Dependent Taurine Uptake Research Articles

Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts

Inhibition of the constitutively active casein kinase 2 (CK2) with 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidasole stimulates the Na(+)-dependent taurine influx via the taurine transporter TauT in NIH3T3 cells. CK2 inhibition reduces the TauT mRNA level and increases the localization of TauT to ER but has no detectable effect on TauT protein expression. On the other hand, CK2 inhibition increases the affinity of TauT towards Na(+ )and reduces the Na(+)/taurine stoichiometry for active taurine uptake. It is suggested that CK2 controls the cellular taurine uptake in unperturbated NIH3T3 cells, i.e., inhibition of CK2 increases the affinity of TauT towards Na(+) and hence Na(+)-dependent taurine uptake.

Estrogen regulation and ion dependence of taurine uptake by MCF-7 human breast cancer cells

It has been reported that estrogen receptor-positive MCF-7 cells express TauT, a Na+-dependent taurine transporter. However, there is a paucity of information relating to the characteristics of taurine transport in this human breast cancer cell line. Therefore, we have examined the characteristics and regulation of taurine uptake by MCF-7 cells. Taurine uptake by MCF-7 cells showed an absolute dependence upon extracellular Na+. Although taurine uptake was reduced in Cl- free medium a significant portion of taurine uptake persisted in the presence of NO3 -. Taurine uptake by MCF-7 cells was inhibited by extracellular β-alanine but not by L-alanine or L-leucine. 17β-estadiol increased taurine uptake by MCF-7 cells: the Vmax of influx was increased without affecting the Km. The effect of 17β-estradiol on taurine uptake by MCF-7 cells was dependent upon the presence of extracellular Na+. In contrast, 17β-estradiol had no significant effect on the kinetic parameters of taurine uptake by estrogen receptor-negative MDA-MB-231 cells. It appears that estrogen regulates taurine uptake by MCF-7 cells via TauT. In addition, Na+-dependent taurine uptake may not be strictly dependent upon extracellular Cl-.

High Affinity Transport of Taurine by the Drosophila Aspartate Transporter dEAAT2

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na(+)/K(+)-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar K(m) and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na(+) ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.

Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation.

Transplacental transfer is the fetus' primary source of taurine, an essential amino acid during fetal life. In intrauterine growth restriction (IUGR), placental transport capacity of taurine is reduced and fetal taurine levels are decreased. We characterized the protein expression of the taurine transporter (TAUT) in human placenta using immunocytochemistry and Western blotting, tested the hypothesis that placental protein expression of TAUT is reduced in IUGR, and investigated TAUT regulation by measuring the Na(+)-dependent taurine uptake in primary villous fragments after 1 h of incubation with different effectors. TAUT was primarily localized in the syncytiotrophoblast microvillous plasma membrane (MVM). TAUT was detected as a single 70-kDa band, and MVM TAUT expression was unaltered in IUGR. The PKC activator PMA and the nitric oxide (NO) donor 3-morpholinosydnonimine decreased TAUT activity (P < 0.05, n = 7-15). However, none of the tested hormones, e.g., leptin and growth hormone, altered TAUT activity significantly. PKC activity measured in MVM from control and IUGR placentas was not different. In conclusion, syncytiotrophoblast TAUT is strongly polarized to the maternal-facing plasma membrane. MVM TAUT expression is unaltered in IUGR, suggesting that the reduced MVM taurine transport in IUGR is due to changes in transporter activity. NO release downregulates placental TAUT activity, and it has previously been shown that IUGR is associated with increased fetoplacental NO levels. NO may therefore play an important role in downregulating MVM TAUT activity in IUGR.

Molecular characterization of taurine transport in bovine aortic endothelial cells

Cultured bovine aortic endothelial (BAE) cells expressed a Na+/Cl−-dependent taurine uptake activity that saturated with an apparent K0.5 of ∼4.9 μM for taurine and was inhibited by β-alanine, guanidinoethane sulfonate, and homotaurine. We isolated a taurine transporter clone from a BAE cell cDNA library that revealed >91% sequence identity at the amino acid level to the previously cloned high-affinity mammalian taurine transporters. The biochemical and pharmacological properties of the bovine taurine transporter cDNA expressed in Xenopus oocyte was similar to those of the high-affinity taurine transporter. Surprisingly, F− blocked taurine uptake in BAE cells with an IC50 of ∼17.5 mM. The endogenous taurine uptake was also inhibited by the protein kinase C activator phorbol 12-myristate 13-acetate, but not by its inactive analog, 4α-phorbol 12,13-didecanoate. The endogenous uptake was stimulated, however, by hypertonic stress and the increase was due to an increase in the Vmax of taurine uptake. Our results provide the first description of a molecular mechanism that may be responsible for maintaining the intracellular taurine content in the endothelial cells.

Intrauterine Growth Restriction Is Associated with a Reduced Activity of Placental Taurine Transporters

Taurine is an essential amino acid during fetal life and appears to be vital for the growth of the fetus and for the development of the CNS. In intrauterine growth restriction (IUGR), fetal plasma concentrations of taurine are reduced, and we tested the hypothesis that this is caused by altered placental transport of taurine. Syncytiotrophoblast microvillous membrane (MVM) and basal membrane (BM) vesicles were isolated from control (fetal weight, 3068+/-191 g; gestational age, 37.0+/-0.7 wk; n=13) and IUGR pregnancies (fetal weight, 1724+/-118 g; gestational age, 35.8+/-0.7 wk; n=11). Uptake of [3H]taurine (0.5 microM) was studied at 22 degrees C using rapid filtration techniques. Sodium stimulated taurine uptake 35-fold in MVM, confirming Na+-dependent transport in this membrane. A Na+-dependent taurine transport could also be demonstrated in BM; however, the activity was only 6% of that in MVM. Na+-independent transport activities were similar in MVM and BM. In IUGR, MVM Na+-dependent taurine transport was reduced by 34% (p < 0.05), whereas Na+-independent uptake was unaltered. In contrast to MVM, Na+-dependent taurine uptake in BM was unaffected by IUGR, whereas Na+-independent transport was decreased by 33% (p < 0.05). The highly polarized distribution of the Na+/taurine cotransporter to the MVM in conjunction with similar Na+-independent transport rates for taurine in MVM and BM provides the basis for net taurine flux from the mother to the fetus. These data suggest that the low plasma concentrations of taurine in IUGR fetuses are caused by a reduced activity of placental taurine transporters.

Compatible organic osmolytes and osmotic modulation of inducible nitric oxide synthetase in RAW 264.7 mouse macrophages.

The influence of osmolarity and compatible organic osmolytes on the phosphorylation of the MAP-kinases Erk-1 and Erk-2 and on the expression of taurine transporter (TAUT) and lipopolysaccharide (LPS)-induced nitric oxide synthetase (iNOS) was studied in RAW 264.7 mouse macrophages. Hypoosmolarity (205 mosmol/l) but not hyperosmolarity (405 mosmol/l) or challenge of the cells with betaine or taurine increased phosphorylation of Erk-1 and Erk-2. Hypoosmotic Erk-phosphorylation was blocked by the MEK-inhibitor PD098059 but was resistant to depletion of extracellular calcium and to inhibition of PLC, PKC, erbstatin-sensitive tyrosine kinases and elevation of intracellular cAMP. Hyperosmolarity stimulated Na+-dependent taurine uptake and led to an increase of TAUT mRNA levels, whereas hypoosmotic exposure diminished both and induced a rapid efflux of the osmolyte from taurine-preloaded cells. The hyperosmotic elevation of TAUT mRNA levels was antagonized upon addition of taurine but not of betaine or myo-inositol. Hyperosmolarity increased the LPS-induced iNOS expression at the mRNA and the protein level. This was suppressed by betaine but not by taurine or myo-inositol. The osmotic regulation of taurine transport and iNOS expression appeared independent of the MEK-Erk pathway and the p38MAPK.

Osmoregulated taurine transport in H4IIE hepatoma cells and perfused rat liver.

The effects of aniso-osmotic exposure on taurine transport were studied in H4IIE rat hepatoma cells. Hyperosmotic (405 mosmol/l) exposure of H4IIE cells stimulated Na+-dependent taurine uptake and led to an increase in taurine transporter (TAUT) mRNA levels, whereas hypo-osmotic (205 mosmol/l) exposure diminished both taurine uptake and TAUT mRNA levels when compared with normo-osmotic (305 mosmol/l) control incubations. Taurine uptake increased 30-40-fold upon raising the ambient osmolarity from 205 to 405 mosmol/l. When H4IIE cells and perfused livers were preloaded with taurine, hypo-osmotic cell swelling led to a rapid release of taurine from the cells. The taurine efflux, but not taurine uptake, was sensitive to 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), suggestive of an involvement of DIDS-sensitive channels in mediating volume-regulatory taurine efflux. Whereas in both H4IIE rat hepatoma cells and primary hepatocytes TAUT mRNA levels were strongly dependent upon ambient osmolarity, mRNAs for other osmolyte transporters, i.e. the betaine transporter BGT-1 and the Na+/myo-inositol transporter SMIT, were not detectable. In line with this, myo-inositol uptake by H4IIE hepatoma cells was low and was not stimulated by hyperosmolarity. However, despite the absence of BGT-1 mRNA, a slight osmosensitive uptake of betaine was observed, but the rate was less than 10% of that of taurine transport. This study identifies a constitutively expressed and osmosensitive TAUT in H4IIE cells and the use of taurine as a main osmolyte, whereas betaine and myo-inositol play little or no role in the osmolyte strategy in these cells. This is in contrast with rat liver macrophages, in which betaine has been shown to be a major osmolyte.

Fasting enhances taurine transport by rat liver plasma membrane vesicles

Hepatic taurine stores are maintained by biosynthesis from the sulfur-containing amino acids, methionine and cysteine, and by uptake via a Na(+)- and Cl(-)-dependent transport system, which is specific for beta-amino acids. We hypothesized that liver stores of taurine are maintained by enhanced hepatic transport during fasting when dietary sources for taurine and its precursors are diminished. Liver plasma membrane vesicles, enriched for the basolateral domain, were prepared from adult male rats fasted for 72 h and from control rats. The maximum velocity for Na(+)-dependent taurine uptake was twofold greater for the fasted group compared with the control group (0.87 +/- 0.09 vs. 0.31 +/- 0.03 nmol.mg protein-1.min-1). The apparent Michaelis constant for taurine was also greater for fasted compared with control (154.0 +/- 0.5 vs. 80.0 +/- 2.0 microM). gamma-Aminobutyric acid, but not alanine or glutamine, abolished the effect of fasting on hepatic taurine transport. To determine the effect of fasting independent of changes in the lipid microenvironment, taurine uptake was measured in proteoliposomes reconstituted by inserting detergent-solubilized membrane proteins into asolectin vesicles. Taurine uptake by proteoliposomes reconstituted from membranes prepared from the fasted group was significantly greater than from the control group. We conclude that Na(+)-dependent taurine transport is enhanced in liver plasma membranes prepared from fasted rats. Our findings imply that enhanced taurine uptake with fasting is due to either an increased number of functional carriers or activation of existing transporters.

Taurine and β-alanine transport in an established human kidney cell line derived from the proximal tubule

The transport mechanisms of taurine and β-alanine by an immortalized human embryonic kidney epithelial cell line (IHKE) were examined. The uptake of these β-amino acids was characterized by two Na +-dependent transport components, whereas an inwardly directed H +-gradient only stimulated amino acid influx to a small extent and in the absence of sodium. Competition experiments revealed that taurine and β-alanine drastically reduced the uptake of one another by the high-affinity Na +-dependent transport system. However, some α-amino acids could also compete with the β-amino acids, but with a low affinity. Examinations of the effect of different anions on the Na +-dependent uptake of taurine at a low amino acid concentration (240 nM) revealed a specific requirement for Cl −, whereas Cl − had no measurable effect at a higher concentration (1.0 mM) of taurine. In addition, activation of taurine transport as a function of Na + and Cl − concentration indicated a probable coupling ratio of 3 Na +/1 Cl −/1 taurine for the high-affinity carrier. Finally, cellular regulation of taurine transport was indicated by the finding that pretreatment with taurine containing media decreased the activity of the taurine transporter(s).

Characterization of a sodium-dependent taurine transporter in rabbit choroid plexus

Taurine, a β-amino acid, plays an important role as a neuromodulator and is necessary for the normal development of the brain. Since de novo synthesis of taurine in the brain is minimal and in vivo studies suggest that taurine does not cross the blood-brain barrier, we examined whether the choroid plexus, the blood-cerebrospinal fluid barrier, plays a role in taurine transport in the central nervous system. The uptake of [3H]taurine into ATP-depleted choroid plexus from rabbit was substantially greater in the presence of an inwardly directed Na + gradient, whereas in the absence of a Na + gradient taurine accumulation was negligible. A transient inside-negative potential gradient enhanced the Na +-driven uptake of taurine into the tissue slices, suggesting that the transport process is electrogenic. Na +-driven taurine uptake was saturable with an estimated V rmmax of 111±20.2 nmol/g per 15 min and a K rmm of 99.8±29.9 μM. The estimated coupling ratio of Na + and taurine was 1.80 ± 0.122. Na +-dependent taurine uptake was significantly inhibited by β-amino acids, but not by α-amino acids, indicating that the transporter is selective for β-amino acids. Na +-dependent taurine uptake showed some selectivity for anions: the accumulation was comparable in the presence of CI −, Br and thiocynate whereas I −, SO 4 2− and gluconate did not stimulate the uptake significantly. Collectively, our results demonstrate that taurine is transported in the choroid plexus via a Na +-dependent, saturable and apparently β-amino acid selective mechanism. This process may be functionally relevant to taurine homeostasis in the brain.

The role of chloride in taurine transport across the human placental brush-border membrane

Taurine, a sulfated ß-amino acid, is conditionally essential during development. A maternal supply of tarine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na +-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na +-dependent, carrier-mediated taurine transport with an apparent K mof 4.00 ± 0.22 μM and a V max of 11.72–0.36 pmol mg 1 protein 20 s 1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential o transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl −. In addition, a Cl −-gradient alone (under Na +-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 ± 0.8 pmol mg −1 protein 20 s −1) and an overshoot compared to Na +, Cl −-equilibrated conditions (0.60 ± 0.06 pmol mg −1 protein 20 s −1). A Cl -gradient (Na +-equilibrated) also to Na +, Cl −-equilibrated conditions (0.60 ± 0.06 pmol mg −1 protein 20 s −1). A Cl −-gradient (Na +-equilibrated) also stimulated uptake of [ 3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl − suggested that 1 Cl − ions is involved in Na +/taurine cotransport. We conclude that Na +-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na +/Cl −/taurine symport.

Sodium-taurine cotransport in reptilian renal brush-border membrane vesicles.

The coupled transport of Na+ with taurine into snake renal brush-border membrane vesicles (BBMV) was studied using 5-s uptake conditions. Taurine transport into snake renal BBMV involved two parallel processes, one saturable (Na(+)-dependent) and one (Na(+)-independent) that behaved like passive diffusion. Below 1 mM taurine concentration, the Na(+)-dependent system accounted for 60% of total taurine uptake. Over both low (0.001-0.80 mM) and high (0.8-5.0 mM) taurine concentration ranges, the Na(+)-dependent taurine uptake within each range showed Michaelis-Menten kinetics, suggesting the presence of two independent saturable Na(+)-dependent transport systems for taurine. The high-affinity, low-capacity system saturated above 100 microM with a Km of 71.4 +/- 45.7 microM and a maximum velocity (Vmax) of 21.9 +/- 3.77 pmol (mg protein)-1 (5 s)-1. The low-affinity, high-capacity system saturated above 1 mM, with a Km of 1.11 +/- 0.63 mM and a Vmax of 252 +/- 47 pmol (mg protein)-1 (5 s)-1. The stoichiometric relationship between external Na+ concentration and taurine uptake (at 10 microM) by the high-affinity BBMV transport system was examined by the activation method under short-circuited conditions. The 5-s rate of taurine transport was a sigmoid function of increasing extravesicular Na+ concentration. Kinetic analysis of the interaction of Na+ with the high-affinity taurine transport system suggested that 3 Na+ ions (3.2 +/- 0.7) may be involved with 1 taurine molecule in the transport event.(ABSTRACT TRUNCATED AT 250 WORDS)

Taurine transport in skate hepatocytes. I. Uptake and efflux.

Taurine is an amino sulfonic acid maintained at relatively high concentrations in skate (Raja erinacea) hepatocytes (65 mmol/l intracellular water). To identify the transport mechanisms involved in intracellular taurine accumulation, uptake and efflux of [14C]taurine was measured in freshly isolated skate hepatocytes. Uptake of 50 microM taurine at 15 degrees C was essentially linear over 3 h when measured in normal elasmobranch Ringer, was diminished by replacement of extracellular Cl- with NO3-, and was almost completely abolished by replacement of Na+ with choline+ or K+. Kinetic studies suggested the presence of two saturable Na(+)-dependent taurine uptake systems [apparent taurine Michaelis constant = 0.089 +/- 0.028 and 4.47 +/- 0.49 mM and maximal velocity (Vmax) = 0.19 +/- 0.07 and 1.65 +/- 0.42 (SE) nmol.microliter-1.15 min-1 for high- and low-affinity components, respectively; n = 4], as well as a small contribution from an Na(+)-independent uptake system. Uptake was inhibited by other beta-amino acids but not by alpha-amino acids, taurocholate, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.5 mM). In contrast to uptake, efflux of [14C]taurine was relatively slow (approximately 10% in 2 h) and was unaffected by isosmotic replacement of extracellular Na+ with choline+ or K+ or by replacing Cl- with NO3-. These findings suggest distinct mechanisms for taurine uptake and efflux in skate hepatocytes; uptake is largely Na+ dependent and requires Cl- for maximal activity, whereas the efflux pathway is independent of transmembrane Na+, K+, or Cl- gradients. Maintenance of high intracellular taurine levels in skate hepatocytes is achieved by active Na(+)-dependent uptake processes and a relatively slow efflux.

Expression of Madin-Darby canine kidney cell Na(+)-and Cl(-)-dependent taurine transporter in Xenopus laevis oocytes

Expression of a Madin-Darby canine kidney (MDCK) cell taurine transporter was examined in Xenopus oocytes that had been injected with poly(A)+ RNA extracted from MDCK cells. Compared with water-injected oocytes, injection of total poly(A)+ RNA resulted in an increase in Na(+)-dependent taurine uptake which was directly related to the amount of RNA injected. The magnitude of expression in poly(A)+ RNA-injected oocytes was 5-10-fold higher than that of water-injected oocytes. Since the Vmax of taurine uptake in MDCK cells is increased by culture in hypertonic medium, we compared oocyte taurine uptake after injection with poly(A)+ RNA from MDCK cells cultured in hypertonic medium with uptake in oocytes injected with poly(A)+ RNA from hypertonic cells elicited twice the taurine uptake elicited by poly(A)+ RNA from isotonic cells. The transporter expressed in oocytes was like that in MDCK cells: it was completely dependent on external sodium and was also anion dependent (Cl- greater than or equal to Br- greater than SCN- much greater than gluconate-). Other beta-amino acids, beta-alanine and hypotaurine, inhibited taurine uptake, but L-alanine and 2-(methylamino) isobutyric acid did not. The apparent Km of the transporter was 7.0 microM. After size fractionation on a sucrose density gradient, poly(A)+ RNA encoding for the MDCK taurine transporter was found in the fraction whose average size was 4.4 kilobases.

Renal transport of taurine in luminal membrane vesicles from rabbit proximal tubule

The uptake of taurine by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule was examined. In pars convoluta, the transport of taurine was characterized by Na +-dependent ( K m1 = 0.086 mM, K m2 = 5.41mM) systems, and one Na +-independent ( K m = 2.87mM) system, which in the presence of an inwardly directed H +-gradient was able to drive the transport of taurine into these vesicles. By contrast, in luminal membrane vesicles from pars recta, the transport of taurine occurred via a dual transport system ( K m1 = 0.012mM, K m2 = 5.62mM), which was strictly dependent on Na +. At acidic pH with or without a H +-gradient, the Na +-dependent flux of taurine was drastically reduced. In both kind of vesicles, competition experiments only showed inhibition of the Na +-dependent high-affinity taurine transporter in the presence of β-alanine, whereas there was no significant inhibition with α-amino acids, indicating a β-amino acid specific transport system. Addition of β-alanine, l-alanine, l-proline and glycine, but not l-serine reduced the H +-dependent uptake of taurine to approx. 50%. Moreover, only the Na +-dependent high-affinity transport systems in both segments specifically required Cl −. Investigation of the stoichiometry indicated 1.8 Na +:1 Cl −:1 taurine (high affinity), 1 Na +:1 taurine (low affinity) and 1 H +:1 taurine in pars convoluta. In pars recta, the data showed 1.8 Na +:1 Cl −:1 taurine (high affinity) and 1 Na +:1 taurine (low affinity).

Effect of HEPES on the Na +, Cl −-dependent uptake of taurine and β-alanine by cultured glial cells. Modulation by composition and osmolarity of medium

The effect of HEPES on the Na +,Cl −-dependent uptake of taurine and β-alanine by cultured glial cells was investigated. Whatever the culture medium used, a similar inhibition of the uptake of taurine by HEPES was found. In a previous paper it was shown that this buffer was able to inhibit the uptake of taurine through a slow and a fast “mechanism”, when present in the culture or in the incubation medium, respectively. Comparison of the effect of HEPES on the uptake of taurine and β-alanine suggested that only the slow “mechanism” was common to the uptake of the β-aminoacid. Therefore, the uptake of the β-aminoacid might be affected by HEPES. On the other hand, a small increase of the osmolarity of the media altered the uptake of taurine, suggesting that caution is needed when interpreting pharmacological experiments.

Perturbation of renal amino acid transport by brush border membrane vesicles in the vitamin D-deficient rat

Vitamin D deficiency is characterized by secondary hyperparathyroidism, phosphaturia, bicarbonaturia, and generalized amino aciduria. While the site at which the phosphaturia ensues has been described to occur at the apical membrane of the renal proximal tubule, no studies are available for amino aciduria. Thus, weanling rats were fed five vitamin D-deficient diets for 4-6 weeks: (i) VLC, 0.02% Ca, 0.3% P; (ii) VLC + 1,25[OH]2D, same + 500 pmole ip for 2 days; (iii) LC, 0.45% Ca, 0.3% P; (iv) HC, 2.5% Ca, 0.3% P; and (v) VLP, 1.2% cA, 0.1% P. The normal diet contained 1.2% Ca, 0.7% P, and 2.5 micrograms% vitamin D. Amino acids, serum 25[OH]D, 1,25[OH]2D, and PTH, using a specific anti-rat PTH antibody, were measured. There were 4.65 +/- 1.1- and 10 +/- 1.39-fold increases in the urinary excretion of taurine and proline, respectively, irrespective of diet. Hypocalcemia, secondary hyperparathyroidism, and increased concentrations of urinary cAMP were demonstrated in all diets, except VLP. Taurinuria and prolinuria manifested at the renal brush border membrane. There was 21-25% and 26-39% attenuation in the peak of the overshoot of Na(+)-dependent uptake of taurine and proline, respectively, that was statistically significant as compared to that of normal diets (P less than 0.01). VLC resulted in a reduction in the Vmax of taurine (VLC, 78.26 +/- 6.88 vs normal, 115.4 +/- 6.26 pmole/mg protein/min, P less than 0.01) and proline (VLC, 402.06 +/- 31.26 vs normal, 589.49 +/- 37.42 pmole/mg protein/15 sec, P less than 0.01) uptake. Acute supplementation with pharmacological doses of 1,25[OH]2D normalized the Vmax of taurine and proline uptake, without affecting their renal excretion. The VLP diet induced and increase in the Km of taurine (VLP, 58.95 +/- 1.88 microM vs normal, 39.75 +/- 2.75 microM P less than 0.01) and proline (VLP, 116.75 +/- 8.87 microM vs normal, 76.82 +/- 7.27 microM P less than 0.01) uptake, without an associated perturbation in the Vmax of uptake. We conclude that the amino aciduria of vitamin D deficiency manifests at the apical membrane of the proximal tubule by an attenuation in the Na(+)-dependent uptake of amino acids. This is associated with a reduction in the initial rate of uptake or number of active transporters in the presence of secondary hyperparathyroidism and hypocalcemia, or a decrease in the affinity of the symport in the presence of P depletion. The data suggest the interplay of multiple factors in the causation of amino aciduria.

Adaptive regulation of taurine transport in two continuous renal epithelial cell lines

Taurine is actively transported by a beta-amino acid transporter located on the proximal tubule apical surface. We have characterized taurine transport into confluent monolayers of two continuous renal epithelial cell lines: LLC-PK1, a cell of porcine proximal tubular origin, and the Madin-Darby canine kidney cell line (MDCK) of distal origin. Taurine uptake is linear up to 90 minutes in LLC-PK1 cells and 180 minutes in MDCK cells. This process is highly dependent upon Na+ as the cation and either Cl- or Br- as the anion. Taurine uptake is inhibited by another beta-amino acid, beta-alanine, to a greater extent than the alpha-analog, L-alanine or other alpha-amino acids. Incubation of cell monolayers with taurine-free medium (0 microM taurine) induces an increase in Na(+)-dependent taurine uptake when compared to cells exposed to standard medium (50 microM taurine). When cells were incubated in medium containing high taurine (500 microM), uptake was decreased as compared to control cells. This adaptive response is evident by 12 hours in both cell lines and is the result of changes in the apparent transport maximum (Jmax) rather than the apparent Km for taurine. The changes in transport observed after manipulation of medium taurine concentration were not associated with differences in taurine efflux. In summary, taurine is transported by a beta-specific, Na-Cl dependent process in both renal epithelial cell lines. Although the factors which regulate taurine transport are not known, an increased transport maximum is observed in cells which have been taurine-starved, and a decreased Jmax is seen in cells supplied with excess taurine.(ABSTRACT TRUNCATED AT 250 WORDS)

Anion dependence of taurine transport by rat renal brush-border membrane vesicles.

The anionic requirements and the stoichiometric relationships of Na+-taurine cotransport into rat renal brush-border membrane vesicles (BBMV) were evaluated. External Cl- (100 mM) or Br- (100 mM) gradients supported the full overshoot of Na+-taurine symport and yielded similar high-affinity transport systems for taurine uptake. No active uptake of taurine was evident in the presence of external (100 mM) NaF, NaI, Na gluconate, or Na p-aminohippurate (PAH). Na+:taurine stoichiometry was 2.18:1 in the presence of Cl- and 1.60:1 in the presence of Br-. When the external anion gluconate was employed, Na+-dependent taurine uptake was negligible over the whole range of Na+ concentrations examined. Cl-:taurine and Br-:taurine stoichiometries in the presence of external Na+ were 0.97:1 and 0.81:1, respectively. External furosemide (1 mM) or bumetanide (1 mM) did not change taurine accumulation and kinetic parameters. The anionic transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (5 x 10(-4) M), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (10(-3) M) and p-chloromercuribenzoate (5 x 10(-4) M) significantly decreased initial rate of taurine uptake by 48, 31, and 31%, respectively. These data suggest that Na+-taurine cotransport into rat renal BBMV is Cl- or Br- dependent and probably operates by means of 2 Na+:1 Cl- or Br-:1 taurine carrier complex. Na+-taurine symport across the rat renal brush-border membrane surface is not affected by diuretics that influence NaCl cotransport but is affected by selected anionic transport inhibitors. An intact anionic binding site may be needed for this translocation process.