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Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts.

Hansen DB, Friis MB, Hoffmann EK, Lambert IH - J. Membr. Biol. (2012)

Bottom Line: Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant.TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions.Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section of Cellular and Developmental Biology, University of Copenhagen, The August Krogh Building, Universitetsparken 13, 2100 Copenhagen Ø, Denmark.

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The reduction in taurine uptake following hypo-osmotic exposure is independent of NOX4 activity. Taurine uptake (nmol g protein−1) was estimated by the tracer technique in NIH3T3 cells exposed to either 4 h iso-osmotic DMEM, low Na+ hypo-osmotic DMEM (200 mOsm) and low Na+ iso-osmotic DMEM (335 mOsm, adjusted to osmolarity by addition of sucrose) or acutely (Acute) to iso-osmotic NaCl, low Na+ hypo-osmotic NaCl and low Na+ iso-osmotic NaCl (335 mOsm, adjusted to osmolarity by addition of sucrose), as described in Materials and Methods. Taurine influx (nmol g protein−1 min−1) was estimated by linear regression of taurine uptake within 15 min. a Taurine uptake following 4 h incubation in the respective DMEM solutions. Data represent three sets of paired experiments. b Taurine influx (4 h and acute) in cells exposed for 4 h to the DMEM solutions (4 h) or acutely to the NaCl medium. Absolute values for controls are 0.056 ± 0.002 nmol g protein−1 min−1 (4 h, n = 3) and 0.222 ± 0.019 nmol g protein−1 min−1 (acute, n = 4). Significance was determined using two-way ANOVA with Bonferroni post test, comparing treatments within the acute and 4 h groups, respectively. c Taurine influx in cells exposed acutely to iso-osmotic and low Na+ iso-osmotic NaCl with or without acute exposure to 0.5 mM H2O2. Absolute values for controls are 0.230 ± 0.016 nmol g protein−1 min−1 (iso-osmotic, n = 3) and 0.089 ± 0.015 nmol g protein−1 min−1 (low Na+ iso-osmotic, n = 3). Significance was determined with Student’s t-test (paired, one-sided) comparing influx with and without ROS/acute with the respective control. d Taurine influx estimated in cells acutely exposed to low Na+ iso-osmotic or low Na+ hypo-osmotic NaCl. NOX4 overexpression was carried out as described in Materials and Methods. BHT (0.5 mM) was present during the influx experiments. Statistical evaluation by two-way ANOVA with Bonferroni post test comparing influx from each treatment with the respective control; e.g., BHT-treated cells were compared with untreated, whereas NOX4-overexpressing cells were compared with mock-treated cells under iso-osmotic and hypo-osmotic conditions. All values are given relative to the respective control ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the respective control; #P < 0.05, ##P < 0.01 compared to low Na+ iso-osmotic
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Fig1: The reduction in taurine uptake following hypo-osmotic exposure is independent of NOX4 activity. Taurine uptake (nmol g protein−1) was estimated by the tracer technique in NIH3T3 cells exposed to either 4 h iso-osmotic DMEM, low Na+ hypo-osmotic DMEM (200 mOsm) and low Na+ iso-osmotic DMEM (335 mOsm, adjusted to osmolarity by addition of sucrose) or acutely (Acute) to iso-osmotic NaCl, low Na+ hypo-osmotic NaCl and low Na+ iso-osmotic NaCl (335 mOsm, adjusted to osmolarity by addition of sucrose), as described in Materials and Methods. Taurine influx (nmol g protein−1 min−1) was estimated by linear regression of taurine uptake within 15 min. a Taurine uptake following 4 h incubation in the respective DMEM solutions. Data represent three sets of paired experiments. b Taurine influx (4 h and acute) in cells exposed for 4 h to the DMEM solutions (4 h) or acutely to the NaCl medium. Absolute values for controls are 0.056 ± 0.002 nmol g protein−1 min−1 (4 h, n = 3) and 0.222 ± 0.019 nmol g protein−1 min−1 (acute, n = 4). Significance was determined using two-way ANOVA with Bonferroni post test, comparing treatments within the acute and 4 h groups, respectively. c Taurine influx in cells exposed acutely to iso-osmotic and low Na+ iso-osmotic NaCl with or without acute exposure to 0.5 mM H2O2. Absolute values for controls are 0.230 ± 0.016 nmol g protein−1 min−1 (iso-osmotic, n = 3) and 0.089 ± 0.015 nmol g protein−1 min−1 (low Na+ iso-osmotic, n = 3). Significance was determined with Student’s t-test (paired, one-sided) comparing influx with and without ROS/acute with the respective control. d Taurine influx estimated in cells acutely exposed to low Na+ iso-osmotic or low Na+ hypo-osmotic NaCl. NOX4 overexpression was carried out as described in Materials and Methods. BHT (0.5 mM) was present during the influx experiments. Statistical evaluation by two-way ANOVA with Bonferroni post test comparing influx from each treatment with the respective control; e.g., BHT-treated cells were compared with untreated, whereas NOX4-overexpressing cells were compared with mock-treated cells under iso-osmotic and hypo-osmotic conditions. All values are given relative to the respective control ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the respective control; #P < 0.05, ##P < 0.01 compared to low Na+ iso-osmotic

Mentions: Taurine uptake in NIH3T3 mouse fibroblasts was previously shown to be totally Na+-dependent and eliminated in the presence of the taurine analog β-alanine (Voss et al. 2004), indicating that taurine uptake in the fibroblasts is mediated by TauT. From Fig. 1a, b it is seen that reduction in the total extracellular Na+ concentration from 150 to 80 mM for 4 h, keeping osmolarity constant with sucrose, results in a significant reduction in the taurine influx in NIH3T3 cells to 56% of the iso-osmotic value (compare “iso-osmotic” to “low Na+ iso-osmotic”). From Fig. 1a, b it is also seen that reduction in the extracellular osmolarity from 335 to 200 mOsm, keeping the extracellular Na+ concentration constant at 80 mM, leads to an additional 50% reduction in taurine uptake (compare “low Na+ iso-osmotic” to “low Na+ hypo-osmotic”). Hence, TauT activity is reduced by 4 h exposure to hypo-osmotic conditions due to reduction in the extracellular Na+ concentration as well as reduction in the extracellular osmolarity, i.e., osmotic cell swelling. This is similar to observations in Ehrlich ascites tumor cells (Hoffmann and Lambert 1983). To test whether reduced expression or membrane localization of TauT is responsible for the decreased taurine uptake under long-term hypo-osmotic conditions (4 h), we compared taurine uptake in cells exposed to 4 h reduction in the extracellular osmolarity with cells exposed acutely to hypo-osmotic conditions. From Fig. 1b it is seen that taurine uptake is reduced to the same extent following 4 h and acute reduction in the extracellular Na+ concentration (compare dark gray bars at 4 h and acute) and in extracellular osmolarity (compare light gray bars at 4 h and acute). As acute and 4 h reduction in Na+ and osmolarity give the same reduction in influx, it is suggested that the reduction in taurine uptake is most likely caused by direct inhibition of TauT. Similarly, it was previously shown in Ehrlich ascites cells that the regulation of the activity of another osmoregulatory transporter, NKCC1, by changes in osmolarity is not related to the number of transport molecules present in the membrane (Hoffmann et al. 1986).Fig. 1


Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts.

Hansen DB, Friis MB, Hoffmann EK, Lambert IH - J. Membr. Biol. (2012)

The reduction in taurine uptake following hypo-osmotic exposure is independent of NOX4 activity. Taurine uptake (nmol g protein−1) was estimated by the tracer technique in NIH3T3 cells exposed to either 4 h iso-osmotic DMEM, low Na+ hypo-osmotic DMEM (200 mOsm) and low Na+ iso-osmotic DMEM (335 mOsm, adjusted to osmolarity by addition of sucrose) or acutely (Acute) to iso-osmotic NaCl, low Na+ hypo-osmotic NaCl and low Na+ iso-osmotic NaCl (335 mOsm, adjusted to osmolarity by addition of sucrose), as described in Materials and Methods. Taurine influx (nmol g protein−1 min−1) was estimated by linear regression of taurine uptake within 15 min. a Taurine uptake following 4 h incubation in the respective DMEM solutions. Data represent three sets of paired experiments. b Taurine influx (4 h and acute) in cells exposed for 4 h to the DMEM solutions (4 h) or acutely to the NaCl medium. Absolute values for controls are 0.056 ± 0.002 nmol g protein−1 min−1 (4 h, n = 3) and 0.222 ± 0.019 nmol g protein−1 min−1 (acute, n = 4). Significance was determined using two-way ANOVA with Bonferroni post test, comparing treatments within the acute and 4 h groups, respectively. c Taurine influx in cells exposed acutely to iso-osmotic and low Na+ iso-osmotic NaCl with or without acute exposure to 0.5 mM H2O2. Absolute values for controls are 0.230 ± 0.016 nmol g protein−1 min−1 (iso-osmotic, n = 3) and 0.089 ± 0.015 nmol g protein−1 min−1 (low Na+ iso-osmotic, n = 3). Significance was determined with Student’s t-test (paired, one-sided) comparing influx with and without ROS/acute with the respective control. d Taurine influx estimated in cells acutely exposed to low Na+ iso-osmotic or low Na+ hypo-osmotic NaCl. NOX4 overexpression was carried out as described in Materials and Methods. BHT (0.5 mM) was present during the influx experiments. Statistical evaluation by two-way ANOVA with Bonferroni post test comparing influx from each treatment with the respective control; e.g., BHT-treated cells were compared with untreated, whereas NOX4-overexpressing cells were compared with mock-treated cells under iso-osmotic and hypo-osmotic conditions. All values are given relative to the respective control ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the respective control; #P < 0.05, ##P < 0.01 compared to low Na+ iso-osmotic
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Fig1: The reduction in taurine uptake following hypo-osmotic exposure is independent of NOX4 activity. Taurine uptake (nmol g protein−1) was estimated by the tracer technique in NIH3T3 cells exposed to either 4 h iso-osmotic DMEM, low Na+ hypo-osmotic DMEM (200 mOsm) and low Na+ iso-osmotic DMEM (335 mOsm, adjusted to osmolarity by addition of sucrose) or acutely (Acute) to iso-osmotic NaCl, low Na+ hypo-osmotic NaCl and low Na+ iso-osmotic NaCl (335 mOsm, adjusted to osmolarity by addition of sucrose), as described in Materials and Methods. Taurine influx (nmol g protein−1 min−1) was estimated by linear regression of taurine uptake within 15 min. a Taurine uptake following 4 h incubation in the respective DMEM solutions. Data represent three sets of paired experiments. b Taurine influx (4 h and acute) in cells exposed for 4 h to the DMEM solutions (4 h) or acutely to the NaCl medium. Absolute values for controls are 0.056 ± 0.002 nmol g protein−1 min−1 (4 h, n = 3) and 0.222 ± 0.019 nmol g protein−1 min−1 (acute, n = 4). Significance was determined using two-way ANOVA with Bonferroni post test, comparing treatments within the acute and 4 h groups, respectively. c Taurine influx in cells exposed acutely to iso-osmotic and low Na+ iso-osmotic NaCl with or without acute exposure to 0.5 mM H2O2. Absolute values for controls are 0.230 ± 0.016 nmol g protein−1 min−1 (iso-osmotic, n = 3) and 0.089 ± 0.015 nmol g protein−1 min−1 (low Na+ iso-osmotic, n = 3). Significance was determined with Student’s t-test (paired, one-sided) comparing influx with and without ROS/acute with the respective control. d Taurine influx estimated in cells acutely exposed to low Na+ iso-osmotic or low Na+ hypo-osmotic NaCl. NOX4 overexpression was carried out as described in Materials and Methods. BHT (0.5 mM) was present during the influx experiments. Statistical evaluation by two-way ANOVA with Bonferroni post test comparing influx from each treatment with the respective control; e.g., BHT-treated cells were compared with untreated, whereas NOX4-overexpressing cells were compared with mock-treated cells under iso-osmotic and hypo-osmotic conditions. All values are given relative to the respective control ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the respective control; #P < 0.05, ##P < 0.01 compared to low Na+ iso-osmotic
Mentions: Taurine uptake in NIH3T3 mouse fibroblasts was previously shown to be totally Na+-dependent and eliminated in the presence of the taurine analog β-alanine (Voss et al. 2004), indicating that taurine uptake in the fibroblasts is mediated by TauT. From Fig. 1a, b it is seen that reduction in the total extracellular Na+ concentration from 150 to 80 mM for 4 h, keeping osmolarity constant with sucrose, results in a significant reduction in the taurine influx in NIH3T3 cells to 56% of the iso-osmotic value (compare “iso-osmotic” to “low Na+ iso-osmotic”). From Fig. 1a, b it is also seen that reduction in the extracellular osmolarity from 335 to 200 mOsm, keeping the extracellular Na+ concentration constant at 80 mM, leads to an additional 50% reduction in taurine uptake (compare “low Na+ iso-osmotic” to “low Na+ hypo-osmotic”). Hence, TauT activity is reduced by 4 h exposure to hypo-osmotic conditions due to reduction in the extracellular Na+ concentration as well as reduction in the extracellular osmolarity, i.e., osmotic cell swelling. This is similar to observations in Ehrlich ascites tumor cells (Hoffmann and Lambert 1983). To test whether reduced expression or membrane localization of TauT is responsible for the decreased taurine uptake under long-term hypo-osmotic conditions (4 h), we compared taurine uptake in cells exposed to 4 h reduction in the extracellular osmolarity with cells exposed acutely to hypo-osmotic conditions. From Fig. 1b it is seen that taurine uptake is reduced to the same extent following 4 h and acute reduction in the extracellular Na+ concentration (compare dark gray bars at 4 h and acute) and in extracellular osmolarity (compare light gray bars at 4 h and acute). As acute and 4 h reduction in Na+ and osmolarity give the same reduction in influx, it is suggested that the reduction in taurine uptake is most likely caused by direct inhibition of TauT. Similarly, it was previously shown in Ehrlich ascites cells that the regulation of the activity of another osmoregulatory transporter, NKCC1, by changes in osmolarity is not related to the number of transport molecules present in the membrane (Hoffmann et al. 1986).Fig. 1

Bottom Line: Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant.TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions.Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section of Cellular and Developmental Biology, University of Copenhagen, The August Krogh Building, Universitetsparken 13, 2100 Copenhagen Ø, Denmark.

Show MeSH
Related in: MedlinePlus