Department of Emergency Medicine, Wright State University School of Medicine, Dayton 45429; and Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45435
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ABSTRACT |
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We investigated mechanisms controlling taurine
synthesis in cultured rat cerebral astrocytes. The mean ± SE rate
of taurine synthesis from extracellular cysteine was 21.2 ± 2.0 pmol · mg protein1 · min
1,
whereas taurine degradation was <1.3% of this rate. Eliminating cellular glutathione and inhibiting glutathione biosynthesis increased taurine synthesis from extracellular cysteine by 39%. In cell homogenates, cysteine dioxygenase (CDO) and cysteine-sulfinate decarboxylase activities were 2.4 ± 0.2 and 8.3 ± 2.8 nmol · mg protein
1 · min
1,
respectively. CDO activity was strongly dependent on cysteine concentration over physiological and pathophysiological ranges of
intracellular cysteine concentration. Growth in hyperosmotic medium
caused a greater increase in culture medium taurine content than that
measured from cells in isosmotic growth medium. Hyperosmotic treatment
transiently increased the rate of cysteine accumulation and cellular
cysteine and glutathione contents but had no effect on the synthesis
rate of taurine from extracellular cysteine. Thus cysteine is
accumulated and then metabolized to taurine through CDO, whose activity
depends on the intracellular cysteine concentration and appears to be
rate limiting for taurine synthesis. Hyperosmotic exposure increases
net taurine production yet has no effect on taurine synthesis from
exogenously applied cysteine. Availability of substrate from
intracellular pools must contribute to maintenance of high
intracellular taurine during hyperosmotic exposure.
cysteine dioxygenase; cysteine-sulfinate decarboxylase; -glutamyltransferase; glutathione; volume regulation
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INTRODUCTION |
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TAURINE BIOSYNTHESIS IN the central nervous system has been described previously (13), but mechanisms that regulate this pathway remain to be defined. Taurine can be synthesized from its amino acid precursor, cysteine, by a variety of mechanisms (13). In the central nervous system, however, taurine synthesis proceeds from cysteine to cysteine sulfinate, hypotaurine, and finally taurine, a sequence commonly called the cysteine-sulfinate decarboxylase (CSD; EC 4.1.1.29) route (42). Contributions to taurine synthesis via cysteamine or cysteate are very minor or insignificant (17). The taurine-synthesizing enzymes, cysteine dioxygenase (CDO; EC 1.13.11.20) and CSD, have been identified and characterized in the rat brain (24, 32). In addition, Kuriyama et al. (17) demonstrated conversion of cysteine to cysteine sulfinic acid, hypotaurine, and taurine in rat whole brain homogenates.
In hyperosmotic hypernatremia, contents of a number of organic osmolytes including polyamines, polyalcohol sugars, and amino acids are elevated in brain tissue (4, 12, 19, 44). In some animal models, accumulation of taurine accounts for as much as 50% of the additional osmolytes needed for brain volume regulation (44). Tissue culture studies of cerebral astrocytes have demonstrated increased cellular contents and rates of influx of taurine (6, 25, 36) and inositol (39) during hyperosmotic exposure. However, in situ, enhanced cellular accumulation of taurine only serves to sequester available taurine intracellularly, as unidirectional transport from blood to brain is not elevated by acute hyperosmotic treatment (40). Thus activation of de novo synthesis of taurine may play an important role in the brain's adaptation to hyperosmotic conditions.
In addition to being a precursor for taurine synthesis, cysteine is a
structural element of proteins and is one of three amino acids in
glutathione (GSH, -glutamylcysteinylglycine). Cysteine, which is in
micromolar concentrations in the brain (37), is a limiting reagent for
the production of GSH during changes in cellular oxidation states (8,
30, 34, 35). GSH, on the other hand, is present in millimolar
concentrations in brain cells (22, 29, 35, 49). GSH serves as an
intracellular reducing agent, a xenobiotic detoxifier, and a free
radical scavenger (46). Studies have shown GSH to be present in higher
concentrations in astrocytes than in neurons (22). Further evidence
suggests that astrocytes provide the substrate for neuronal GSH
synthesis by releasing cysteine into the extracellular space for
accumulation by neurons (35). Release of GSH from cultured astrocytes
also may supply neurons with GSH directly or perhaps may serve as a nonneuroactive precursor to glutamate (49).
Conversely, extracellular GSH may serve as a source of cellular
cysteine. Hanigan and Ricketts (11) demonstrate that cysteine-dependent fibroblasts containing -glutamyltransferase (
-GT; EC 2.3.2.2), a
cell surface enzyme capable of hydrolyzing the
-glutamyl bond of GSH
or transferring the
-glutamyl moiety to an acceptor amino acid, can
use extracellular GSH both to replenish intracellular GSH stores and to
provide a source of cysteine. This manner of GSH and cysteine cycling
may be present in astrocytes, which also express
-GT activity (22,
31). Thus alterations in GSH metabolism and cellular content may, in
turn, affect taurine biosynthesis by regulating the availability of
their common precursor, cysteine. Finally, activities of
sodium-dependent and sodium-independent cysteine/cystine transporters
(8, 34, 35) may regulate cellular pools of free cysteine in astrocytes
and thus the biosynthesis pathways for GSH and taurine.
In this study, we examine rates of various transport and biochemical processes involved in the production of taurine from extracellular cysteine in cultured astrocytes from the rat cerebral cortex. Previous studies demonstrated that taurine synthesis is enhanced in astrocytes exposed to hyperosmotic conditions (5, 6). To further understand the regulation of taurine synthesis in vivo, we also explore mechanisms that contribute to this enhanced synthesis during hyperosmotic exposure.
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MATERIALS AND METHODS |
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Cell culture. Primary astrocyte cultures were prepared from the cerebral cortices of 2- to 4-day-old Sprague-Dawley (Harlan Sprague-Dawley, Indianapolis, IN) rat pups by a modification of methods described previously (5, 26, 27). Cells were grown on 35- or 100-mm plastic culture dishes at an initial plating density of ~60,000 cells/cm2. Astrocytes were plated in Eagle's minimum essential medium (MEM; GIBCO Life Technologies, Grand Island, NY) containing 20% (vol/vol) newborn calf serum (GIBCO Life Technologies), antibiotics (50 U/ml penicillin, 50 mg/ml streptomycin), four times the normal concentration of MEM vitamins, twice the normal concentration of MEM amino acids, and 1.6 mg/ml additional sodium bicarbonate. Plating medium was replaced 1-2 days later with an identical medium but containing only 10% (vol/vol) newborn calf serum. This formulation contained ~200 µM cysteine and no GSH or taurine from MEM; however, newborn calf serum may contain these constituents. Measured concentrations of taurine in fresh growth medium were 2-5 µM. Growth medium was changed twice weekly as astrocytes reached confluency over 2-3 wk. At the final medium change, some dishes received culture medium containing additional NaCl to raise the osmolality from the control value (300 mosmol/kg) to 350, 400, or 450 mosmol/kg. To avoid medium evaporation and elevations in medium osmolality, 35-mm dishes were placed into a covered 100-mm petri dish containing an open reservoir of distilled water. Experiments were performed 0, 1, 8, 24, or 48 h later. Medium osmolality increased by <10 mosmol/kg over 48 h in the 35-mm dishes. In 100-mm dishes, the osmolality increase over this period ranged from 7 mosmol/kg in 300 mosmol/kg medium (2%) to 26 mosmol/kg in 450 mosmol/kg medium (6%). Before analysis, cells were rinsed free of culture medium using 37°C phosphate-buffered saline (PBS) containing (in mM) 2.7 KCl, 1.0 CaCl2, 0.5 MgCl2, 2.7 Na2HPO4, 0.5 KH2PO4, 5.5 glucose (pH 7.3), and sufficient NaCl to match the osmolality of the culture medium.
Cell volume. Astrocyte cell volumes were measured by determining the 3-O-[methyl-3H]methyl-D-glucose ([3-3H]OMG) space using a method modified from that described by Bender et al. (7). Cultures were incubated for 5 min at 37°C with glucose-free PBS containing 0.8 µCi/ml [14C]sucrose (400-700 mCi/mmol; NEN, Wilmington, DE), 0.8 µCi/ml 3-O-[methyl-3H]methyl-D-glucose (60-90 Ci/mmol; NEN), 1 mM 3-OMG, and sufficient NaCl to match the osmolality of the culture medium. Cells were then rinsed with an ice-cold solution of 10 mM tris(hydroxymethyl)aminomethane (Tris) (pH 7.3), 0.5 mM Ca(NO3)2, 1 mM phloretin to block glucose transport, and sufficient sucrose to match the osmolality of the culture medium. Cells then were scraped into 0.6 M HClO4. This solution was centrifuged, and the pellet was dissolved in 1 M NaOH. The activities of 14C and 3H in the deproteinized acid extract and the protein content of the NaOH solution were determined by liquid scintillation spectroscopy and the method of Lowry et al. (21), respectively. Measured 3-O-[methyl-3H]methyl-D-glucose was corrected for extracellular radiotracer using [14C]sucrose as a marker for the volume of extracellular solution remaining on the dishes.
Taurine, cysteine, and GSH contents. Astrocyte taurine content was measured by high-performance liquid chromatography (HPLC) in deproteinized cell extracts of 0.6 M HClO4 as previously described (6). Culture medium taurine concentrations were determined with a Waters model 420-AC fixed-wavelength (338-nm excitation, 425-nm cutoff emission; Waters, Milford, MA) fluorescence detector using mobile phase consisting of 28% methanol plus 72% (vol/vol) phosphate buffer (pH 5.5) flowing at 1.2 ml/min.
Cellular cysteine content was determined by a modification of methods described previously by Richie and Lang (33). Deproteinized cell extracts of 0.6 M HClO4 were separated using a Bioanalytical Systems (BAS, West Lafayette, IN) chromatograph equipped with a BAS Biosphere ODS (4.6 × 250 mm, 5 µm) column. Mobile phase flowed at 1.0 ml/min and contained 96% 100 mM monochloroacetic acid (pH 3), 4% methanol, and 2 mM heptane sulfonic acid (Alltech, Deerfield, IL) as an ion-pairing reagent. Cellular cysteine was detected with a Au/Hg electrochemical detector at +0.15 V vs. an Ag/AgCl reference electrode. GSH content was determined by modification of methods described by Jocelyn (15). GSH was extracted by scraping astrocytes into 3.6 mM EDTA and 3.4 M NaCl in 1.1% (wt/vol) glacial metaphosphoric acid. The suspension was centrifuged, and protein was measured in the cellular pellet as described above. A 450-µl aliquot of supernatant was mixed with 225 µl of 0.5 mM 5,5'-dithio-bis(2-nitrobenzoic acid) in 1% (wt/vol) sodium citrate and 1.8 ml of 300 mM Na2HPO4. Absorbance was read at 412 nm and compared with GSH standards prepared in the same manner.Conversion of extracellular
[35S]cysteine to cellular
[35S]taurine.
Each culture dish received 2 ml PBS containing 1 µCi/ml
[35S]cysteine
(>1,000 Ci/mmol; Amersham Life Science, Arlington Heights, IL), 1 mM
unlabeled cysteine, and sufficient NaCl to match the osmolality of the
culture medium. After 60 min at 37°C, the reaction was terminated
by removing the PBS and rapidly rinsing three times with ice-cold 10 mM
Tris (pH 7.3) plus sufficient sucrose to match the osmolality of the
culture medium. The cells were then scraped from the dish into 1 ml of
distilled water. The suspension was frozen at 70°C and
lyophilized, and the dried residue was resuspended in 100 µl of 0.6 M
HClO4. After centrifugation, amino
acid contents were determined in this deproteinized extract by HPLC
(6). The eluate was collected in fractions at 12-s intervals at times corresponding to the elution of the taurine peak.
[35S]taurine was
measured in the eluate fractions by liquid scintillation counting.
Protein was determined in the pellet remaining after centrifugation
(21).
Taurine degradation. Taurine degradation was determined for astrocyte cultures grown in isosmotic conditions. Cells were exposed to 0.5 µCi/ml [3H]taurine in culture medium for 2 h before changing the medium to fresh isosmotic culture medium. At 24 or 48 h later, the medium was removed and cells were rinsed in ice-cold 10 mM Tris (pH 7.3) plus sufficient sucrose to match the osmolality of the culture medium. Astrocytes were scraped into 1 ml of 0.6 M HClO4 and centrifuged. Protein was determined in the pellet after solubilization in 1 M NaOH (21). Taurine content of the medium and cell supernatant was determined in triplicate by HPLC after derivitization to a fluorescent product as described previously (6). Fractions were collected for radioactivity counting during elution of the derivitized taurine reaction product. Integrated radioactivity in the samples corresponding to derivitized taurine was expressed relative to the content of radioactivity in the cell supernatant. Because the derivitization reaction with taurine is incomplete, this ratio for each sample was compared with a similar ratio determined using [3H]taurine in a standard cocktail of taurine plus seven other amino acids.
Cysteine influx. Each culture dish was incubated in PBS, which was osmotically matched to the culture medium with NaCl and which also contained 0.5 µCi/ml [14C]cysteine (>250 mCi/mmol; Amersham Life Science) and 1 mM unlabeled cysteine. With this high concentration of cysteine [2-20 times the Michaelis-Menten binding constant (Km) for various cysteine transporters (3, 34)], changes in uptake rate would preferentially indicate changes in the maximal transport rate rather than the Km of the transporter. Pilot studies revealed uptake was linear for at least 3 min at 37°C (data not shown). In this study, cysteine accumulation was measured after 2 min of exposure to the radioisotope. After uptake of cysteine, culture dishes were rapidly rinsed three times with ice-cold 10 mM Tris (pH 7.3) containing sufficient sucrose to match the osmolality of the incubation PBS and culture medium. Cells were scraped from the dish in 0.6 M HClO4, and the suspension was centrifuged. The supernatant was removed for radioactive counting. The pellet was solubilized in 1 M NaOH for determination of protein content (21). In some studies, the PBS was removed and the cells were rinsed with the sucrose solution immediately after radiolabeled cysteine was added to the culture dish. [14C]cysteine contents of cells treated in this manner were subtracted from the contents of cells incubated for 2 min.
Enzyme activities. CDO activity was determined by a modification of methods described by Daniels and Stipanuk (9). Cells were scraped from the culture dish in 50 mM KH2PO4 (pH 7) and sonicated briefly. Protein was measured in an aliquot of the resulting astrocyte-buffer homogenate (21); 900 µl of the remaining homogenate were added to 2.1 ml of an ice-cold incubation mixture to give final concentrations of (in mM) 4 NAD+, 0.5 Fe(NH4)2(SO4)2, 5 NH2OH · HCl, 50 KH2PO4 (pH 7), and 5 cysteine plus 3 µCi/ml [35S]cysteine (<1,000 Ci/mmol, Amersham Life Science). The final reaction mixture osmolality was ~150 mosmol/kg. In some studies, the substrate concentration dependence of CDO activity was determined over the range of 0.5-10 mM cysteine. In other studies, the osmolality of the reaction mixture was raised to 300 or 450 mosmol/kg by adding additional KCl. All reactions were carried out at 37°C in 25-ml flasks placed in a shaking water bath for 30 min. The reaction was terminated with the addition of 1.5 ml of 2 mM cysteine sulfinate in 15% (wt/vol) trichloroacetic acid. The entire contents of each flask were transferred to a centrifuge tube and spun for 10 min at 2,000 g. The resulting supernatant was applied to a 0.6 × 5-cm Dowex 50-X8 (200-400 mesh, hydrogen form) column. [35S]cysteine sulfinate was eluted from the column with 10 ml 0.5% polyethylene lauryl ether (Brij 35) in water, and total radioactivity was determined. Blank reactions were performed using astrocyte homogenates that had been inactivated in a boiling water bath for 2 min. Results from these studies were subtracted from activities measured with experimental cell homogenates.
CSD activity was determined by a modification of methods previously described (9, 47). The substrate for the CSD reaction, [14C]cysteine sulfinate, was prepared enzymatically, and the specific activity of the carboxyl moiety was determined as described by Daniels and Stipanuk (9). Cells were scraped from the dish, and protein content was determined as described above for CDO; 750 µl of the remaining astrocyte-buffer homogenate were added to an equal amount of ice-cold incubation mixture, giving final concentrations of (in mM) 30 [14C]cysteine sulfinate (~0.0167 µCi/ml), 0.75 pyridoxal 5' phosphate, 0.5 dithiothreitol, and 250 KH2PO4 (pH 7). The mixture was placed in a sealed 25-ml flask with a center well containing 300 µl of 2-aminoethanol-ethylene glycol (1:2 vol/vol) and folded filter paper. After 60 min at 37°C, the reaction was terminated by injecting 1 ml of 10% (wt/vol) trichloroacetic acid into the reaction mixture. The reaction flask was left for an additional 60 min to completely trap all evolved [14C]CO2. Contents of the center well were then removed for radioactivity counting. Results from blank reactions, carried out using heat-inactivated cell homogenates, were subtracted from results from experimental cell homogenates. A commercial kit (Sigma Diagnostics, St. Louis, MO) was used to determineData analysis. Data were analyzed by analysis of variance (Systat, Chicago, IL). Post hoc analysis using Dunnett's test for multiple comparisons with a control population was used as appropriate. Differences between experimental groups were accepted as significant only when P < 0.05. Kinetic parameters were calculated using nonlinear (Quasi-Newton) curve-fitting procedures (Systat), assuming Michaelis-Menten kinetics.
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RESULTS |
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Total culture taurine content. Total taurine content of the culture dish (medium plus cells) was significantly elevated 1, 8, 24, and 48 h after changing to a fresh culture medium (Table 1). As we previously described, at each time point the majority of total culture taurine was in the growth medium (6). The percentage of taurine contained in the medium varied from a maximum of 83% at 1 h to a minimum of 61% at 24 h. Taurine cellular content (per mg protein) and calculated intracellular concentration (in mM) decreased 1 h after the medium change but returned to the initial value by the 48-h time point. Whereas total culture taurine content increased by 42%, total protein content of the culture dish increased by only 22% over the 48-h experimental period (Table 1).
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Taurine synthesis.
The mean ± SE rate of conversion of extracellular
[35S]cysteine to
[35S]taurine in
astrocytes was 21.2 ± 2.0 pmol · mg
protein1 · min
1.
Cells treated with BSO and CDNB to eliminate GSH synthesis and intracellular GSH content converted extracellular cysteine to taurine
at a significantly (P < 0.02) faster
rate of 29.5 ± 2.3 pmol · mg
protein
1 · min
1.
Hyperosmotic treatment did not effect the rate of conversion of
extracellular cysteine to taurine in untreated cells but significantly (P < 0.01) decreased this rate in
cells treated with BSO and CDNB (24 h, 400 mosmol/kg, 13.9 ± 2.0 pmol · mg
protein
1 · min
1).
Taurine degradation.
The fractional recovery of radioactivity eluted as derivitized
[3H]taurine by HPLC
was 0.262 ± 0.020 (mean ± SE) using a standard cocktail of
amino acids. This fractional recovery is less than unity because of
degradation of the fluorescent product during the time before elution
from the column (14, 20). By comparison, 24 and 48 h after cells were
loaded with radioactive taurine, 0.299 ± 0.012 and 0.302 ± 0.013 of the total radioactivities in the cell and medium supernatants,
respectively, were recovered as radioactive derivitized taurine. These
fractional recoveries are not significantly different from that
measured in the standard cocktail. However, because of uncertainties in
the actual mean values, we performed regression analysis using the
fractional recovery from the standard cocktail as the baseline (0 h)
value. This analysis yielded a mean (±SE) rate of change in taurine
fractional recovery of 0.020 ± 0.011 per day. Using the calculated
intercept at 0 h (0.268) and the lower bound of the 95% confidence
interval for the rate of change in fractional recovery (0.00349
per day), we computed a maximal taurine degradation rate of 1.3% per
day.
Cellular cysteine and GSH contents. Cellular cysteine and GSH contents increased significantly within 1 h of medium change and returned to initial values by 48 h (Table 1). Calculated cysteine concentration increased more than sixfold during the experimental period following the medium change. Hyperosmotic treatment caused even larger increases in cysteine and GSH contents 8 and 24 h after the medium change than that observed in control cultures measured at the same time point (Fig. 1).
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Cysteine influx. The rate of cysteine accumulation did not change over the 48-h experimental period in control cultures (Fig. 2). However, after 1 and 8 h, cells grown in a 400 and 450 mosmol/kg culture medium had rates of cysteine accumulation that were 12-23% greater than those measured in control cultures. The rate of cysteine uptake returned to (400 mosmol/kg) or went below (350, 450 mosmol/kg) that of control cultures 24 and 48 h after the medium change.
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Biosynthetic enzyme activities.
CDO activity measured directly from homogenates of isosmotic control
cultures was 2.37 ± 0.23 nmol · mg
protein1 · min
1
before medium change. This activity varied over the subsequent 48-h
experimental period, reaching a maximal value of 3.55 ± 0.34 nmol · mg
protein
1 · min
1
24 h after the medium change. CSD activity (mean ± SE) in
homogenates of cells grown in 300 mosmol/kg culture medium was 8.25 ± 1.46 nmol · mg
protein
1 · min
1.
-GT activity was constant in control cultures throughout the experimental period, with a mean ± SE value of 0.778 ± 0.041 nmol · mg
protein
1 · min
1.
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LDH efflux. Release of LDH from cells into the culture medium provides a measure of cell viability (16). LDH activity of control cells scraped and sonicated in their culture medium was 21.4 nmol NADH/min, a value 6.2 times the activity measured in fresh culture medium. LDH activity of medium removed from cultures in each experimental condition (isosmotic, hyperosmotic, and following BSO and CDNB treatments) was slightly less than, or not different from, the activity measured in fresh culture medium (data not shown).
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DISCUSSION |
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These studies examined several biochemical and physiological mechanisms
that may regulate taurine synthesis and accumulation in astrocytes.
From our data, we calculate a mean rate of taurine production of 19 pmol · mg
protein1 · min
1
by dividing the increase in total culture taurine over 48 h by the
average protein content per dish (0.21 mg). This value is similar to
that measured from the conversion of extracellular [35S]cysteine to
cellular taurine. This rate of taurine synthesis from extracellular
cysteine would permit complete replacement of astrocyte taurine in 2 days. In contrast, degradation is <3% over a 48-h period. Our direct
measurements of
[35S]taurine
production from
[35S]cysteine and
quantification of CDO and CSD activities represent the first
demonstration that cultured astrocytes are capable of synthesizing
taurine from extracellular cysteine. These results support the glial
localization of CSD shown by Almarghini et al. (1) and suggest
astrocytes are major contributors to organic osmolyte synthesis in the
brain. Comparing relative rates of cysteine accumulation and CDO and
CSD activities suggests CDO is rate limiting for taurine synthesis.
Taurine synthesis from extracellular cysteine is enhanced if turnover
of the GSH pathway is eliminated, suggesting catabolism of endogenous
unlabeled GSH contributes to the pool of cysteine utilized by CDO.
Finally, this rate of taurine synthesis from extracellular cysteine is
supported by a robust rate of cysteine accumulation.
Similar to our previous data (6), total culture taurine (medium + cells) and cellular taurine concentration increase following exposure
to hyperosmotic medium. The calculated rate of increase of total
culture taurine over the 48-h experimental period for cells in 450 mosmol/kg medium is 26 pmol · mg
protein1 · min
1.
This value is 37% higher than that calculated for control cells maintained in 300 mosmol/kg medium (18 pmol · mg
protein
1 · min
1).
By comparison, maximal taurine degradation in isosmotic conditions (1.3% per day) is 0.24 pmol · mg
protein
1 · min
1.
Thus inhibition of taurine degradation by hyperosmotic exposure cannot
explain the increase in total culture taurine. These results strongly
suggest hyperosmotic exposure leads to increased taurine synthesis. A
similar increase in taurine synthesis of astrocytes in situ would
contribute to the elevated taurine content observed in brains of
animals exposed to systemic hyperosmolality (4, 45). Although
intracellular taurine concentrations increase in our cell culture
system, a significant portion of the total culture taurine appears in
the extracellular fluid, a volume much larger than the intracellular
space. Similar efflux of newly synthesized taurine in situ would
rapidly elevate the extracellular taurine concentration, thus further
limiting cellular taurine loss and increasing cellular reuptake.
Taurine is a major contributor to the increase in total brain amino acids in animal models of hypernatremia (4, 44), as well as in astroglial cultures exposed to hyperosmotic medium (25, 36). This increase in situ may be due to increased synthesis, increased influx, or decreased transport of taurine out of brain parenchyma. In the present studies, total culture taurine content was increased for each hyperosmotic condition, indicating taurine synthesis is accelerated throughout the range of osmolalities tested. We anticipate astroglial taurine synthesis is similarly increased in comparable models of hypernatremia, in which experimental serum osmolalities range from 335 to 400 mosmol/kg (4, 19, 44).
Taurine flux from blood to brain does not increase during acute hyperosmotic hypernatremia (40). Enhanced taurine uptake (6, 36), and decreased taurine efflux by astrocytes in situ (6), may cause intracellular taurine sequestration; however, these changes in cellular transport cannot directly increase brain taurine content. Increased glial accumulation of taurine may explain the decrease in extracellular taurine concentration described in hyperosmotic animals (18). Depending on the mechanism of brain taurine efflux, this decrease in extracellular taurine concentration may lead to decreased efflux. The contribution that changes in de novo synthesis make to the increase in total brain taurine must await quantitative data regarding the rate of taurine efflux across the blood-brain barrier of normal and hypernatremic animals.
In previous studies, we have shown that enhanced taurine uptake and inhibition of the taurine efflux pathway contribute to elevated intracellular taurine concentrations following prolonged (>24 h) hyperosmotic exposure (6). The results presented here suggest enhanced de novo taurine synthesis also occurs with hyperosmotic exposure. However, activities of key enzymes in the taurine biosynthetic pathway are not altered by hyperosmotic exposure. In addition, no change in CDO activity was measured when the reaction mixture osmolality was raised to match that which the cells experienced during the experimental treatment period, suggesting that enzyme activity is not directly altered by hyperosmotic conditions inside the cell. Although these data indicate that the quantity of enzyme is unaltered by hyperosmolality, concentrations of substrates or enzymatic cofactors or enzyme phosphorylation states (41) not measured in these studies may be altered by the hyperosmotic treatment and thus may modify taurine biosynthesis in the cell. In particular, elevated intracellular cysteine concentrations caused by cell shrinkage and increased uptake would enhance, by kinetic means, the reaction velocity of CDO.
CDO activity is highly dependent on the concentration of cysteine in the reaction mixture. Thus taurine synthesis may be regulated by the substrate availability from the extracellular space or mobilization of intracellular cysteine pools. Cellular cysteine levels increased from 4.2 to 35.6 nmol/mg protein during hyperosmotic exposure (Fig. 1A), corresponding to intracellular cysteine concentrations ranging from <1 mM in isosmotic culture medium at 0 h to 5.6 mM in cultures treated in 450 mosmol/kg medium for 8 h. From Fig. 3, this elevation in cysteine concentration should increase CDO activity over sixfold. However, the production of intracellular taurine from extracellular cysteine is not increased in hyperosmotically treated cells. Conversion of extracellular [35S]cysteine to [35S]taurine is probably not limited by the cysteine influx rate, since, in all experimental conditions, cysteine uptake is rapid enough to equilibrate the radiolabel with the total intracellular cysteine content within the 60-min reaction period.
Cysteine utilized by CDO may be derived from extracellular or
intracellular pools. We found an increased synthesis rate of taurine
from extracellular cysteine when the -glutamyl cycle was inhibited
by BSO and the remaining GSH was eliminated by CDNB, suggesting
cysteine derived from GSH catabolism is shunted to taurine
biosynthesis. Conversely, cysteine taken up from the extracellular space may be preferentially used by the
-glutamyl cycle for GSH synthesis or another pathway. However, even with the GSH pathway eliminated, hyperosmotic treatment did not enhance taurine synthesis from extracellular cysteine.
Mechanisms surrounding the transient changes in cysteine and GSH contents in control (300 mosmol/kg) cultures cannot be explained by the results of this study. In previous experiments, we have shown significant decreases in intracellular taurine levels following a medium change (6). This decrease may be due to net efflux into fresh culture medium containing low concentrations of taurine or swelling-induced efflux caused by decreased osmolality of fresh growth medium. The reason for the increases in cysteine and GSH contents after 1 and 8 h is less apparent but may be a cellular response to the medium change. Nonetheless, taurine, cysteine, and GSH levels in control cells all returned to initial values by 24-48 h.
The transport system responsible for cysteine accumulation is not clear from our data, as both a sodium-independent, cystine-specific pathway and a faster sodium-dependent pathway for cysteine transport have been described in cultured astrocytes (8, 35). Although both cysteine and cystine may be present in the influx medium, Bannai (2) indicates that 90% or more of the substrate is present in the reduced form (cysteine) at the pH used during these experiments. Increased uptake in hyperosmotic conditions may be due to activation of one of these transporters, or, alternatively, increased concentrations of NaCl in the hyperosmotic experimental culture medium and influx incubation medium may enhance sodium-dependent cysteine uptake. Sodium dependence may contribute to the osmolality dependence of cysteine uptake at 1 and 8 h of hyperosmotic exposure but cannot account for decreases in accumulation that occur after 8 h. The transient increase in cysteine transport induced by hyperosmolality is similar to that described for taurine accumulation in these and other cell types (6, 28, 39). Additional experiments are necessary to describe the sodium dependence of cysteine uptake and the mechanisms responsible for increased transport rates in hyperosmotically treated astrocytes.
Elevations in cellular cysteine match the time course of increases in cysteine uptake following exposure to hyperosmotic conditions, suggesting enhanced rates of cysteine uptake directly lead to elevated intracellular cysteine levels. Because accumulation was measured with a high extracellular cysteine concentration, changes in uptake rate represent alterations in the maximal velocity of the transporter. Similar increases in uptake would also be expected at the lower physiological concentrations of cysteine that are likely to occur in situ. However, with the high extracellular concentration of cysteine used in these uptake experiments, any change in uptake due to an alteration in the transporter Km would not be detected. Sagara et al. (34, 35) and Bannai and Tateishi (3) showed that astrocyte GSH levels depend on the concentrations of cysteine or cystine in the culture medium. At all time points, astrocyte GSH contents are at least two times larger than the cellular free cysteine pool. In addition, cysteine uptake rates return to control values by 24 h while cellular cysteine levels remain elevated. This difference may represent a lag period between the initial accumulation and subsequent metabolism of cysteine to other cellular compounds.
Total -GT activity is not altered by exposing astrocytes to
hyperosmotic culture conditions, but the enzyme rate of reaction within
the cell may be decreased directly by hyperosmolality. These results
may explain increases measured in GSH contents and suggest amino acid
transport mediated by
-GT turnover (23) does not contribute to the
observed accumulation of amino acids during exposure to hyperosmotic
conditions (25). In addition, our data indicate that the increase in
cellular cysteine in hyperosmotic astrocytes is not due to enhanced
metabolism of GSH. Osmolality dependence of enzymatic activity also has
been described for pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate
dehydrogenase (EC 1.1.1.49), and LDH (EC 1.1.1.27), as well as others,
and may be related to changes in protein conformation due to altered
ionic strength (38, 48). In addition, the synthesis of sorbitol, another organic osmolyte, has been shown to increase in renal cells
subjected to increased osmolality due to an enhancement of aldose
reductase (EC 1.1.1.21) activity (43).
In summary, the accumulation and maintenance of high levels of intracellular taurine in astrocytes occur by several mechanisms. We previously reported that rapid taurine accumulation is enhanced by hyperosmotic exposure, whereas high intracellular taurine concentrations are maintained by decreases in taurine efflux (6). The present results suggest intracellular cysteine concentrations regulate taurine synthesis through the kinetic properties of CDO. During hyperosmotic exposure, increased levels of intracellular cysteine are metabolized to GSH and to taurine via CDO. Cellular GSH may be another accessible pool of cellular cysteine in astrocytes as it is in other cell types (11). This mechanism is supported, in part, by the increased rate of taurine synthesis from extracellular cysteine in GSH-depleted cells and the decline of cellular GSH during enhanced taurine synthesis in hyperosmotic cells.
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ACKNOWLEDGEMENTS |
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We sincerely thank Dr. Martha Stipanuk and her laboratory staff for their generous assistance and advice on cysteine metabolism. We also thank the Wright State University Biomedical Science Program and the Department of Emergency Medicine for financial assistance.
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FOOTNOTES |
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This work was supported by a grant awarded to J. E. Olson from the National Institutes of Health (NS-23218).
Address for reprint requests: J. E. Olson, Dept. of Emergency Medicine, Wright State Univ. School of Medicine, 3525 Southern Boulevard, Dayton, OH 45429.
Received 23 May 1997; accepted in final form 4 December 1997.
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REFERENCES |
---|
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---|
1.
Almarghini, K.,
A. Remy,
and
M. Tappaz.
Immunocytochemistry of the taurine biosynthetic enzyme, cysteine sulphinate decarboxylase in the cerebellum: evidence for a glial localization.
Neuroscience
43:
111-119,
1991[Medline].
2.
Bannai, S.
Transport of cystine and cysteine in mammalian cells.
Biochim. Biophys. Acta
779:
289-306,
1984[Medline].
3.
Bannai, S.,
and
N. Tateishi.
Role of membrane transport in metabolism and function of glutathione in mammals.
J. Membr. Biol.
89:
1-8,
1986[Medline].
4.
Bedford, J. J.,
and
J. P. Leader.
Response of the rat to anisosmotic conditions.
Am. J. Physiol.
264 (Regulatory Integrative Comp. Physiol. 33):
R1164-R1179,
1993
5.
Beetsch, J. W.,
and
J. E. Olson.
Taurine transport in rat astrocytes adapted to hyperosmotic conditions.
Brain Res.
163:
10-15,
1993.
6.
Beetsch, J. W.,
and
J. E. Olson.
Hyperosmotic exposure alters total taurine quantity and cellular transport in rat astrocyte cultures.
Biochim. Biophys. Acta
1290:
141-148,
1996[Medline].
7.
Bender, A. S.,
J. T. Neary,
T. Blicharska,
L.-O. B. Norenberg,
and
M. D. Norenberg.
Role of calmodulin and protein kinase C in astrocytic cell volume regulation.
J. Neurochem.
58:
1874-1882,
1992[Medline].
8.
Cho, Y.,
and
S. Bannai.
Uptake of glutamate and cystine in C-6 glioma cells and in cultured astrocytes.
J. Neurochem.
55:
2091-2097,
1990[Medline].
9.
Daniels, K. M.,
and
M. H. Stipanuk.
The effect of dietary cysteine level on cysteine metabolism in rats.
J. Nutr.
112:
2130-2141,
1982[Medline].
10.
Griffith, O. W.,
and
A. Meister.
Potent and specific inhibition of glutathione synthesis by butathionine sulfoximine (S-n-butyl homocysteine sulfoximine).
J. Biol. Chem.
254:
7558-7560,
1979[Abstract].
11.
Hanigan, M. H.,
and
W. A. Ricketts.
Extracellular glutathione is a source of cysteine for cells that express -glutamyl transpeptidase.
Biochemistry
32:
6302-6306,
1993[Medline].
12.
Heilig, C. W.,
M. E. Stromski,
J. D. Blumenfeld,
J. P. Lee,
and
S. R. Gullans.
Characterization of the major brain osmolytes that accumulate in salt-loaded rats.
Am. J. Physiol.
257 (Renal Fluid Electrolyte Physiol. 26):
F1108-F1116,
1989
13.
Huxtable, R. J.
Taurine in the central nervous system and the mammalian actions of taurine.
Prog. Neurobiol.
32:
471-533,
1989[Medline].
14.
Jacobs, W. A.
o-Phthalaldehyde-sulfite derivatization of primary amines for liquid chromatography-electrochemistry.
J. Chromatogr. A
392:
435-441,
1987.
15.
Jocelyn, D. C.
Spectrophotometric assay of thiols.
In: Methods in Enzymology: Sulfur and Sulfur Amino Acids, edited by W. B. Jakoby,
and O. W. Griffith. Orlando, FL: Academic, 1987, vol. 143, p. 44-67.
16.
Koh, J. Y.,
and
D. W. Choi.
Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay.
J. Neurosci. Methods
20:
83-90,
1987[Medline].
17.
Kuriyama, K.,
S. Ida,
and
S. Ohkuma.
Alteration of cerebral taurine biosynthesis in spontaneously hypertensive rats.
J. Neurochem.
42:
1600-1606,
1984[Medline].
18.
Lehmann, A.
Effects of microdialysis-perfusion with anisoosmotic media on extracellular amino acids in the rat hippocampus and skeletal muscle.
J. Neurochem.
53:
525-535,
1989[Medline].
19.
Lien, Y.-H.,
J. I. Shapiro,
and
L. Chan.
Effects of hypernatremia on organic brain osmolytes.
J. Clin. Invest.
85:
1427-1435,
1990[Medline].
20.
Lindroth, P.,
and
K. Mopper.
High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde.
Anal. Chem.
51:
1667-1684,
1979.
21.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
22.
Makar, T. K.,
M. Nedergaard,
A. Preuss,
A. S. Gelbard,
A. S. Perumal,
and
J. L. Cooper.
Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes play an important role in antioxidative processes in the brain.
J. Neurochem.
62:
45-53,
1994[Medline].
23.
Meister, A.
Glutathione metabolism and its selective modification.
J. Biol. Chem.
263:
17205-17208,
1988
24.
Misra, C. H.,
and
J. W. Olney.
Cysteine oxidase in the brain.
Brain Res.
97:
117-126,
1975[Medline].
25.
Olson, J. E.,
and
M. D. Goldfinger.
Amino acid content of rat cerebral astrocytes adapted to hyperosmotic conditions in vitro.
J. Neurosci. Res.
27:
241-246,
1990[Medline].
26.
Olson, J. E.,
and
D. Holtzman.
Respiration in rat cerebral astrocytes from primary culture.
J. Neurosci. Res.
5:
497-506,
1980[Medline].
27.
Olson, J.,
and
D. Holtzman.
Respiration and cell volume of primary cultured cerebral astrocytes in media of various osmolarities.
Brain Res.
246:
273-279,
1982[Medline].
28.
Paredes, A.,
M. McManus,
H. Moo Kwon,
and
K. Strange.
Osmoregulation of Na+-inositol cotransporter activity and mRNA levels in brain glial cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C1282-C1288,
1992
29.
Raps, S. P.,
J. C. K. Lai,
L. Hertz,
and
J. L. Cooper.
Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons.
Brain Res.
493:
398-401,
1989[Medline].
30.
Ratan, R. R.,
T. H. Murphy,
and
J. M. Baraban.
Macromolecular synthesis inhibitors prevent oxidative stress-induced apoptosis in embryonic cortical neurons by shunting cysteine from protein synthesis to glutathione.
J. Neurosci.
14:
4385-4392,
1994[Abstract].
31.
Reichelt, K. L.,
and
E. Poulsen.
-Glutamyl aminotransferase and transglutaminase in subcellular fractions of rat cortex and in cultured astrocytes.
J. Neurochem.
59:
500-504,
1992[Medline].
32.
Remy, A.,
S. Henry,
and
M. Tappaz.
Specific antiserum and monoclonal antibodies against the taurine biosynthesis enzyme cysteine sulfinate decarboxylase: identity of brain and liver enzyme.
J. Neurochem.
54:
870-879,
1990[Medline].
33.
Richie, J. P.,
and
C. A. Lang.
The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection.
Anal. Biochem.
163:
9-15,
1987[Medline].
34.
Sagara, J.,
K. Miura,
and
S. Bannai.
Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspension.
J. Neurochem.
61:
1667-1671,
1993[Medline].
35.
Sagara, J.,
K. Miura,
and
S. Bannai.
Maintenance of neuronal glutathione by glial cells.
J. Neurochem.
61:
1671-1676,
1993.
36.
Sánchez-Olea, R.,
J. Morán,
and
H. Pasantes-Morales.
Changes in taurine transport evoked by hyperosmolality in cultured astrocytes.
J. Neurosci. Res.
32:
86-92,
1992[Medline].
37.
Slivka, A.,
and
G. Cohen.
Brain ischemia markedly elevates levels of the neurotoxic amino acid, cysteine.
Brain Res.
608:
33-37,
1993[Medline].
38.
Somero, G. N.
Proteins, osmolytes, and fitness of internal milieu for protein function.
Am. J. Physiol.
251 (Regulatory Integrative Comp. Physiol. 20):
R197-R213,
1986
39.
Strange, K.,
F. Emma,
A. Paredes,
and
R. Morrison.
Osmoregulatory changes in myo-inositol content and Na+/myo-inositol cotransport in rat cortical astrocytes.
Glia
12:
35-43,
1994[Medline].
40.
Stummer, W.,
A. L. Betz,
P. Shakui,
and
R. F. Keep.
Blood-brain barrier taurine transport during osmotic stress and in focal cerebral ischemia.
J. Cereb. Blood Flow Metab.
15:
852-859,
1995[Medline].
41.
Tang, X. W.,
C.-C. Hsu,
J. V. Schloss,
M. D. Faiman,
E. Wu,
C.-Y. Yang,
and
J.-Y. Wu.
Protein phosphorylation and taurine biosynthesis in vivo and in vitro.
J. Neurosci.
17:
6947-6951,
1997
42.
Tappaz, M.,
K. Almarghini,
F. Legay,
and
A. Remy.
Taurine biosynthesis enzyme cysteine sulfinate decarboxylase (CSD): the long and tricky trail to identification.
Neurochem. Res.
17:
849-859,
1992[Medline].
43.
Trachtman, H.
Cell volume regulation: a review of cerebral adaptive mechanisms and implications for clinical treatment of osmolal disturbances. I.
Pediatr. Nephrol.
5:
743-750,
1991[Medline].
44.
Trachtman, H.,
R. Barbour,
J. A. Sturman,
and
L. Finberg.
Taurine and osmoregulation: taurine is a cerebral osmoprotective molecule in chronic hypernatremic dehydration.
Pediatr. Res.
23:
35-39,
1988[Abstract].
45.
Trachtman, H.,
S. Futterweit,
and
R. del Pizzo.
Taurine and osmoregulation. IV. Cerebral taurine transport is increased in rats with hypernatremic dehydration.
Pediatr. Res.
32:
118-124,
1992[Abstract].
46.
Viña, J. R.,
A. Devesa,
C. Garcia,
I. Puertes,
and
E. O'Connor.
Maintenance of GSH content in primary astrocyte cultures under oxidative stress conditions (Abstract).
Biochem. Soc. Trans.
21:
74S,
1992.
47.
Worden, J. A.,
and
M. H. Stipanuk.
A comparison by species, age, and sex of cysteine sulfinate decarboxylase activity and taurine concentration in liver and brain of animals.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
82B:
233-239,
1985.
48.
Yancey, P. H.,
M. E. Clark,
S. C. Hand,
R. D. Bowlus,
and
G. N. Somero.
Living with water stress: evolution of osmolyte systems.
Science
217:
1214-1222,
1982[Medline].
49.
Yudkoff, M.,
D. Pleasure,
L. Cregar,
Z.-P. Lin,
I. Nissim,
J. Stern,
and
I. Nissim.
Glutathione turnover in cultured astrocytes: studies with [15N]glutamate.
J. Neurochem.
55:
137-145,
1990[Medline].