cells, many NDI-causing AQP2 mutants are retained in the endoplasmic reticulum. The author's laboratory has found that "chemical chaperones," that is, small compounds that promote protein folding in vitro, are able to correct defective AQP2 trafficking in cell culture models. The transgenic mouse and mammalian cell models are thus beginning to provide clues about the role of aquaporins in normal physiology and disease.     

Characterization of lineage restricted forms of a Xenopus CD45 homologue

The leukocyte common antigen, also known as CD45, is a structurally heterogenous molecule ranging in molecular weight from 180 to 220 kDa. CD45 belongs to a family of high molecular weight, cell surface glycoproteins expressed on all hematopoietic lineages with the exception of mature erythrocytes. In higher vertebrates, the highly conserved cytoplasmic domain of CD45 exhibits protein tyrosine phosphatase activity and has been implicated in lymphocyte activation through dephosphorylation of critical tyrosine residues on substrates associated with signal transduction pathways. The monoclonal antibody CL21 recognizes a high molecular weight determinant expressed on the surface of Xenopus leukocytes which was postulated to be a CD45 homologue. In order to determine if lymphocyte subpopulations expressed different molecular weight variants, splenic B cells were identified and isolated on the basis of surface IgM and the CL21 determinant expressed by these cells was compared to the determinant expressed by thymocytes. Immunoprecipitation revealed that IgM + B cells expressed a 220 kDa molecular weight variant whereas thymocytes and IgM-cells expressed a 180 kDa variant. Bone marrow myeloid cells, isolated on the basis of light scatter properties, expressed a determinant which ranged from 150 to 160 kDa. Dephosphorylation experiments utilizing p-nitrophenyl phosphate, 32P-labeled Raytide [tyr(P)], or Kemptide [ser(P)] as substrates demonstrated that immunoprecipitated CL21 antigen exhibited tyrosine specific phosphatase activity which was inhibited by sodium orthovanadate. Thus, data based on the presence of enzymatic activity and lineage restricted molecular weight variants support the hypothesis that the CL21 determinant is the amphibian homologue of mammalian CD45, and suggest that both structural and functional elements of CD45 have been conserved during vertebrate evolution.