Journal of Neurochemisrry Raven Press, Ltd., New York Q 1992 International Society for Neurochemistry
Biotin Transport in Primary Culture of Astrocytes: Effect of Biotin Deficiency Pilar Rodriguez-Pombo and Magdalena Ugarte Depurtumenio de Biologia Molecular, Facultad de Ciencias, Universidud Autbnoma de Madrid, Mudrid, Spain
~
Abstract: The uptake of radioactive biotin has been studied in glial cell cultures from dissociated cerebral hemispheres of newborn rats. We describe saturable kinetics for the biotin uptake at biotin concentrations of 60 M. The uptake appeared temperature sensitive, Na+ independent, nonsensitive to valinomycin, and not affected by metabolic inhibitors such as sodium fluoride or azide. Lipoic acid and biocytin were effective in inhibiting the biotin uptake. These findings are consistent with biotin uptake by the primary culture of astrocytes as a process of facilitated dif-
fusion. Moreover, biotin uptake in astrocytes grown in biotinrestricted conditions was significantly higher compared with the control. This increase appeared mediated through a pronounced increase (10-fold) in the V,,, of the biotin uptake without any change in the apparent K,. Key Words: Astrocytes-Biotin-Transport-Biotin deficiency. RodriguezPombo P. and Ugarte M. Biotin transport in primary culture of astrocytes: Effect of biotin deficiency. J. Neurochern. 58, 1460-1463 (1992).
Biotin is an essential water-soluble vitamin that is required for normal cellular function, growth, and development. Biotin acts as a prosthetic group for enzymes in which the biotin moiety functions as a mobile carboxyl camer in several different carboxylation reactions in the brain and in other tissues. Biotin deficiency in humans leads to serious clinical abnormalities, including growth retardation, skin abnormalities, and neurologic disorders (Sweetmanand Nyhan, 1986). Several authors have speculated that there is a homeostatic system in the CNS that regulates the concentration of total biotin in the brain (Bhagavan and Coursin, 1970; Murthy and Mistry, 1977). The regulatory mechanism to maintain the biotin levels in the brain, however, has not been sufficiently clarified. Because biotin is not synthesized in the body, biotin must enter the brain from blood. Spector and Mock ( 1987), using an in situ rat brain perfusion technique, have described the transfer of biotin through the cerebral capillaries of the blood-brain barrier by a saturable camer-mediated transport system with a half-maximal saturation concentration of 100 pM. Transport studies on the intact nervous system are difficult to evaluate owing to the cellular heterogeneity of the tissue. In the last few years, separate cultures of
neuronal and glial cells have been developed (Sensenbrenner, 1977), providing a model system to study transport at the cellular level. Recently, we reported the validity of primary cultures of astrocytes from rats as an experimental approach to the study of biotin deficiency in nervous tissue (Rodriguez-Pombo et al., 1992). Here, we report on the uptake of [3H]biotinby rat brain astrocytes and the effect of biotin deficiency on this uptake.
Received May 17, 1991 ; revised manuscript received September 10, 1991; accepted September 19, 1991. Address correspondence and reprint requests to Dr. M. Ugarte at Departamento de Biologia Molecular, Facultad de Ciencias, Universidad Autbnoma, 28049 Madrid, Spain.
Abbreviations used: MEM, modified Eagle’s minimal essential medium; KRP solution, Krebs-Ringer phosphate solution.
MATERIALS AND METHODS Materials Cell culture dishes were obtained from Costar Europe. Modified Eagle’s minimal essential medium (MEM) and fetal calf serum were from Flow Laboratories. Avidin, azide, biotin, biocytin, choline chloride, lipoic acid, L-glutamate, sodium fluoride, and valinomycin were from Sigma. All other chemicals were of analytical quality. d-[8,9-’H]Biotin (specificradioactivity, 35 Ci/mmol) was purchased from New England Nuclear, and ~-[U-’~C]glutamate (specific radioactivity, 285 mCi/mmol) was from Amersham.
-
Cell culture Primary astroglial cell cultures were prepared by aseptically isolating cerebral hemispheres of newborn Wistar rats ac-
1460
BIOTIN TRANSPORT IN ASTROCYTES cording to a modification of a previously described method (Booher and Sensenbrenner, 1972). The tissue was freed of meninges and capillary vessels. Mechanical dissociation was achieved by sequential passage through two needles of decreasing diameter (0.9 and 0.7 mm) in the presence of 20% serum-enriched medium [MEM containing 2 mMglutamine, 200,000 U/L of penicillin, 100 mg/L of streptomicin, and 20% (vol/vol) heat-inactivated fetal calf serum]. The resulting cell suspension was seeded into multidish culture plates at a density of 2.5 X lo5 cell/cm2. The culture dishes were incubated at 37°C with a humidified atmosphere of 5% (vol/ vol) COz in air. The medium was changed on day 2 and subsequently every 3-4 days. The serum content was reduced to 10% (wt/vol) at the end of the first week. At this time, biotin-deficient astrocytes were obtained by growing the cells in 10%serum-enriched medium supplemented with 50 U/L of avidin. Cells were used in transport experiments on day 2 1. In our laboratory, the cells were identified as astrocytes by the presence of glial fibrillary acidic protein.
Functional evaluation of astrocytes To determine the suitability of our primary culture of astrocytes for transport studies, we studied the kinetic parameters and Na+ dependence of L-glutamate uptake in control and biotindeficient astrocytes in accordance with the method described below. The biotin deficiency did not greatly modify the kinetic constants and Na*-dependent transport of ~-glutamate. The kinetic constants determined were a K, of 200 p M and a V,,, of 25 nmol/min/mg of protein (data not shown). The replacement of sodium with lithium chloride reduced by 10% the uptake of 10 pM L-glutamate. These observations are in agreement with those of Schousboe et al. (1977).
Uptake studies Cellular uptake was examined in cells plated in 24-well trays, the growth medium was discarded, and cells were rinsed four times with 1 ml of Krebs-Ringer phosphate (KRP) solution (20 mM NaC1, 4.75 mM KCl, 1.2 mM MgS04, 1.2 mMCaCI2, 10 mMNa2HP04, and 20 mMglucose, pH 7.4), unless othewise stated, and allowed to equilibrate for 30 rnin at 37°C in 0.25 ml of the same solution. d-[8,9-3H]Biotinat concentrations ranging from 7 to 420 nM or L-[U14C]glutamateat concentrations between 1OW5 and M was added to the wells in triplicate. At the appropriate time ( I rnin for biotin or 4 rnin for L-glutamate), the medium was removed by suction, and cells were quickly washed five times with I ml of ice-cold KRP solution. After air-drying, cells were dissolved in 0.25 ml of 1 M NaOH overnight at 4°C. A portion (100 pl) of each sample was combined with scintillation cocktail. The radioactivity content was measured by liquid scintillation counting. An aliquot of the remaining NaOH-solubilized cells was used for protein quantification (Lowry et al., 1951). Blank values were obtained by incubation of the cultures at the time closest to 0. For measurement of nonspecific (diffusional)biotin uptake, samples were incubated in reaction mixture containing an excess amount (50 p M ) of biotin in addition to the labeled substrate. Net specific uptake was calculated by subtracting the nonspecific from the total uptake and expressed as femtomolesper minute per milligram of protein.
Statistics Unless otherwise stated in the table and figure legends, data are mean ? SD values from triplicate determinations
1461
for at least two different cultures. The V,, and apparent K , values were calculated using an iterative, nonlinear, leastsquare fitting program. Differencesbetween mean values were evaluated using Student's two-tailed t test.
RESULTS Characterization of biotin transport Uptake of [3H]biotinby primary culture of astrocytes was studied at two different temperatures, 37 and 4°C (Fig. 1). The rate of uptake decreased as the temperature was lowered. The time course of [3H]biotinuptake at 37°C was linear with time up to 1 rnin and peaked after 5 rnin of incubation. At 4°C the time course of [3H]biotinwas linear with time at least until 10 min. Kinetic studies were performed by measuring the rates of uptake of [3H]biotinat different concentrations of substrate. The lower value was imposed by the sensitivity of the radiochemical method used. As is shown in Fig. 2, the range of the saturation for the biotin uptake at a substrate concentration was between 7 and 60 nM. These saturation kinetics was even clearer when values were corrected for the rates corresponding to the nonspecific component experimentally measured as described above. Kinetic constants of the resultant saturable process were then determined using a fitting curve program and found to be a K, of I 1.7 nM and a V,,, of 63 fmol/min/mg of protein. At [3H]biotin concentrations between 60 and 420 nM, the uptake was linear and corresponded to a nonsaturable system (data not shown). NaCl replacement with KCI or choline chloride or incubation of astrocytes in the absence of CaCI2 after M EGTA, a wash with KRP solution containing to eliminate traces of Ca2+,did not greatly modify the uptake of 28 nM [3H]biotin(data not shown). The influence of membrane potential on biotin uptake by astrocytes was tested by examining the effect of 5 p M valinomycin in the presence of an outwardly directed K+ gradient. The inside negative membrane potential failed to stimulate the initial 20 nM [3H]biotin uptake (data not shown). The energy dependence of biotin transport was studied by measuring the intracellular uptake by as-
-
Time (min)
FIG. 1. Effect of temperature on the time course of [3H]biotin uptake by primary culture of astrocytes. Cells were incubated for different times in KRP solution containing 57 nM [3H]biotin at 37 (0)or 4°C (0).
J. Neurochem., Vol. 58. No. 4, 1992
P. RODRIGUEZ-POMBO AND M. UG'4RTE
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FIG. 3. Effect of biotin deficiency on biotin uptake into cultured astrocytes. Cells cultured in control (C)or biotin-restrictedconditions (D) were incubated for 1 min at 37°C in KRP solution containing 14 nM t3H]biotin. [Biotin] (nM)
FIG. 2. Biotin uptake into cultured astrocytes at biotin concentrations ranging from 7 to 60 nM. Cells were incubated for 1 min at 37°C in KRP solution with radioactive biotin at the indicated concentration (0;total uptake) or radioactive biotin plus 50 pA4 unlabeled biotin (0;unspecific uptake).The specific biotin uptake (dotted line) was obtained by subtracting the unspecific uptake from the total uptake at each concentrationof biotin.
trocytes pretreated with azide or sodium fluoride at 1 mM for 60 min. None of the inhibitors had an effect on the process (data not shown). Table 1 shows the effect on the saturable biotin uptake of biotin analogues with variations in the valeric acid side chain, such as biocytin, or in the ring, such as lipoic acid. A very significant reduction of the net biotin uptake was observed in the presence of lipoic acid a lesser, but also significant, reduction in the biotin uptake was observed in the presence of biocytin. Effect of biotin deficiency on biotin uptake When the cells were maintained in medium restricted in biotin for 14 days, the total uptake of 14 nM [3H]biotin was 10-fold higher than the levels measured in control cells (Fig. 3). To determine whether the enhancement in biotin transport was associated with variations in the K,,, or VmaXof the saturable uptake, we studied the kinetic properties of [3H]biotin uptake in biotin-deficient astrocytes in the range of [3H]biotin concentrations between 6 and 320 M ( F i g . 4). A saturable system could be observed. Kinetic constants were determined by fitting the data to b' = Vmax
where the V,,,
[1/(1
K,,, is 18 nM. The rate constant of the unsaturable component kdir (0.87 fmol/min/mg of protein) was calculated by computer analysis (see Statistics). DISCUSSION The present study examines the transport of biotin across the plasma membrane of cultured astrocytes. Purity and suitability of the primary culture of astrocytes used in this study have been confirmed by immunocytochemical and functional criteria. Uptake of biotin by astrocytes was saturable at low concentrations (7-60 M) but linear at >60 nM biotin. An uptake mechanism with a double component had been extensively described; however, the K,,, values reported for the biotin transport in several tissues and studies were different (Dakshinamurti et al., 1987; Said et al., 1987; Spencer and Roth, 1988). This apparent discordance could be attributed to different methodological approaches. In rats, the plasma concentration of biotin was -20 nM, and cerebral biotin content was higher than in liver or serum in vivo (Bhagavan and Coursin, 1970). In this sense, the K , value described here for the biotin uptake in astrocytes, 11.7 M, would be in the range of physiological concentrations of biotin. Biotin transport in astrocytes appears to be a temperature-sensitive process, and the linearity observed with time at 4°C was compatible with a passive diffusion process. Metabolic inhibitors such as sodium
+ K m / s ) ] + kdiffs I
is 760 fmol/min/mg of protein and the
TABLE 1. Efect of structural analogues on the saturable biotin uptake in primary culture of astrocytes ~~
~
Structural analogue
Net biotin uptake (fmol/min/mg of protein)
None Biotin (50 FLM) Biocytin (50 p M ) Lipoic acid (50 p M )
51.7 2 5.4
0 33.7 f 2 18.5 f 5
Inhibition (%)
0 100" 3s6 65'
Cells were incubatedfor I min at 37°C in KRP solution containing 28 nM 13H]biotinand the compounds indicated. Corrections were made for the unsaturable component of the uptake. p < 0.0005, ' p < 0.05, ' p < 0.005 (Student's two-tailed i test).
J. Neurochem.. c'ol. 58. No. 4. l Y Y 2
-
0 100
200
300
[ e i o i i n ] (nM)
FIG. 4. Biotin uptake into biotin-deficientastrocytes as a function of biotin concentration. Cells cultured in biotin-restricted conditions were incubated for 1 min at 37°C in KRP solution with radioactive biotin at the indicated concentrations (0;total uptake). The specific biotin uptake (dotted line) was obtained by subtracting the unspecific uptake from the total uptake.
BIOTIN TRANSPOR T IN A STR OC YTES
fluoride or azide, however, had no effect on biotin transport, demonstrating that the process is not ATP dependent. Moreover, biotin uptake is unaffected by Na+ gradient replacement by K' or choline+;therefore, secondary active transport involving these ions appears unlikely. Biotin uptake was not an electrogenic process, demonstrated by the noneffect of valinomycin. To assess the specificity of the biotin transport in astrocytes, two structural analogues have been tested. Lipoic acid was a very effective inhibitor of the initial rate of biotin uptake, suggesting that the thiophane ring is necessary for recognition by the cell. The significant inhibition observed with biocytin suggests that the valeric acid side chain must also be accounted for to describe this process of recognition. The specificity discovered for the biotin transport system in astrocytes appears similar to that reported for the biotin uptake by another mammalian cell (Cohen and Thomas, 1982; Gore and Hoinard, 1987). In conclusion, biotin transport by astrocytes exhibits characteristics that are compatible with a facilitated diffusion; there are saturation kinetics toward substrate, uptake is apparently not energy dependent, and substrate specificity is considerable. Finally, the present study examines the effect of biotin deficiency on the uptake of biotin in astrocytes from rats. Biotin uptake in biotin-deficient astrocytes was 10-fold higher than in control cells. This increase could not be explained by a change in the cellular membrane permeability because we have demonstrated that Na+ gradient-dependent transport of L-glutamate in biotindeficient astrocytes was similar to that in control cells and comparable with that previously reported (Schousboe et al., 1977). The similarity found in the K, of the biotin transport process for control and biotin-deficient cells combined with the greatly increased V,,, obtained in the deficient cells could suggest a change in the number of the functional transport carriers in biotin-deficient astrocytes. However, the mechanism responsible for this change in V,,, requires further investigation. Whether this enhancement is due to an increase in the number of the carrier proteins, as has been proposed for bacterial cells (Prakash and Eisenberg, 1974), remains to be clarified. Moreover, these results suggest the capability of astrocytes to adapt to the challenge of deficiency of an essential nutrient. That capability may be especially important when deficiency in biotinidase occurs, as is the case for late-onset multiple carboxylase deficiency disease.
1463
Acknowledgment: We are indebted to Prof. F. Valdivieso for valuable discussions of the results in the progress of this study and for critical reading o f the manuscript. We also acknowledge the excellent technical assistance of Ms. Carmen Hernandez. This work was supported by an institutional grant from the Fondo de Investigaciones Sanitarias.
REFERENCES Bhagavan H. N. and Coursin D. B. (1 970) Depletion of biotin from brain and liver in biotin deficiency. J. Neurochem. 17,289-290. Booher J. and Sensenbrenner M. (1972) Growth and cultivation of dissociated neurons and dial cells from embryonic chick, rat, and human brain in flask cultures. Neurobiology 2, 97-105. Cohen N. D. and Thomas M. (1982) Biotin transport into fully differentiated 3T3-LI cells. Biochem. Biophys. Res Commun. 108, 1508-1 5 16. Dakshinarnurti K., Chauhan J., and Ebrahim H. (1987) Intestinal absorption ofbiotin and biocytin in the rat. Biosci. Rep. 7,667673. Gore J. and Hoinard C. (1987) Evidence for facilitated transport of biotin by hamster enterocytes. J . Nutr. 117, 527-532. Hansson E. (1985) Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures. Neurochem. Rex 10,667-675. Lowry 0.H., Rosebrough N. J., Farr A. L., and Randall R. J. ( I 95 1) Protein measurement with the Folin phenol reagent. J. Bid. Chem. 193,265-275. Murthy P. N. S. and Mistry S. P. (1 977) Biotin. Prog. Food Nutr. Sci. 2, 405-455. Prakash 0. and Eisenberg M. A. (1974) Active transport of biotin in Escherichia coli K-12. J. Bacteriol. 120, 785-79 I . Rodriguez-Pombo P., Sweetman L., and Ugarte M. (1992) Primary cultures of astrocytes from rat as a model for biotin deficiency in nervous tissue. Mol. Chem. Neuropathol. (in press). Said H. M. and Redha R. (1987) A camer-mediated system for transport of biotin in rat intestine in vitro. Am. J. Physiol. 252, G5255. Schoushoe A., Svenneby G., and Hertz L. (1977) Uptake and metabolism of glutamate in astrocytes from dissociated mouse brain hemispheres. J. Neurochem. 29, 999-1005. Sensenbrenner M. (1977) Dissociated brain cells in primary culture, in Cell Tissue and Organ Cultures in Neurobiology (Federoff S. and Hertz L., eds), pp. 191-2 13. Academic Press, New York. Shank R. P., Bennett G. S., Freytag S. O., and Campbell G. L. (1985) Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res. 329, 364-367. Spector R. and Mock D. (1987) Biotin transport through the bloodbrain bamer. J. Neurochem. 48, 400-404. Spencer P. D. and Roth K. S. (1988) On the uptake of biotin by the rat renal tubule. Biochem. Med. Metab. Biol. 40, 95-100. Sweetman L. and Nyhan W. L. (1986) Inheritable biotin-treatable disorders and associated phenomena. Annu. Rev. Niitr. 6, 3 17343. Yu A. C. H., Drejer J., Hertz L., and Schousboe A. (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J. Neurochem. 41, 1484-1487.
J . Neurochem., Vol. 58. No. 4, 1992
Biotin Transport in Primary Culture of Astrocytes: Effect of Biotin Deficiency Pilar Rodriguez-Pombo and Magdalena Ugarte Depurtumenio de Biologia Molecular, Facultad de Ciencias, Universidud Autbnoma de Madrid, Mudrid, Spain
~
Abstract: The uptake of radioactive biotin has been studied in glial cell cultures from dissociated cerebral hemispheres of newborn rats. We describe saturable kinetics for the biotin uptake at biotin concentrations of 60 M. The uptake appeared temperature sensitive, Na+ independent, nonsensitive to valinomycin, and not affected by metabolic inhibitors such as sodium fluoride or azide. Lipoic acid and biocytin were effective in inhibiting the biotin uptake. These findings are consistent with biotin uptake by the primary culture of astrocytes as a process of facilitated dif-
fusion. Moreover, biotin uptake in astrocytes grown in biotinrestricted conditions was significantly higher compared with the control. This increase appeared mediated through a pronounced increase (10-fold) in the V,,, of the biotin uptake without any change in the apparent K,. Key Words: Astrocytes-Biotin-Transport-Biotin deficiency. RodriguezPombo P. and Ugarte M. Biotin transport in primary culture of astrocytes: Effect of biotin deficiency. J. Neurochern. 58, 1460-1463 (1992).
Biotin is an essential water-soluble vitamin that is required for normal cellular function, growth, and development. Biotin acts as a prosthetic group for enzymes in which the biotin moiety functions as a mobile carboxyl camer in several different carboxylation reactions in the brain and in other tissues. Biotin deficiency in humans leads to serious clinical abnormalities, including growth retardation, skin abnormalities, and neurologic disorders (Sweetmanand Nyhan, 1986). Several authors have speculated that there is a homeostatic system in the CNS that regulates the concentration of total biotin in the brain (Bhagavan and Coursin, 1970; Murthy and Mistry, 1977). The regulatory mechanism to maintain the biotin levels in the brain, however, has not been sufficiently clarified. Because biotin is not synthesized in the body, biotin must enter the brain from blood. Spector and Mock ( 1987), using an in situ rat brain perfusion technique, have described the transfer of biotin through the cerebral capillaries of the blood-brain barrier by a saturable camer-mediated transport system with a half-maximal saturation concentration of 100 pM. Transport studies on the intact nervous system are difficult to evaluate owing to the cellular heterogeneity of the tissue. In the last few years, separate cultures of
neuronal and glial cells have been developed (Sensenbrenner, 1977), providing a model system to study transport at the cellular level. Recently, we reported the validity of primary cultures of astrocytes from rats as an experimental approach to the study of biotin deficiency in nervous tissue (Rodriguez-Pombo et al., 1992). Here, we report on the uptake of [3H]biotinby rat brain astrocytes and the effect of biotin deficiency on this uptake.
Received May 17, 1991 ; revised manuscript received September 10, 1991; accepted September 19, 1991. Address correspondence and reprint requests to Dr. M. Ugarte at Departamento de Biologia Molecular, Facultad de Ciencias, Universidad Autbnoma, 28049 Madrid, Spain.
Abbreviations used: MEM, modified Eagle’s minimal essential medium; KRP solution, Krebs-Ringer phosphate solution.
MATERIALS AND METHODS Materials Cell culture dishes were obtained from Costar Europe. Modified Eagle’s minimal essential medium (MEM) and fetal calf serum were from Flow Laboratories. Avidin, azide, biotin, biocytin, choline chloride, lipoic acid, L-glutamate, sodium fluoride, and valinomycin were from Sigma. All other chemicals were of analytical quality. d-[8,9-’H]Biotin (specificradioactivity, 35 Ci/mmol) was purchased from New England Nuclear, and ~-[U-’~C]glutamate (specific radioactivity, 285 mCi/mmol) was from Amersham.
-
Cell culture Primary astroglial cell cultures were prepared by aseptically isolating cerebral hemispheres of newborn Wistar rats ac-
1460
BIOTIN TRANSPORT IN ASTROCYTES cording to a modification of a previously described method (Booher and Sensenbrenner, 1972). The tissue was freed of meninges and capillary vessels. Mechanical dissociation was achieved by sequential passage through two needles of decreasing diameter (0.9 and 0.7 mm) in the presence of 20% serum-enriched medium [MEM containing 2 mMglutamine, 200,000 U/L of penicillin, 100 mg/L of streptomicin, and 20% (vol/vol) heat-inactivated fetal calf serum]. The resulting cell suspension was seeded into multidish culture plates at a density of 2.5 X lo5 cell/cm2. The culture dishes were incubated at 37°C with a humidified atmosphere of 5% (vol/ vol) COz in air. The medium was changed on day 2 and subsequently every 3-4 days. The serum content was reduced to 10% (wt/vol) at the end of the first week. At this time, biotin-deficient astrocytes were obtained by growing the cells in 10%serum-enriched medium supplemented with 50 U/L of avidin. Cells were used in transport experiments on day 2 1. In our laboratory, the cells were identified as astrocytes by the presence of glial fibrillary acidic protein.
Functional evaluation of astrocytes To determine the suitability of our primary culture of astrocytes for transport studies, we studied the kinetic parameters and Na+ dependence of L-glutamate uptake in control and biotindeficient astrocytes in accordance with the method described below. The biotin deficiency did not greatly modify the kinetic constants and Na*-dependent transport of ~-glutamate. The kinetic constants determined were a K, of 200 p M and a V,,, of 25 nmol/min/mg of protein (data not shown). The replacement of sodium with lithium chloride reduced by 10% the uptake of 10 pM L-glutamate. These observations are in agreement with those of Schousboe et al. (1977).
Uptake studies Cellular uptake was examined in cells plated in 24-well trays, the growth medium was discarded, and cells were rinsed four times with 1 ml of Krebs-Ringer phosphate (KRP) solution (20 mM NaC1, 4.75 mM KCl, 1.2 mM MgS04, 1.2 mMCaCI2, 10 mMNa2HP04, and 20 mMglucose, pH 7.4), unless othewise stated, and allowed to equilibrate for 30 rnin at 37°C in 0.25 ml of the same solution. d-[8,9-3H]Biotinat concentrations ranging from 7 to 420 nM or L-[U14C]glutamateat concentrations between 1OW5 and M was added to the wells in triplicate. At the appropriate time ( I rnin for biotin or 4 rnin for L-glutamate), the medium was removed by suction, and cells were quickly washed five times with I ml of ice-cold KRP solution. After air-drying, cells were dissolved in 0.25 ml of 1 M NaOH overnight at 4°C. A portion (100 pl) of each sample was combined with scintillation cocktail. The radioactivity content was measured by liquid scintillation counting. An aliquot of the remaining NaOH-solubilized cells was used for protein quantification (Lowry et al., 1951). Blank values were obtained by incubation of the cultures at the time closest to 0. For measurement of nonspecific (diffusional)biotin uptake, samples were incubated in reaction mixture containing an excess amount (50 p M ) of biotin in addition to the labeled substrate. Net specific uptake was calculated by subtracting the nonspecific from the total uptake and expressed as femtomolesper minute per milligram of protein.
Statistics Unless otherwise stated in the table and figure legends, data are mean ? SD values from triplicate determinations
1461
for at least two different cultures. The V,, and apparent K , values were calculated using an iterative, nonlinear, leastsquare fitting program. Differencesbetween mean values were evaluated using Student's two-tailed t test.
RESULTS Characterization of biotin transport Uptake of [3H]biotinby primary culture of astrocytes was studied at two different temperatures, 37 and 4°C (Fig. 1). The rate of uptake decreased as the temperature was lowered. The time course of [3H]biotinuptake at 37°C was linear with time up to 1 rnin and peaked after 5 rnin of incubation. At 4°C the time course of [3H]biotinwas linear with time at least until 10 min. Kinetic studies were performed by measuring the rates of uptake of [3H]biotinat different concentrations of substrate. The lower value was imposed by the sensitivity of the radiochemical method used. As is shown in Fig. 2, the range of the saturation for the biotin uptake at a substrate concentration was between 7 and 60 nM. These saturation kinetics was even clearer when values were corrected for the rates corresponding to the nonspecific component experimentally measured as described above. Kinetic constants of the resultant saturable process were then determined using a fitting curve program and found to be a K, of I 1.7 nM and a V,,, of 63 fmol/min/mg of protein. At [3H]biotin concentrations between 60 and 420 nM, the uptake was linear and corresponded to a nonsaturable system (data not shown). NaCl replacement with KCI or choline chloride or incubation of astrocytes in the absence of CaCI2 after M EGTA, a wash with KRP solution containing to eliminate traces of Ca2+,did not greatly modify the uptake of 28 nM [3H]biotin(data not shown). The influence of membrane potential on biotin uptake by astrocytes was tested by examining the effect of 5 p M valinomycin in the presence of an outwardly directed K+ gradient. The inside negative membrane potential failed to stimulate the initial 20 nM [3H]biotin uptake (data not shown). The energy dependence of biotin transport was studied by measuring the intracellular uptake by as-
-
Time (min)
FIG. 1. Effect of temperature on the time course of [3H]biotin uptake by primary culture of astrocytes. Cells were incubated for different times in KRP solution containing 57 nM [3H]biotin at 37 (0)or 4°C (0).
J. Neurochem., Vol. 58. No. 4, 1992
P. RODRIGUEZ-POMBO AND M. UG'4RTE
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FIG. 3. Effect of biotin deficiency on biotin uptake into cultured astrocytes. Cells cultured in control (C)or biotin-restrictedconditions (D) were incubated for 1 min at 37°C in KRP solution containing 14 nM t3H]biotin. [Biotin] (nM)
FIG. 2. Biotin uptake into cultured astrocytes at biotin concentrations ranging from 7 to 60 nM. Cells were incubated for 1 min at 37°C in KRP solution with radioactive biotin at the indicated concentration (0;total uptake) or radioactive biotin plus 50 pA4 unlabeled biotin (0;unspecific uptake).The specific biotin uptake (dotted line) was obtained by subtracting the unspecific uptake from the total uptake at each concentrationof biotin.
trocytes pretreated with azide or sodium fluoride at 1 mM for 60 min. None of the inhibitors had an effect on the process (data not shown). Table 1 shows the effect on the saturable biotin uptake of biotin analogues with variations in the valeric acid side chain, such as biocytin, or in the ring, such as lipoic acid. A very significant reduction of the net biotin uptake was observed in the presence of lipoic acid a lesser, but also significant, reduction in the biotin uptake was observed in the presence of biocytin. Effect of biotin deficiency on biotin uptake When the cells were maintained in medium restricted in biotin for 14 days, the total uptake of 14 nM [3H]biotin was 10-fold higher than the levels measured in control cells (Fig. 3). To determine whether the enhancement in biotin transport was associated with variations in the K,,, or VmaXof the saturable uptake, we studied the kinetic properties of [3H]biotin uptake in biotin-deficient astrocytes in the range of [3H]biotin concentrations between 6 and 320 M ( F i g . 4). A saturable system could be observed. Kinetic constants were determined by fitting the data to b' = Vmax
where the V,,,
[1/(1
K,,, is 18 nM. The rate constant of the unsaturable component kdir (0.87 fmol/min/mg of protein) was calculated by computer analysis (see Statistics). DISCUSSION The present study examines the transport of biotin across the plasma membrane of cultured astrocytes. Purity and suitability of the primary culture of astrocytes used in this study have been confirmed by immunocytochemical and functional criteria. Uptake of biotin by astrocytes was saturable at low concentrations (7-60 M) but linear at >60 nM biotin. An uptake mechanism with a double component had been extensively described; however, the K,,, values reported for the biotin transport in several tissues and studies were different (Dakshinamurti et al., 1987; Said et al., 1987; Spencer and Roth, 1988). This apparent discordance could be attributed to different methodological approaches. In rats, the plasma concentration of biotin was -20 nM, and cerebral biotin content was higher than in liver or serum in vivo (Bhagavan and Coursin, 1970). In this sense, the K , value described here for the biotin uptake in astrocytes, 11.7 M, would be in the range of physiological concentrations of biotin. Biotin transport in astrocytes appears to be a temperature-sensitive process, and the linearity observed with time at 4°C was compatible with a passive diffusion process. Metabolic inhibitors such as sodium
+ K m / s ) ] + kdiffs I
is 760 fmol/min/mg of protein and the
TABLE 1. Efect of structural analogues on the saturable biotin uptake in primary culture of astrocytes ~~
~
Structural analogue
Net biotin uptake (fmol/min/mg of protein)
None Biotin (50 FLM) Biocytin (50 p M ) Lipoic acid (50 p M )
51.7 2 5.4
0 33.7 f 2 18.5 f 5
Inhibition (%)
0 100" 3s6 65'
Cells were incubatedfor I min at 37°C in KRP solution containing 28 nM 13H]biotinand the compounds indicated. Corrections were made for the unsaturable component of the uptake. p < 0.0005, ' p < 0.05, ' p < 0.005 (Student's two-tailed i test).
J. Neurochem.. c'ol. 58. No. 4. l Y Y 2
-
0 100
200
300
[ e i o i i n ] (nM)
FIG. 4. Biotin uptake into biotin-deficientastrocytes as a function of biotin concentration. Cells cultured in biotin-restricted conditions were incubated for 1 min at 37°C in KRP solution with radioactive biotin at the indicated concentrations (0;total uptake). The specific biotin uptake (dotted line) was obtained by subtracting the unspecific uptake from the total uptake.
BIOTIN TRANSPOR T IN A STR OC YTES
fluoride or azide, however, had no effect on biotin transport, demonstrating that the process is not ATP dependent. Moreover, biotin uptake is unaffected by Na+ gradient replacement by K' or choline+;therefore, secondary active transport involving these ions appears unlikely. Biotin uptake was not an electrogenic process, demonstrated by the noneffect of valinomycin. To assess the specificity of the biotin transport in astrocytes, two structural analogues have been tested. Lipoic acid was a very effective inhibitor of the initial rate of biotin uptake, suggesting that the thiophane ring is necessary for recognition by the cell. The significant inhibition observed with biocytin suggests that the valeric acid side chain must also be accounted for to describe this process of recognition. The specificity discovered for the biotin transport system in astrocytes appears similar to that reported for the biotin uptake by another mammalian cell (Cohen and Thomas, 1982; Gore and Hoinard, 1987). In conclusion, biotin transport by astrocytes exhibits characteristics that are compatible with a facilitated diffusion; there are saturation kinetics toward substrate, uptake is apparently not energy dependent, and substrate specificity is considerable. Finally, the present study examines the effect of biotin deficiency on the uptake of biotin in astrocytes from rats. Biotin uptake in biotin-deficient astrocytes was 10-fold higher than in control cells. This increase could not be explained by a change in the cellular membrane permeability because we have demonstrated that Na+ gradient-dependent transport of L-glutamate in biotindeficient astrocytes was similar to that in control cells and comparable with that previously reported (Schousboe et al., 1977). The similarity found in the K, of the biotin transport process for control and biotin-deficient cells combined with the greatly increased V,,, obtained in the deficient cells could suggest a change in the number of the functional transport carriers in biotin-deficient astrocytes. However, the mechanism responsible for this change in V,,, requires further investigation. Whether this enhancement is due to an increase in the number of the carrier proteins, as has been proposed for bacterial cells (Prakash and Eisenberg, 1974), remains to be clarified. Moreover, these results suggest the capability of astrocytes to adapt to the challenge of deficiency of an essential nutrient. That capability may be especially important when deficiency in biotinidase occurs, as is the case for late-onset multiple carboxylase deficiency disease.
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Acknowledgment: We are indebted to Prof. F. Valdivieso for valuable discussions of the results in the progress of this study and for critical reading o f the manuscript. We also acknowledge the excellent technical assistance of Ms. Carmen Hernandez. This work was supported by an institutional grant from the Fondo de Investigaciones Sanitarias.
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J . Neurochem., Vol. 58. No. 4, 1992
