Taurine Transporter Activity Research Articles

Hypertonicity stimulates taurine uptake and transporter gene expression in Caco-2 cells

The osmoregulation of taurine transport in intestinal epithelial cells was investigated using human intestinal Caco-2 cells. The activity of taurine transport in the Caco-2 cells was increased by hypertonic conditions. This hypertonicity-induced up-regulation was dependent on both the culturing time and the osmotic pressure. Hypertonicity did not affect the activity of l-leucine, l-lysine, or l-glutamic acid transport, suggesting that osmoregulation was specific to taurine transport. The intracellular taurine content of Caco-2 cells was also increased by culturing in a hypertonic medium. These hypertonicity-induced changes in the intracellular taurine content and transport activity were reversible. A kinetic analysis of taurine transport in the control and hypertonic cells suggested that the up-regulation was associated with an increase in the amount of the taurine transporter. The mRNA level of the taurine transporter in hypertonic cells was markedly higher than that in the control cells, indicating that this osmotic regulation was due to the increased expression of the taurine transporter gene.

Effects of steroid hormones and cyclosporine A on taurine-transporter activity in the RAW264.7 cell line.

The activity of the taurine transporter is affected by various extracellular stimuli, such as ions, hormones and stress. To assess the effects of steroid hormones and cyclosporine A (CsA) on taurine-transporter activity, the murine monocytic cell line, RAW264.7, was stimulated with dexamethasone (DM), triamcinolone (TA), cortisone (CS), hydrocortisone (HCS), prednisone (PSN), prednisolone (PSL) and methylprednisolone (MPSL) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of the cell with TPA led to a significant reduction in taurine-transporter activity. However, in the case of the stimulation of the cells with steroid hormones in the presence of TPA, all of the hormones reversed the TPA-induced reduction in the taurine-transporter activity. Treatment of the cells with CsA led to a significant reduction in taurine-transporter activity, but ionomycin (IM) alone did not affect taurine-transporter activity. However, IM reversed the TPA- and CsA-induced reduction in taurine-transporter activity. These results showed that both IM and the glucocorticoid hormones reversed TPA-induced reductions in taurine-transporter activity but only IM reversed the CsA-induced reduction of transporter activity in the RAW264.7 cell line.

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.

Molecular characterization and in situ localization of a mouse retinal taurine transporter.

Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na+- and Cl(-)-dependent [3H]taurine transport activity in Xenopus laevis oocytes that saturated with an average Km of 13.2 microM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl- was varied, a single saturation process with an average apparent equilibrium constant (K(Cl-)) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na+ was varied (apparent equilibrium constant, K(Na+) approximately 54.8 mM). Analyses of the Na+- and Cl(-)-concentration dependence data suggest that at least two Na+ and one Cl- are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > beta-alanine > L-diaminopropionic acid > guanidinoethane sulfonate > beta-guanidinopropionic acid > chloroquine > gamma-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4alpha-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.

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 μM taurine or 500 μM taurine for 24 h. Down-regulation was observed after 12 h exposure to high taurine (500 μM) 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.

Role of conserved peptide in taurine transporter inactivation modulated by protein kinase C.

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.

Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes

The goal was to investigate the role of protein kinases in modulating taurine transporter activity in Xenopus laevis oocytes expressing the mouse retinal Na +/C −/taurine transporter. The currents generated by the taurine transporter were studied with a two-electrode voltage clamp and we recorded the maximal current ( I max ), presteady-state charge transfer Q, and membrane capacitance C m . 8-BR-cAMP, a membrane-permeable activator of the cAMP-dependent protein kinase (PKA), decreased I max (41%), Q (41%) and C m (10%). Similarly, 1 μM sn-1,2-dioctanoylglycerol (DOG), an activator of the Ca 2+ I diacylglycerol-dependent protein kinase (PKC), decreased I max (56%), Q (37%), and C m (9%). Calyculin A, a specific inhibitor of protein phosphatases 1 and 2A, also produced effects similar to those of 8-Br-cAMP and DOG, and decreased I max (64%), Q (38%), and C m (10%). We conclude that the taurine transporter is regulated by activators of PKA and PKC, and regulation occurs largely by changes in the number of transporters in the plasma membrane.

Regulation of expression of taurine transport in two continuous renal epithelial cell lines and inhibition of taurine transporter by a site-directed antibody.

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. (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.

Dexamethasone recovers phorbol-ester induced reduction of taurine transportation in mouse macrophage cell line, RAW 264.7.

The human taurine transporter gene has 12 transmembrane domains and its molecular weight is 69.6 kDa. The long cytoplasmic carboxy and amino termini might function as regulatory attachment sites for other proteins. Six potential protein kinase C phosphorylation sites have been reported in human taurine transporter. In this report, we studied the effects of phorbol 12-myristate 13-acetate (PMA) and glucocorticoid hormone on the taurine transportation in RAW 264.7, a mouse macrophage cell line. When the cells were incubated with [3H]taurine in the presence or absence of Na+ ion for 40 min at 37°C, [3H]taurine uptake rate was 780-times higher in the Na+-containing buffer than in the Na+-deficient buffer, indicating that this cell line expresses taurine transporter protein on the cell surface. THP1, human promonocyte cell line, also showed a similar property. The [3H]taurine uptake rate was not influenced by the inflammatory-inducing cytokines such as interleukin-1, gamma-interferon or interleukin-1 + gamma-interferon, but was decreased by PMA in the RAW 264.7 cell line. This suggests that activation of protein kinase C inhibits taurine transporter activity directly or indirectly. The inhibition of [3H]taurine uptake by PMA was time-dependent. Maximal inhibition occurred with 1 h stimulation with PMA. Increasing the treatment time beyond 1 h reduced the inhibition of [3H]taurine uptake, due to the depletion or inactivation of protein kinase C. The cell line also showed concentration-dependent [3H]taurine uptake by PMA stimulation. Phorbol ester (1 μM) caused 23% inhibition. The inhibition was significant even at a concentration as low as 10 nM PMA. The reduced [3H]taurine uptake could be reversed by treatment with glucocorticosteroid hormone. Dexamethasone recovered the reduced taurine uptake induced by phorbol ester, the maximum effect being seen at 1 h. This may suggest that macrophage cells require higher taurine concentration in a stressed state, for the secretion of glucocorticoid hormone is increased by hypothalamo-pituitary-adrenocortical axis activation in the blood stream.

Regulation of taurine transport by Escherichia coli heat-stable enterotoxin and guanylin in human intestinal cell lines.

The human colon carcinoma cell lines Caco-2 and HT-29 take up taurine actively. Treatment of Caco-2 cells with Escherichia coli heat-stable enterotoxin (STa) or with guanylin inhibited taurine uptake by approximately 40%. In contrast, neither STa nor guanylin changed the uptake of taurine in HT-29 cells. The inhibition in Caco-2 cells was associated with a decrease in the maximal velocity as well as in the affinity of the transporter. STa caused a 21-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels in Caco-2 cells with no change in cAMP levels. Neither cGMP nor cAMP levels were affected by STa treatment in HT-29 cells. Experiments with protein kinase inhibitors suggested that protein kinase A may mediate the observed effects of STa on taurine uptake. In accordance with this suggestion, treatment of Caco-2 cells with cholera toxin, which elevated intracellular cAMP levels, was found to inhibit taurine uptake. The steady state levels of the taurine transporter mRNA transcripts were not altered as a result of STa treatment. Studies with Caco-2 cells grown on permeable filters revealed that STa acts from the apical side. The taurine uptake from the apical side was inhibited by STa, but the taurine uptake from the basolateral side remained unaffected. It is suggested that the activity of the intestinal taurine transporter may be regulated by protein kinase A at a posttranslational level and that the intestinal absorption of taurine may be impaired during infection with enterotoxigenic strains of E. coli.

Substrate-specific regulation of the taurine transporter in human placental choriocarcinoma cells (JAR)

Exposure of the JAR human placental choriocarcinoma cells to taurine leads to a marked decrease in the activity of the taurine transporter in these cells. The ability to induce this adaptive response is not unique to taurine but is shared by other substrates of the transporter as well. Compounds such as betaine and α-aminoisobutyric acid which are not substrates for the transporter do not produce this effect. The change in the taurine transporter activity induced by taurine exposure is however unique to the taurine transporter because the activities of many other transport systems remain unaffected under these conditions. The adaptive regulation is not associated with any change in the dependence of the transporter activity on Na + and Cl −, in the Na +/Cl −/taurine stoichiometry and in the affinities of the transporter for Na + and Cl −. The decrease in the transporter activity caused by taurine exposure is due to a decrease in the maximal velocity of the transporter, and to a lesser extent, in the substrate affinity of the transporter. The decrease in the transporter activity observed in intact cells is demonstrable in plasma membrane vesicles after isolation from control and taurine-exposed cells. Cycloheximide and actinomycin D block the adaptive response in intact cells to a significant extent, but not completely. Northern blot analysis of mRNA from control and taurine-exposed cells shows that taurine exposure causes a significant decrease in the steady state levels of the taurine transporter mRNA. It is concluded that the activity of the taurine transporter in JAR cells is subject to substrate-specific adaptive regulation and that transcriptional as well as posttranscriptional events are involved in this regulatory process.

Cyclic AMP-dependent up-regulation of the taurine transporter in a human retinal pigment epithelial cell line.

This investigation was undertaken to study the role of cAMP in the regulation of the taurine transporter expressed in a human retinal pigment epithelial (HRPE) cell line. Treatment of the HRPE cells with cholera toxin for 24 h was found to stimulate the taurine transporter activity, as measured by taurine transport into the cells in the presence of NaCl, to a significant extent. The stimulation was 50-60% at 100 ng/ml cholera toxin. This stimulation was specific to the taurine transporter since the transport of two other amino acids (leucine and alanine), which are not substrates for the taurine transporter, was not affected by cholera toxin under similar conditions. Exposure of the cells to cholera toxin for a time period > 4 h was needed to elicit the stimulatory effect. The cholera toxin-induced stimulation of the taurine transporter activity was associated with an increase in the maximal velocity of the transport system. The affinity of the transporter for taurine was not altered by the treatment. The stimulatory effect was markedly blunted when the treatment of the cells with cholera toxin was done in the presence of actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of translation. The increase in the taurine transporter activity induced by cholera toxin was associated with a 2.6-fold increase in the steady state levels of the transporter mRNA. Measurement of cyclic nucleotides in control and cholera toxin-treated cells revealed that the toxin caused a 20-fold increase in the cellular levels of cAMP, the levels of cGMP remaining unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional characterization and chromosomal localization of a cloned taurine transporter from human placenta

A cDNA clone highly related to the rat brain taurine transporter has been isolated from a human placental cDNA library. Transfection of this cDNA into HeLa cells results in a marked elevation of taurine transport activity. The activity of the cDNA-induced transporter is dependent on the presence of Na+ as well as Cl-. The Na+/Cl-/taurine stoichiometry for the cloned transporter is 2:1:1. The transporter is specific for taurine and other beta-amino acids, including beta-alanine, and exhibits high affinity for taurine (Michaelis-Menten constant approximately 6 microM). The clone consists of a coding region 1863 bp long (including the termination codon), flanked by a 376 bp-long 5' non-coding region and a 625 bp-long 3' non-coding region. The nucleotide sequence of the coding region predicts a 620-amino acid protein with a calculated M(r) of 69,853. Northern-blot analysis of poly(A)+ RNA from several human tissues indicates a complex expression pattern differing across tissues. The principal transcript, 6.9 kb in size, is expressed abundantly in placenta and skeletal muscle, at intermediate levels in heart, brain, lung, kidney and pancreas and at low levels in liver. Cultured human cell lines derived from placenta (JAR and BeWo), intestine (HT-29), cervix (HeLa) and retinal pigment epithelium (HRPE), which are known to possess Na(+)- and Cl(-)-coupled taurine transport activity, also contain the 6.9 kb transcript. Somatic cell hybrid and in situ hybridization studies indicate that the cloned taurine transporter is localized to human chromosome 3 p24-->p26.

Molecular identity and calmodulin-mediated regulation of the taurine transporter in a human retinal pigment epithelial cell line.

The molecular identity and calmodulin-mediated regulation of the taurine transporter were investigated in a human retinal pigment epithelial cell line (HRPE). Reverse transcription-polymerase chain reaction amplification of HRPE cell mRNA using primer specific for a taurine transporter cloned from human placenta yielded a product of expected size (approximately 0.9 kb) which hybridized to the placental cDNA probe under high stringency conditions. The nucleotide sequence of the product was identical to the sequence of the portion of the placental taurine transporter cDNA flanked by the specific primers. The taurine transporter expressed in the HRPE cell line thus appears to be identical to the transporter cloned from the placenta. Treatment of the HRPE cells with a selective calmodulin antagonist CGS 9343 B (CGS) led to a marked decrease in taurine transport activity. This effect could be reproduced with W-7, another calmodulin antagonist. The inhibition caused by CGS occurred rapidly (t1/2 approximately 10 min). Treatment of the cells with CGS did not affect the transport of leucine, and amino acid not recognized by the taurine transporter as a substrate. The CGS-induced inhibition of taurine transport was accompanied by a decrease in the maximal velocity of the transporter with no detectable change in the substrate affinity. The steady state levels of the transporter mRNA however remained unaffected by CGS treatment. It is concluded that the HRPE cell line expressed a taurine transporter identical to the transporter describe in the human placenta and that the function of this transporter is regulated by calmodulin-dependent processes.

Expression of taurine transporter and its regulation by diet in Xenopus laevis oocytes following injection of rat kidney cortex mRNA.

NaCl-dependent taurine transport adapts to changes in the dietary intake of sulfur amino acids. The renal adaptive response is expressed by enhanced NaCl-dependent taurine cotransport by brush border membrane vesicles after a low taurine diet and reduced transport after a high taurine diet as compared to a normal taurine diet. In order to determine if this adaptive regulation is dependent on new protein synthesis, the Xenopus laevis oocyte expression system was utilized to define the translational regulation of taurine transporter activity. Poly(A)+ RNA was isolated from kidney cortex of Sprague Dawley rats fed either a low, normal or high taurine diet for 28 days. Injection of poly(A)+ RNA resulted in a time- and dose-dependent increase in NaCl-taurine co-transport. Taurine uptake was stimulated about 2-10-fold after injection of poly(A)+ RNA (10-40 ng) as compared to H2O-injected oocytes. Taurine uptake by oocytes was sodium- and anion-dependent (Cl- > Br- > SCN- > I-). The Km and Vmax of the taurine transporter were 22.5 microM and 8.35 pmol/h/oocyte respectively, similar to the Km of 17.0 microM found in rat brush border membrane vesicles. Because the adaptive response involves an augmented or reduced Vmax of the transporter, taurine uptake by oocytes injected with poly(A)+ RNA from rats fed each diet was examined. Poly(A)+ RNA from rats fed a low taurine diet elicited twice the taurine uptake elicited from rats fed a normal taurine diet and more than three times the uptake from high taurine-fed rats. Northern blot analysis after hybridization with an RNA probe for the taurine transporter cDNA from MDCK cells (obtained from Dr. Uchida) indicated that the molecular size of taurine transporter mRNA is about 1.9 kb and is regulated by diet. Expression of taurine transporter by the oocytes injected with 30 ng of capped transcript from pNCT was significantly reduced by taurine in the medium. In conclusion, taurine uptake by oocytes after injection of mRNA is similar to brush border membrane vesicles taurine transport. The long-term adaptive response is regulated at the level of mRNA, and the short-term adaptive response is regulated at the level of protein synthesis or secretion. We speculate that the renal adaptive response to altered dietary sulfur amino acid intake is both transcriptionally and translationally regulated.

Regulation of taurine transport in human colon carcinoma cell lines (HT-29 and Caco-2) by protein kinase C

The effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester known to stimulate protein kinase C, on taurine transport was studied in the human colon carcinoma cell line HT-29. PMA (1 microM) was found to inhibit taurine uptake in confluent monolayers of this cell line by approximately 70% after pretreatment of the cells with the compound for 1 h (IC50 = 42.7 +/- 2.6 nM). The inhibitory effect of PMA on the taurine transporter could be confirmed by using beta-alanine, another substrate for the transporter. Kinetic analysis of taurine uptake indicated that the PMA effect was associated with a decrease in the maximal velocity (954 +/- 26 vs. 676 +/- 28 pmol.10 min-1.mg of protein-1) and an increase in the Michaelis-Menten constant (9.8 +/- 0.5 vs. 13.3 +/- 1.0 microM). The inhibition of taurine uptake could be blocked by cotreatment of the cultures with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. Treatment of the cells with PMA did not alter the uptake of leucine and lysine, stimulated the uptake of aspartic acid, and inhibited the uptake of proline. The PMA effect on taurine uptake was not prevented by cycloheximide, actinomycin D, colchicine, or cytochalasin D. Comparison of the taurine transport activity in HT-29 cells with that in Caco-2, another human colon carcinoma cell line, revealed that the latter cell line also expressed the taurine transporter but at a much reduced level (about one-fifth compared with HT-29).(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and functional reconstitution of the human placental taurine transporter

The taurine transporter from purified human placental brush-border membranes was solubilized and reconstituted into proteoliposomes in a functional form. Solubilization was done with 2.5% cholate in the presence of 4 M urea. The proteins in the solubilizate were precipitated with 6% poly(ethylene glycol) and the precipitated proteins were reconstituted into proteoliposomes with an asolectin/protein ratio of 10:1. Under these experimental conditions, the taurine transport activity in the proteoliposomes was maximal. SDS-PAGE analysis of proteins, however, revealed that the proteoliposomes still contained a majority of the proteins originally present in the brush-border membranes. Uptake of taurine in the reconstituted proteoliposomes was obligatorily dependent on the presence of Na + as well as Cl −. Substitution of Na + with other monovalent cations such as K + and Li + reduced the taurine transport activity drastically. Similarly, substitution of Cl with other monovalent anions such as SCN −, F −, I and NO 3 − could support the transport activity only to a maximum of 30% of the control activity. In the presence of Cl −, the uptake rate was sigmoidally related to Na + concentration, resulting in a Na +/taurine coupling ratio of 2:1. The apparent dissociation constant for Na + was about 195 mM. In the presence of Na +, the uptake rate was hyperbolically related to Cl − concentration, indicating a Cl −/taurine coupling ratio of 1:1. The apparent dissociation constant for Cl − was about 205 mM. The NaCl-dependent taurine uptake was stimulated by an inside-negative membrane potential, showing that the uptake process was electrogenic. The uptake system was specific for β-amino acids. The affinity of the system for taurine was high with an apparent dissociation constant of 2.7 ± 0.1 μM. It is concluded that the taurine transporter can be dislodged from the placental brush-border membranes and reconstituted in a catalytically active from in proteoliposomes with no significant change in its characteristics.

Regulation of taurine transporter activity in LLC-PK1 cells

Taurine transporter activity increases after exposure of cultured renal epithelial cells to taurine-free medium for 24 h and decreases after incubation in high (500 microM) taurine. This adaptive response mimics that observed in rat kidney after manipulation of dietary taurine. In order to elucidate potential mechanisms involved in the regulation of beta-amino acid transporter activity, the role of RNA transcription, protein synthesis, and protein import (trafficking), as well as protein kinase C activation, on the control of taurine transport was examined in the continuous proximally derived LLC-PK1 renal cell line. Inhibition of RNA transcription with actinomycin D did not alter the up-regulatory and down-regulatory adaptive responses. Inhibition of protein synthesis with cycloheximide prevented the increased taurine transport in response to taurine-free medium as well as the decrease in taurine transport after exposure to high taurine. Colchicine prevented the response to taurine-free medium but had no effect on the response to high-taurine medium. Exposure of confluent cell monolayers to the active phorbol esters, phorbol 12-myristate 13-acetate and phorbol 12,13 dibutyrate, resulted in a reduction in taurine uptake. The effect was seen within minutes of exposure but was not observed in the presence of the inactive phorbol 4-alpha. This inhibitory action was blocked by staurosporin, an inhibitor of protein kinase C (PKC). Treatment of cells with the diacylglycerol kinase inhibitor R59022, which results in increased intracellular diacylglycerol, a natural stimulant of PKC, also inhibited taurine uptake, providing further evidence for a specific effect of PKC activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-induced inhibition of taurine transport in brush-border membrane vesicles from rabbit small intestine

The influence of Ca 2+ on the activity of the taurine transport system was investigated in rabbit small intestinal brush-border membrane vesicles. Preincubation of the brush-border membrane vesicles with Ca 2+ prepared by the Mg 2+-aggregation method markedly decreased the NaCl gradient-dependent uptake of taurine in these vesicles. Uptake of glucose and alanine, both dependent on a Na + gradient, were also decreased by Ca 2+-treatment, but their reduction was very small compared with that of taurine uptake. The inhibitory effect of Ca 2+ was dose- and time-dependent. The inhibition was reduced by the presence of ethylene glycol-bis(β-amino ethyl ether)- N, N, N′- N′-tetraacetic acid during treatment of the membrane vesicles with Ca 2+. Neomycin partially protected the taurine transporter activity from the Ca 2+-induced inhibition, but indomethacin did not. 5-Nitro-2-(3-phenylpropylamino)benzoate, a Cl −-channel blocker, did not increase taurine uptake in the Ca 2+-treated membrane vesicles. It is concluded that the Ca 2+-induced inhibition of taurine uptake in rabbit intestinal brush-border membrane vesicles is not due to accelerated dissipation of the ion gradient driving forces across the membrane but rather to a direct effect on the transporter, most likely mediated by the activation of the membrane-bound phospholipase C.

Tyrosine residues are essential for the activity of the human placental taurine transporter

Treatment of human placental brush-border membrane vesicles with four tyrosine group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tetranitromethane and p-nitrobenzenesulfonyl fluoride, inhibited NaCl gradient-driven taurine uptake in these vesicles without affecting the vesicle integrity. The relative potency of these reagents to inhibit the transporter was in the following order: tetranitromethane > NBD-Cl > p-nitrobenzenesulfonyl fluoride > N-acetylimidazole. The inhibition by N-acetylimidazole was reversible with hydroxylamine and the inhibition by NBD-Cl was reversible with 2-mercaptoethanol. Kinetic analysis of taurine uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the inhibition was primarily due to a reduction in the maximal velocity. There was no change in the affinity of the transporter for taurine in control and treated vesicles. The transporter could be protected from the N-acetylimidazole-induced inhibition by Na +. The dependence of taurine uptake rate on extravesicular Na + concentration was sigmoidal and analysis of the data revealed that two Na + ions were involved per transport of one taurine molecule. It is concluded that tyrosine residues are essential for optimal transport function of the human placental taurine transporter and that these critical tyrosine residues are located at or near the Na +-binding site of the transporter.