Andrea M. Budreau

Adaptive regulation of MDCK cell taurine transporter (pNCT) mRNA: transcription of pNCT gene is regulated by external taurine concentration.

NaCl-dependent taurine transporter (pNCT) activity of MDCK cells (Madin-Darby canine kidney) is up- or down-regulated by medium taurine manipulation. In this study we found that the abundance of pNCT mRNA was up- or down-regulated after cells were incubated in media containing 0 microM taurine or 500 microM taurine for 24 h. Down-regulation was observed after 12 h exposure to high taurine (500 microM) and mRNA abundance was appreciably reduced after 72 h exposure. Nuclear run-off assays show that the gene for pNCT is induced at the transcriptional level by taurine. Addition of cycloheximide blocked the adaptive response and reduced transcription of pNCT mRNA in MDCK cells. Cycloheximide had virtually no effect on pNCT mRNA stability, suggesting that ongoing protein synthesis is required for adaptive regulation of pNCT gene transcription.

Molecular cloning and functional expression of an LLC-PK1 cell taurine transporter that is adaptively regulated by taurine

Studies have shown that the renal tubular epithelium adapts to alterations in the sulfur amino acid composition of the diet. The renal adaptive response has been described in man, mouse, rat, dog, and pig. The observed phenomenon involves increased or decreased initial rate activity of the NaCl-dependent taurine transporter at the brush border membrane surface of the proximal tubule following dietary manipulation of taurine. A cDNA encoding a taurine transporter has been isolated from LLC-PK1 cells, designated pTAUT, and its functional properties have been examined in Xenopus laevis oocytes. The nucleotide sequence of the clone predicts a 621-amino acid protein with about 90% homology to other cloned taurine transporter cDNAs. When expressed in oocytes the transporter displays a Km of 25 microM and is dependent on the presence of external sodium and chloride, characteristics similar to taurine uptake by LLC-PK1 cells. The abundance of pTAUT mRNA and protein were up-regulated in cells cultured in taurine-free medium as compared with cells cultured in medium containing 500 microM taurine. Activation of PKC by PMA had no effect on adaptive regulation of pTAUT mRNA and protein, indicating that down-regulation of LLC-PK1 cell taurine transport activity by PMA occurs at the post-translational level.

Characteristics of Taurine Transport in Cultured Renal Epithelial Cell Lines: Asymmetric Polarity of Proximal and Distal Cell Lines

Taurine transport was determined in two continuous, renal epithelial cell lines: LLC-PK1 derived from the proximal tubule of the pig, and the Madin-Darby canine kidney cell (MDCK) from the distal tubule of the dog. In LLC-PK1, taurine transport is maximal at the apical surface, whereas in MDCK cells, transport is greatest at the basolateral surface. Transport is highly dependent on both sodium and chloride in the external medium, and is specific for beta-amino acids. The apical and basolateral surfaces of both cell lines show an adaptive response to extracellular taurine concentration, but only the basolateral surface of the MDCK cell responds to hyperosomolality by increased taurine accumulation. Thus, differential control of the beta-amino acid transport system by substrate and external tonicity exists. The role of the beta-amino acid transport system may differ according to the origin of the cell: in the proximal renal tubular cell, net transepithelial reabsorption of filtered taurine increases the body pool. By contrast, taurine accumulation by distal tubular cells may form a mechanism of cell volume regulation in response to osmotic stress.

Regulation of Expression of Taurine Transport in Two Continuous Renal Epithelial Cell Lines and Inhibition of Taurine Transporter by a Site-Directed Antibody

UNLABELLED The renal tubular epithelium adapts to changes in the sulfur amino acid composition of the diet, particularly in terms of reabsorption of taurine. The adaptive response is expressed by enhanced or decreased NaCl-dependent taurine transport by rat renal brush border membrane vesicles (BBMV). Taurine transport activity in two cultured renal epithelial cell lines (MDCK and LLC-PK1) is up- or down-regulated by extracellular taurine concentration as the result of reciprocal changes in the Vmax of the transporter. In MDCK cells, abundance of taurine transporter mRNA (pNCT mRNA) was up- or down-regulated after incubation in media containing 0, 50, or 500 microM taurine. Decreased mRNA was observed in both cell lines after 12 h, and it was appreciably reduced after 72 h exposure to 500 microM taurine. Northern blot analysis of mRNA from LLC-PK1 cells using pNCT cDNA as a riboprobe showed that two transcripts, 9.6 kb and 7.2 kb, were expressed; the abundance of mRNA was increased or decreased after incubation in taurine-free or high taurine medium, respectively. Down-regulation was observed primarily in the 7.2 kb transcript after 24 h incubation. Rapid up-regulation occurred in the 9.6 kb transcript within 12 h of transfer from high to low taurine. Nuclear run-off assays showed that the gene for pNCT is induced at the transcriptional level by taurine. Regulation of expression of the taurine transporter was also studied by injection of pNCT cRNA into Xenopus laevis oocytes. Expression of transport activity was significantly reduced (64%) when oocytes were incubated in 50 microM taurine as compared to 0 microM taurine. Transport activity was totally blocked when pNCT cRNA-injected oocytes were exposed to an active phorbol ester, PMA (10(-6) M). Inhibition of uptake was reversed by staurosporine, an inhibitor of protein kinase C activity. An inactive phorbol ester, 4 alpha-phorbol, had no effect on taurine transport. A polyclonal antibody directed a highly conserved intracellular segment between homologous transmembrane domains VI and VII inhibited taurine transport activity in both pNCT cRNA-injected oocytes and BBMV. Incubation of oocytes with 10 micrograms/ml antibody (Ab) reduced taurine uptake to 46% of control, and 20-80 micrograms/ml Ab reduced uptake to 20% of control. In BBMV, active taurine uptake (10 microM) was inhibited approximately 30% by 10 pg Ab/mg protein, whereas none specific IgG had no significant effect. Proline uptake (20 microM) by BBMV was not inhibited by the Ab, nor was GABA uptake (50 microM). Two pNCT proteins, approximately 70 kD and approximately 30 kD, were detected by Western blot, and the abundance of both was regulated by medium taurine. IN CONCLUSION (i) regulation of taurine transport activity in LLC-PK1 cells by medium taurine occurs at a level of mRNA transcription; (ii) regulation of pNCT occurs at both transcriptional and translational levels; (iii) pNCT expression is regulated by protein kinase C-dependent phosphorylation; and (iv) the intracellular segment between domains VI and VII may be required for activation of the taurine transporter; this segment may function as a gate in taurine transport.

Does the Taurine Transporter Gene Play a Role in 3p-Syndrome?

Taurine has been shown to be essential for the development and survival of mammalian cells, especially cells of the cerebellum and retina18. Notably, taurine concentration reaches the mM range in the CNS20. The highest intracellular concentration of taurine is found in neonatal and early postnatal brain, suggesting a developmental role. Sturmans group found that the surviving F1 offspring of taurine-deficient female cats have a large number of neurologic defects, including degeneration of retinal pigmented epithelium and ocular tapetum, delayed cerebellar granule cell division and migration, and abnormal cerebral cortical development. Taurine also appears to optimize the proliferation and differentiation of human fetal cerebral brain cells in culture. Addition of taurine to the medium improves neuron growth, neurite expansion, and neuronal survival2. Taurine is considered a conditionally essential amino acid in man, as the enzymatic activity of cysteine sulfonic acid decarboxylase (CSAD), which catalyzes the formation of taurine from cysteine, is low in humans, and even lower or absent in term and preterm infant liver28. This ontogeny of catalytic enzyme activity is also true in other primates21. In children with short gut syndrome, taurine is an essential component of total parenteral nutrition (TPN) solutions to prevent a defined retinopathy, which is reversed only by taurine administration. Human milk also contains high concentrations of taurine11. We have shown previously that taurine is a semi-conditionally essential amino acid in very low birth weight premature infants, especially those who cannot ingest food and are obliged to be fed by TPN28. Plasma taurine values in preterm infants fall to almost non-detectable values,

IDENTIFICATION OF SER-322 AS SPECIFIC SITE IN TAURINE TRANSPORTER (pNCT) THAT IS PHOSPHORYLATED BY PKC • 1657

IDENTIFICATION OF SER-322 AS SPECIFIC SITE IN TAURINE TRANSPORTER (pNCT) THAT IS PHOSPHORYLATED BY PKC • 1657

Molecular Cloning of Promoter Region of Taurine Transporter Gene That Is Regulated by p53 Tumor Suppressor Gene

Molecular Cloning of Promoter Region of Taurine Transporter Gene That Is Regulated by p53 Tumor Suppressor Gene

Cloning and Characterization of the Promoter Region of the Rat Taurine Transporter Gene: Transcriptional Regulation by Taurine and Estradiol 1809

Expression of the renal taurine transporter gene is regulated by intracellular taurine content. In certain cell lines, transcription is regulated by taurine availability. We attempted to define the nature of the signal for this adaptive response. A clone containing a segment of the 5′- upstream region of rat taurine transporter genomic DNA was obtained by screening a P1 library using PCR probes derived from rat brain taurine transporter cDNA (rB16a). The DNA (≈1.6 kb pairs) was isolated and characterized. The 5′-flanking region of the gene contains typical eukaryotic promoter elements, including a TATA box, four consensus binding sites for SP1, and two estrogen receptor half-site sequences (AGGTCA). The DNA fragment was also analyzed by transient transfection. When linke to a firefly luciferase reporter gene, it enhanced transcription 100-fold in two renal cell lines, and was influenced by the presence or absence of taurine. Promoter activity was also enhanced by treatment with β-17 estradiol in human breast cancer cells, and in rat cardiomyocytes, but not in renal cells.

ROLE OF CONSERVED PEPTIDE IN TAURINE TRANSPORTER INACTIVATION MODULATED BY PROTEIN KINASE C. † 2153

Activation of protein kinase C (PKC) by the active phorbol ester 12-myristate 13-acetate (PMA, 100 nM) or phorbol-12, 13-dibutyrate (100 nM) reduced taurine uptake by 80% in oocytes given injections with cRNA from and expressing the Madin-Darby canine kidney cell taurine transporter pNCT. Inhibition of PKC by calphostin C or staurosporine increased taurine uptake by 20% and 400%, respectively. The inhibitory effect of PMA on taurine uptake was blocked by calphostin C, a specific inhibitor of PKC. Modulation by PMA mainly affected the apparent affinity K(m) (from 5.6 microM to 18.1 microM) with minimal effect on the maximal velocity (25% decrease) of the transporter. A polyclonal antibody (AbS4) directed against a conserved intracellular segment (S4) of the Madin-Darby canine kidney cell taurine transporter enhanced taurine uptake by pNCT cRNA-treated oocytes. The effect of AbS4 was blocked by incubation with the corresponding peptide antigen. Preimmune IgG and peptide antigen had no effect on taurine transporter activity expressed in oocytes. Modulation seemed to occur through phosphorylation of a consensus PKC site located on segment S4 of the transporter, because downregulation of the transporter by PMA (100 nM) was abolished by preinjection of AbS4 (12 ng/ oocyte). In contrast, downregulation of the transporter by PMA could not be restored by AbS4 when pNCT-expressing oocytes were pretreated with PMA (50 nM). In conclusion, the peptide segment recognized by this antibody appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation.