Biocytin

Biocytin and biotin uptake into NB2a neuroblastoma and C6 astrocytoma cells
Barbara Baur, Terttu Suormala*, E. Regula Baumgartner

Abstract

Uptake of biocytin and biotin was investigated in cultured transformed variants of neuronal (NB2a neuroblastoma) and glial (C6 astrocytoma) CNS cells. NB2a cells took up both compounds but biocytin was transported more efficiently than biotin in the nanomolar concentration range. In NB2a cells a single transport mechanism was found for biocytin with different kinetic parameters in the presence 5 pmol / min / mg). Two transport systems (K m1 17 mM, Vmax1 53 pmol / min / mg; K m2 314 mM, Vmax2 360 pmol / min / mg) were identified for biotin with only system 1 being Na -dependent. Biocytin uptake was competitively inhibited by excess biotin but not vice versa. Inhibition studies with structural analogs indicated different specificities for biotin and biocytin uptake. Biocytin uptake into C6 cells was hardly detectable whereas biotin was taken up by diffusion (k D 0.6 ml / min / mg) and a single saturable mechanism (K m 70 mM, Vmax 119 pmol / min / mg) at high extracellular Na . High extracellular K enhanced biotin diffusion into C6 cells. Inhibition studies with structural analogs revealed a less specific biotin uptake mechanism in C6 than in NB2a cells. Biocytin normalized deficient biotin-dependent propionyl-CoA carboxylase activity within 4 h in biotin-deficient NB2a cells whereas in C6 cells reactivation was ,20% thereby confirming that biocytin is only poorly transported into C6 cells. Specific biocytin uptake into NB2a cells is to our knowledge the first demonstration of a carrier-mediated transport mechanism for this compound. Neuronal biocytin uptake might contribute to the pathogenesis of biotinidase deficiency where biocytin is present in elevated levels.  2002 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters, and receptors
Topic: Uptake and transporters

Keywords: Biotin; Biocytin; Uptake; Biotinidase deficiency; Cultured CNS cells; NB2a neuroblastoma; C6 astrocytoma

1. Introduction biocytin is found in plasma and urine at a similar con-
centration to that of biotin in healthy subjects [1,2,20].
The vitamin biotin is required as essential cofactor for We have shown earlier that in biotinidase deficiency the the four biotin-dependent carboxylase enzymes in man. decrease of carboxylase activities occurs more rapidly in Biotin deficiency leads to multiple carboxylase deficiency, the brain than in other organs and is associated with severe which results in life-threatening metabolic derangement brain damage [3,12]. This is in accordance with the [1,24]. The closely related metabolite biocytin is derived observation that neurological abnormalities are often the from endogenous degradation of biotin-dependent carbox- first manifestation of this disorder. Biotinidase seems to be ylases and is not detectable in body fluids of healthy important for biotin homeostasis in the CNS since in individuals due to rapid cleavage by biotinidase. This acquired biotin deficiency the brain is better conserved enzyme is responsible for endogenous biotin recycling and from biotin depletion than other organs [6]. Indeed, liberation of dietary protein-bound biotin. However, in considerable biotinidase activity is present in several types biotinidase deficiency, a human congenital disorder, of brain cells indicating that biotin recycling is functional within the CNS [22]. Because biocytin is uniquely found

112 B. Baur et al. / Brain Research 925 (2002) 111 – 121
Biocytin does not inhibit biotin transport across the (Hoffmann-La Roche, Basel, Switzerland). Cell culture blood –brain barrier [4,19]. However, it might interfere media and antibiotic solution were purchased from Gibco. with biotin metabolism in certain cell populations of the Foetal calf serum (FCS) was from Amimed and newborn CNS. Biocytin has recently found growing application in calf serum (NBCS) from Biospa, Germany. OptiPhase the field of neuroanatomical studies where it is used as a HiSafe II scintillation cocktail was from LKB. All other neuronal tract-tracing marker [15]. When injected intraven- chemicals were obtained from commercial sources and tricularly, biocytin is taken up by certain subpopulations of were of analytical grade.containing biocytin can be detected by avidin –biotin methodology. Neurons are completely stained whereas labelling of glial cells is equivocal. In contrast, biotin did not produce neuronal or glial labelling. This suggests that biocytin does not need to be metabolized to biotin for uptake and that this metabolite may have special functions in the CNS. These findings prompted us to investigate and characterize both biotin and biocytin uptake in neuronal (NB2a neuroblastoma) and astroglial (C6 astrocytoma) types of transformed CNS cells. Biotin uptake mechanisms have been described in a variety of tissues including the endothelium of the blood brain barrier [4] and astrocytes [17]. Reports on direct measurement of biocytin uptake are few. Biocytin was not taken up by cultured endothelial cells of the blood –brain barrier [4] and two other studies investigating intestinal biocytin absorption in the rat came to contradictory conclusions. One report proposed a car- rier-mediated process for biocytin uptake into rat intestinal everted sacs [11]. This suggestion, however, was disproved by another study using the same experimental model in which intestinal biocytin absorption was shown to occur by simple physical diffusion [18]. Another, but indirect, study

2.2. Methods

2.2.1. Culture of NB2 a neuroblastoma and C6 astrocytoma cells
NB2a neuroblastoma are immature neuronal cells orig- inating from mouse cortex and C6 astrocytoma are trans- formed rat astrocytes. Both cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) sup- plemented with 10% FCS, 15 mM glucose and 4 mM glutamine in 24-well culture dishes (Costar) for uptake measurements. Stem cultures of both cell lines were subcultured twice a week. Biotin deficiency was induced by growing the cells in 25-cm culture flasks in a low- biotin medium (DMEM with 10% NBCS, final biotin concentration 0.1 nM) for 5 days in order to measure reactivation of the biotin-dependent mitochondrial prop- ionyl-CoA carboxylase (PCC) [21]. Maximal PCC activity was determined in parallel cultures grown in the same medium supplemented with 1 mM biotin.

2.2.2. Uptake measurements

provided evidence that biocytin is taken up by axons in rat Cells were incubated in 24-well dishes maintained at brain [10]. 378C in Hank’s balanced salt solution (1.3 mM CaCl 2 , 5.4
Recent investigations in our laboratory showed that mM KCl, 0.4 mM KH 2 PO 4 , 0.5 mM MgCl 2 , 0.4 mM expression of biotinidase and carboxylase activities as well MgSO 4 , 147 mM NaCl, 4.2 mM NaHCO 3 , 3.4 mM as sensitivity to biotin depletion in both NB2a and C6 cells K 2 HPO 4 , 5.6 mM glucose), pH 7.4, buffered with 10 mM are comparable to their non-transformed counterparts, HEPES (HBSS –HEPES) unless otherwise stated. The cells validating the use of NB2a and C6 cells as a model for were washed three times with 1 ml prewarmed HBSS – investigations of biotin metabolism in brain [22]. HEPES and preincubated in a further 0.5 ml for 10 –30

2. Materials and methods

min. Uptake was initiated by addition of 0.3 ml / well HBSS –HEPES containing 0.2 mCi / ml C-sucrose, 1 –2 mCi / ml H-biotin (21 –42 nM) or 0.2 –0.4 mCi / ml (5 –10 nM) H-biocytin and other constituents according to the

2.1. Materials experimental design. With exception of concentration
dependence and inhibition studies, the experiments were
D-[8,9- H]biotin (45 –50 Ci / mmol) was purchased from performed without addition of unlabelled biotin or Du Pont de Nemours-New England Nuclear, [U- C]suc- biocytin. Uptake was terminated by the addition of 1 rose (560 mCi / mmol) was from Amersham. [Biotin- ml / well ice-cold HBSS –HEPES and immediate removal H]biocytin (39 Ci / mmol) was custom synthesized by of the solution. After three further washes the cells were Amersham. Unlabeled biocytin, desthiobiotin, left to solubilize in 300 ml 1 M NaOH for 2 h at room diaminobiotin, biotin methylester, lipoic acid, pantothenic temperature. An aliquot of 200 ml was neutralized with acid and ouabain were obtained from Sigma. Unlabeled 200 ml 1 M HCl, 10 ml scintillation cocktail was added biotin, biotinyl-p -aminobenzoic acid (biotinyl-PABA) and and radioactivity was measured in a Packard Tricarb 4,49-diisothiocyanostilbene-2,29-disulfonic acid (DIDS) C1900 scintillation spectrometer. Protein was determined were from Fluka (Buchs, Switzerland). Neurobiotin (N -(2- using the Bio-Rad protein assay kit with bovine serum aminoethyl)-biotinamide) was obtained from Vector Lab- albumin as standard [8]. Uptake was corrected for non- oratories. Bisnorbiotin was synthesized by Dr. W. Holick, specific adsorption as measured by C-sucrose association

2.3. Statistics

Unless specified each experiment was performed in triplicate on at least three different occasions. Values are expressed as mean6standard error of the mean (S.E.M.). Parameters of saturation kinetics were estimated with a 3 H 14 C non-linear regression program using a Gauss –Newton where cpm sample and cpm sample are cpm originating algorithm ( MINIM 2.0.1. by R.D. Purves). Data were tested from H-biotin or H-biocytin and C-sucrose, respective- for differences from controls using Student’s two-tailed ly, from the samples; and are total cpm of H-biotin or t -test with levels of significance as indicated.
H-biocytin and C-sucrose, respectively, in the incubation solution; V is the incubation volume; c is the biotin or
biocytin concentration of the incubation solution and mg is 3. Results the protein content per well.
C-Sucrose cpm in the samples was about 0.5 nCi / mg The main features of biocytin and biotin uptake into and remained constant up to 60 min of incubation showing NB2a neuroblastoma and C6 astrocytoma cells are summa- that C-sucrose is not taken up by NB2a or C6 cells to a rized in Table 1. Detailed results of the individual experi- significant extent. ments are presented below.

2.2.3. Structural analogs of biotin and biocytin
A panel of structural analogs were examined for their inhibitory potency on biocytin and biotin uptake. Authentic biotin consists of an ureido ring fused with a tetrahydro- thiophene ring and contains a valeric acid side chain with a free carboxyl group. In biocytin the carboxyl group is blocked by an amido bond linked to the e-aminogroup of a lysine residue. Desthiobiotin, the precursor of bacterial biotin synthesis, and diaminobiotin both have alterations within the ring structure. Lipoic acid and pantothenic acid are structural analogs with a free carboxyl group. Neuro- biotin [N -(2-aminoethyl)biotinamide] is widely used as intracellular tracer to morphologically identify neurons [13], a field of application where also biocytin is employed [15]. Biotinyl-PABA contains a carboxyl group blocked by a p -aminobenzoic acid residue and is used as an artificial substrate in the colorimetric determination of biotinidase activity [14]. Bisnorbiotin is an endogenous degradation product of biotin generated by mitochondrial b-oxidation and in which the side-chain is shortened by two methylene groups.

2.2.4. Reactivation of propionyl-CoA carboxylase ( PCC)

3.1. Time dependence and cation requirements of biocytin and biotin uptake

Time-dependent biocytin and biotin uptake was assayed in the presence of high extracellular concentrations of different cations with Cl as the accompanying anion. The physiological situation of a high extracellular Na con- centration (156 mM) is represented by the incubation in normal HBSS –HEPES. The presence of ouabain reduces Na / K -ATPase activity which leads to a collapse of transmembrane Na – and K -gradients thereby inhibiting Na -dependent uptake mechanisms.
The uptake of 5.5 nM biocytin or 21 nM biotin into NB2a cells is shown in Fig. 1A and B, respectively. For both compounds uptake was highest when the NB2a cells were incubated in HBSS –HEPES. Addition of 1 mM ouabain significantly reduced time-dependent uptake, and incubation in HBSS –HEPES with NaCl replaced by cholineCl, LiCl or KCl led to a further reduction of intracellular biocytin or biotin accumulation.
The uptake of biocytin into NB2a cells incubated in normal HBSS –HEPES was much higher and thus more efficient than that of biotin although assayed at four times lower concentration. Further, biotin uptake was linear up to activity in biotin-deficient cells by biotin and biocytin 5 min and increased little between 10 and 60 min of Reactivation of deficient PCC activity with biotin and incubation whereas biocytin accumulation was linear at biocytin and assay of PCC activity in crude cell homoge- least up to 10 min and increased considerably up to 60 nates was performed as described [21], except that the min.
incubation medium used for reactivation was HBSS – C6 cells showed different results for biotin and biocytin HEPES. Briefly, culture medium was removed, cell layers uptake when incubated under identical conditions. Biocytin were rinsed with PBS and the incubation medium was was only very poorly transported and significant accumula- added. After equilibration at 378C in a waterbath, reactiva- tion over background values was not observed until after tion was started by the addition of biotin or biocytin. 60 min of incubation in HBSS –HEPES (49.662.5 fmol / Reactivation was terminated by rapid removal of the mg; results not shown). Uptake of 21 nM biotin into C6 medium and immediate harvesting of cells by trypsiniza- cells is shown in Fig. 2. In the presence of high external tion. Cells were washed with PBS, centrifuged at 148C, KCl (156 mM) uptake was highly stimulated compared to and pellets stored at 2708C until assayed for PCC activity. NaCl conditions. Biotin uptake was decreased when LiCl or cholineCl constituted the main salt or in the presence of relation to increasing biocytin or biotin concentrations in 1 mM ouabain. In contrast to the findings in NB2a cells, the presence of high extracellular NaCl, KCl or cholineCl. biotin uptake into C6 cells was clearly higher with K than The kinetic parameters were calculated using computerized with Na and appeared to deviate from linearity at non-linear regression analysis.
incubation times shorter than 1 min. In NB2a cells biocytin uptake was measured between
2.5 nM and 1 mM and biotin uptake between 1 and 600

3.2. Kinetics of biocytin and biotin uptake

mM. Biocytin uptake displayed somewhat different kinetic parameters depending on which chloride salt was present

Initial uptake rates (1 min incubation) were measured in (Fig. 3A). The experimental values fitted best to a single

Fig. 1. Time-dependence and cation requirements of biocytin and biotin uptake into NB2a cells. Cells were preincubated for 70 min in the respective incubation medium without radiolabelled tracer and then incubated with either 5.5 nM H-biocytin (A) or 21 nM H-biotin (B) either in HBSS –HEPES ( d), HBSS –HEPES supplemented with 1 mM ouabain ( m ) or in HBSS –HEPES were NaCl was replaced by KCl ( s), cholineCl ( j) or LiCl ( h). Values are means6S.E.M. of 3 –5 experiments. Inset in (A): magnification of the plot for biocytin uptake during the first 5 min of incubation.

Fig. 2. Time-dependence and cation requirements of biotin uptake into C6 cells. Cells were preincubated for 70 min in the respective incubation medium without radiolabelled tracer and then incubated with 21 nM H-biotin either in HBSS –HEPES ( d), HBSS –HEPES supplemented with 1 mM ouabain ( m ) or in HBSS –HEPES were NaCl was replaced by KCl ( s), cholineCl ( j) or LiCl ( h). Values are means6S.E.M. of 3 –4 experiments. Inset: magnification of the plot for biotin uptake during the first 5 min of incubation without the condition of high extracellular KCl.

saturation mechanism according to Michaelis –Menten kinetics without a diffusion component, under all three conditions. At high external NaCl concentration, a K m value of 0.4260.04 mM and a Vmax value of 20.360.9 pmol / min / mg were calculated. Replacement of NaCl by cholineCl resulted in lower values of K m 0.1160.02 mM and Vmax 5.460.2 pmol / min / mg. At high extracellular KCl concentration the K m of biocytin uptake was 1.760.1 mM and the Vmax 31.860.9 pmol / min / mg.
The kinetics of biotin uptake into NB2a cells are shown in Fig. 3B. In the presence of high extracellular NaCl concentration two saturation mechanisms were identified, one with a K m1 value of 1767 mM and a Vmax1 value of 53616 pmol / min / mg and the second with a K m2 of 314651 mM and a Vmax2 of 360610 pmol / min / mg. When NaCl was replaced by KCl only a single mechanism was evident with a K m of 464627 mM and a Vmax of 345611 pmol / min / mg. Similar results to those found in the presence of KCl were obtained at high extracellular cholineCl concentration (results not shown).
Biotin uptake into C6 cells was assayed at a con- centration range of 1 –300 mM and showed a saturable component in the presence of high external NaCl (Fig. 4). Biotin accumulation varied with the external biotin con- centration in a linear manner at high extracellular KCl or cholineCl concentrations indicating a diffusion process,

Fig. 3. Saturation of biocytin and biotin uptake into NB2a cells. Cells were incubated for 1 min at 378C in media with varying biocytin concentrations and 10 nM H-biocytin (A) or varying biotin concentrations and 42 nM H-biotin (B). Incubation media were HBSS –HEPES ( d), or HBSS –HEPES were NaCl was replaced by KCl ( s) or cholineCl ( j).Values are means6S.E.M. of 3 –6 experiments. The curves were derived from experimental data by computerised non-linear regression analysis.

Fig. 4. Saturation of biotin uptake into C6 cells. Cells were incubated for Fig. 5. Inhibition of biocytin uptake into NB2a cells by biotin. Dixon plot 1 min at 378C in media varying biotin concentrations and 42 nM of inhibition of biocytin uptake by biotin. Uptake of biocytin was assayed H-biotin. Incubation media were HBSS –HEPES ( d), or HBSS –HEPES in concentrations of 0.03 mM ( h) or 0.3 mM ( d) together with 10 nM were NaCl was replaced by KCl ( s) or cholineCl ( j).Values are H-biocytin for 1 min at 378C in HBSS –HEPES containing 0, 5, 15 or 25

means6S.E.M. of three experiments. The curves were derived from experimental data by computerised non-linear regression analysis. Inset: difference of uptake between high extracellular NaCl and CholineCl conditions ( h).

which was, however, more than three-fold higher with KCl

mM biotin. Values are means6S.E.M. of four experiments.

extracellular NaCl or KCl concentrations and in C6 cells in the presence of high extracellular NaCl or cholineCl.
Table 2 summarizes the effects of structural analogs at 3 mM on the uptake of 0.3 mM biocytin in NB2a cells. At

as the main external salt compared to cholineCl. Uptake in high extracellular NaCl concentration the strongest inhibi- the presence of NaCl fitted best to a single saturation tion was obtained with lipoic acid which significantly mechanism with a K m value of 70621 mM and Vmax value reduced biocytin accumulation to 10% of the control, i.e. of 119614 pmol / mg / min and a diffusion component with more pronounced than obtained by isotopic dilution with a k D value of 0.6360.05 ml / mg / min. In the presence of unlabelled biocytin (20% of control). Lipoic acid and KCl or cholineCl diffusion constants (k D ) of 2.2460.03 isotopic dilution with unlabelled biocytin significantly and 0.6760.03 ml / min / mg, respectively, were calculated. decreased biocytin uptake also at high extracellular KCl to
16% and 30% of the control, respectively. Only Na –

3.3. Competitive inhibition of biocytin uptake into NB2 a neuroblastoma cells by biotin

A Dixon plot (Fig. 5) shows that biocytin uptake into NB2a cells is competitively inhibited by unlabelled biotin. An inhibition constant K i of 1761 mM was calculated in the presence of high extracellular NaCl concentration. When NaCl was replaced by KCl the inhibition constant was 2563 mM (results not shown). Biotin uptake was not inhibited by excess biocytin (results not shown).

dependent biocytin uptake was inhibited by neurobiotin, lysine –HCl, biotinyl-PABA and, more pronounced, bisnor- biotin. Biocytin accumulation in the presence of these compounds was significantly reduced to 74, 74, 81 and 68% of the control value, respectively.
The effects of the same compounds on biotin uptake in NB2a cells are shown in Table 3. In these experiments, the concentration of biotin was 15 mM and inhibitor con- centration 300 mM. The inhibition pattern showed some differences to that obtained with biocytin. At high extracel- lular concentrations of both NaCl and KCl, desthiobiotin, lipoic acid and isotopic dilution with unlabelled biotin

3.4. Inhibition of biocytin and biotin uptake by significantly reduced biotin uptake. In the presence of structural analogs NaCl the reduction corresponded to 23, 25 and 26% of
control values, respectively. When NaCl was replaced by
The inhibitory effect of several compounds on initial KCl the reduction was 43, 58 and 53%, respectively. Only rates of biocytin and biotin uptake was examined over 1 in the presence of NaCl a significant inhibition by pan- min at biocytin or biotin concentrations in their K m range. tothenic acid (18% of control) and bisnorbiotin (60% of In NB2a cells uptake was assayed in the presence of high control) was observed. Diaminobiotin inhibited biotin NB2a cells were incubated for 1 min in HBSS –HEPES or in HBSS –HEPES in which NaCl was replaced by KCl containing 0.3 mM biocytin and 10 nM H-biocytin (control) and 3 mM inhibitor as indicated. Some of the analogs were assayed in different experimental sets and thus have their own controls. The controls were set to 100% and absolute values were (a) 9.461.0, (b) 4.760.3, (c) 6.160.9, (d) 2.460.2 pmol biocytin / mg / min6S.E.M. Values are means of 3 –4 experiments and were compared to controls using Student’s unpaired two-tailed t -test; Ns, not significant. *, Isotopic dilution of uptake only in the presence of high extracellular KCl acid under NaCl and cholineCl conditions was not sig- concentration (71% of control). nificantly different from the control, it seems that only
The influence of the structural analogs on biotin uptake diffusion of biotin is enhanced by lipoic acid. None of the into C6 cells is shown in Table 4. In these experiments structural analogs inhibited biotin uptake at high extracel- biotin concentration was 60 mM and inhibitor concen- lular cholineCl concentration.
tration was 450 mM. In the presence of a high extracellular Biotin uptake in the presence of high external KCl NaCl concentration uptake was significantly inhibited by concentration was not inhibited by structural analogs all of the compounds assayed except biocytin, (results not shown).
diaminobiotin and biotin methylester. The most potent inhibitors were pantothenic acid and bisnorbiotin, which

3.5. Inhibition of KCl-activated biotin uptake into C6

reduced intracellular biotin accumulation to 54 and 57%, astrocytoma cells
respectively. An unexpected finding was the apparent
stimulation of biotin uptake by lipoic acid (124% of The effects of the anion channel inhibitor DIDS and the control) which was even more pronounced in the presence Na / K -ATPase inhibitor ouabain on KCl-activated of high extracellular cholineCl concentration (186% of biotin uptake into C6 cells are shown in Table 5. The control). Since the difference between results for lipoic presence of DIDS and ouabain, separately and in combina-NB2a cells were incubated for 1 min in HBSS –HEPES or in HBSS –HEPES in which NaCl was replaced by KCl containing 15 mM biotin and 42 nM

H-biotin (control) and 300 mM inhibitor as indicated. Some of the analogs were assayed in different experimental sets and thus have their own controls.

The controls were set to 100% and absolute values were (a) 58.362.7, (b) 52.762.4, (c) 18.261.6, (d) 23.061.7 pmol biotin / mg / min6S.E.M. Values are means of 3 –4 experiments and were compared to controls using Student’s unpaired two-tailed t -test. Ns, not significant. *, Isotopic dilution of H-biotin with 315 mM cold biotin.

H-biotin (control) and 450 mM inhibitor as indicated. The controls were set to 100% and absolute values were (a) 11963, (b) 5162 pmol

biotin / mg / min6S.E.M. Values are means of three experiments and were compared to controls using Student’s unpaired two-tailed t -test. Ns, not significant. *, Isotopic dilution of H-biotin with 510 mM cold biotin.

tion inhibited biotin uptake. Uptake was only slightly Measurement of reactivation at different concentrations reduced in the presence of ouabain (P ,0.05) and the revealed that a physiological concentration of biotin or reduction was more pronounced when DIDS was present biocytin (2 nM) was not sufficient to reactivate PCC (P ,0.001). No significant synergistic effect was observed within a 4-h incubation period in both cell types. A biotin when both compounds were added. concentration of 20 nM resulted in a clear increase of PCC activity from 3 to 16% and from 3 to 34% of maximal
3.6. Reactivation of PCC activity in biotin-deficient activity in NB2a and C6 cells, respectively. Similarly, 20 NB2 a neuroblastoma and C6 astrocytoma cells nM biocytin was able to reactivate PCC to 9% of maximal
activity in NB2a cells whereas no reactivation was found
The time dependence of reactivation of deficient PCC in C6 cells (results not shown).
activity in NB2a or C6 cells by preincubation with a
pharmacological concentration (1 mM) of biocytin or
biotin is shown in Fig. 6. Reactivation by biotin was
similar in both cell types with PCC activity increasing
from 3 to 80% of maximal activity within 1 –2 h. The
reactivation of PCC by biocytin was delayed compared
with that by biotin in both cell lines albeit to a different
extent. The delay in NB2a cells was only moderate with
80% of maximal activities being reached between 2 and 4
h, whereas in C6 cells only 33% of maximal activity was
reached after 6 h of incubation.

Table 5
Inhibition of KCl-activated biotin uptake into C6 cells
Inhibitor (1 mM) fmol / mg / 5 min % of P values
6S.E.M. control
Control 245611 100
C6 cells were preincubated for 30 min in KCl –HBSS –HEPES with or Fig. 6. Time-dependent reactivation of deficient PCC-activity by 1 mM without inhibitor as indicated. Uptake was measured for 5 min at 378C in biotin or biocytin. Biotin deficient NB2a ( d, s) or C6 ( j,h ) cells were the same media substituted with 21 nM H-biotin. Values are incubated with 1 mM biotin ( d, j) or biocytin ( s, h) for the time means6S.E.M. of three experiments. Values were compared to control periods indicated. Then cells were harvested and enzyme activity was using Student’s unpaired two-tailed t -test. measured. Values are means6S.D. of a representative experiment.

4. Discussion nism for both compounds. On the other hand, the different
inhibition pattern in the presence of structural analogs and
This study provides evidence for specific uptake mecha- the cationic modulation of only biocytin uptake might nisms for biocytin and biotin in NB2a neuroblastoma and indicate that binding sites for biotin and biocytin are not for biotin in C6 astrocytoma cells with different charac- the same.
teristics. The main difference between the two cell strains In C6 cells most of the structural analogs significantly was the finding of specific biocytin uptake only in NB2a inhibited Na -dependent biotin uptake into C6 cells. cells which is to our knowledge the first demonstration of Biotin transport in C6 cells is thus of lower specificity than specific biocytin uptake into a tissue. that in NB2a cells. None of the structural analogs tested
In NB2a cells, both biocytin and biotin were taken up inhibited biotin uptake in the presence of high external most efficiently in the presence of high extracellular NaCl choline supporting the concept of non-specific diffusion concentration (156 mM) which represents the physiologi- under this condition. An unexpected finding was the cal condition. In C6 cells, biocytin uptake was detectable apparent stimulation of biotin uptake in the presence of only after prolonged incubation and therefore could not be lipoic acid, which was even more pronounced when Na further investigated. Biotin uptake into C6 cells was was replaced by choline . Lipoic acid is otherwise known partially Na -dependent but not as pronounced as in NB2a as a potent inhibitor of biotin transport mechanisms in a cells.The inhibition of uptake in the presence of ouabain or variety of tissues and has been reported to share a Na – by cationic replacement points towards a partial depen- dependent multivitamin transport mechanism with biotin dence on metabolic energy since the maintenance of the and pantothenic acid. This transporter has been cloned physiologic extracellular to intracellular sodium gradient is from rat placenta and was localized in other rat tissues mediated by Na / K -ATPase at the expense of ATP including brain [16]. Inhibition of biotin uptake by both hydrolysis. lipoic acid and pantothenic acid is, however, in agreement
In NB2a neuroblastoma, the saturation kinetics for with our findings in NB2a cells.
biocytin uptake were in a lower concentration range than The characteristics of the biotin uptake mechanism in for biotin. Biocytin uptake exhibited a higher affinity and C6 cells do not agree with a study performed in primary was more efficient than biotin uptake with substrates in the cultures of rat brain astrocytes [17]. These authors calcu- low nanomolar concentration range. For biocytin, ex- lated a K m of 17 nM and found no effect of Na perimental values fitted best to a single transport mecha- replacement by choline or K . Further, they observed a nism with different kinetic parameters depending on which significant inhibition of biotin uptake by biocytin. These main cation was present in the incubation medium. Similar apparent contradictions to our results have to be explained cationic modulation of substrate uptake into neurons has by methodological differences and by the different ex- also been reported for the human dopamine transporter [9]. perimental models used.
Two transport mechanisms were identified for biotin We ourselves observed K -stimulated biotin influx in uptake into NB2a cells in the presence of high external C6 cells which was slightly reduced in the presence of Na . Replacement of Na by K revealed a single ouabain and more potently in the presence of DIDS. This mechanism which might be identical with the second increased biotin uptake can be explained by enhanced transport system found under high Na conditions. diffusion rather than by activation of an uptake mechanism Biocytin uptake is competitively inhibited by excess biotin since saturation of biotin uptake was not observed in this but not vice versa. The inhibition constant, K i , of biotin condition. It is conceivable that the enhanced biotin was found to be identical to the K m1 of its uptake system. diffusion in the presence of high extracellular K is due to
In C6 cells, biotin uptake showed saturation in the the K clearance mechanism in astrocytes which is known presence of high external Na and initial rates fitted best to to be sensitive to ouabain and DIDS [23].
a single uptake mechanism combined with a diffusion The reactivation of deficient PCC activity by biotin or process. Replacement of Na by choline abolished biocytin in biotin-deficient NB2a and C6 cells supports our saturable uptake and intracellular biotin accumulation was findings from the uptake studies. The complete recovery of entirely due to diffusion. In the presence of high external PCC activity with a pharmacological biotin concentration K biotin uptake showed no saturation but the diffusion (1 mM) and the clear increase of PCC activity with a near rate was more than threefold increased. to physiological concentration (20 nM) confirm that biotin
Biocytin and biotin uptake in NB2a cells and biotin is readily internalized by both cell types and subsequently uptake in C6 cells showed a different inhibition pattern in bound to apocarboxylases. The reactivation by biocytin the presence of the same structural analogs indicating was delayed relative to that by biotin, which can be different specificities. Bisnorbiotin was the only compound explained by the necessity for intracellular cleavage of examined which inhibited all three uptake systems moder- biocytin by biotinidase prior to incorporation of the biotin ately. moiety into apocarboxylases. However, PCC activity was
The competitive inhibition of biocytin uptake in NB2a fully restored by 1 mM biocytin in NB2a cells within 6 h cells by excess biotin suggests a common transport mecha- but increased to only 33% of maximal in C6 cells. These findings support the idea of effective transport of biocytin the Roche Research Foundation, Basel, Switzerland and into NB2a cells but undetectable transport under identical the Fonds zur Forderung¨ von Lehre und Forschung der assay conditions in C6 cells. This difference cannot be Freiwillige Akademischen Gesellschaft, Basel, Switzer- explained by a difference in the level of biotinidase land.
activity since C6 cells have about tenfold higher specific biotinidase activity than NB2a cells [22] which may partially compensate for the poor biocytin uptake capacity in these cells.
Extrapolating our findings to the physiological situation it appears that biocytin and biotin entry into neuronal and biotin entry into glial cells are not limited under normal circumstances. The uptake systems have K m values well above the corresponding physiological concentrations, which is in the low nanomolar range for biotin in human and mammalian plasma [7]. The CSF concentration of biotin is even lower and is reported to be 15% of the human plasma concentration [5]. Biocytin on the other hand is not detectable in body fluids of healthy individuals but it is found in plasma and urine of biotinidase deficient patients. In this condition plasma biocytin concentration is in the range of 0.6 –0.9 nM [2] and urinary biocytin excretion is similar to the excretion of biotin in healthy individuals [20]. There are no reports concerning biocytin concentrations in CSF. However, it is highly unlikely that this metabolite occurs in detectable amounts under normal conditions since considerable biotinidase activity is present in several types of CNS cells including neurons and astrocytes [22]. Further, biocytin was not detectable in CSF of untreated patients with biotinidase deficiency [detection limit 2 nM; unpublished observation].
Whether the herein-described uptake mechanism for biocytin in neuronal cells is of physiological significance remains to be established.
It is, however, conceivable that in the condition of untreated biotinidase deficiency where biocytin cannot be hydrolyzed to biotin neuronal biocytin uptake might be detrimental. In the other known defect in biotin metabo- lism, holocarboxylase synthetase (HCS) deficiency, biocytin is not detectable. Also HCS deficiency results in deficiencies of the biotin-dependent carboxylases and patients with this disorder present clinically very similar except that neurological abnormalities are not among the first manifesting symptoms [1,24]. In biotinidase de- ficiency, the levels of biotin are pathologically decreased or undetectable whereas those of biocytin are increased. Predominantly biocytin might thus accumulate in neurons and could contribute to the progressive neurological im- pairment characteristic of this disorder by a yet unknown mechanism.

Acknowledgements

We acknowledge the help of Dr. B. Fowler in the preparation of this manuscript. This work was supported by grants of the Novartis Foundation, Basel, Switzerland,

References

[1] E.R. Baumgartner, T. Suormala, Multiple carboxylase deficiency: Inherited and acquired disorders of biotin metabolism, Intern. J. Vit. Nutr. Res. 67 (1997) 377 –384.
[2] E.R. Baumgartner, T. Suormala, H. Wick, J. Bausch, J.-P. Bonjour, Biotinidase deficiency: Factors responsible for the increased biotin requirement, J. Inher. Metab. Dis. 8 (Suppl. 1) (1985) 59 –64.
[3] E.R. Baumgartner, T. Suormala, H. Wick, A. Probst, U. Blauenstein, C. Bachmann, M. Vest, Biotinidase deficiency: a cause of subacute necrotizing encephalomyelopathy (Leigh syndrome). Report of a case with lethal outcome, Pediatr. Res. (1989) 260 –266.
[4] B. Baur, E.R. Baumgartner, Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood –brain barrier, Brain Res. 858 (2000) 348 –355.
[5] C. Bernouilli, Entwicklung einer sensitiven Methode zur Biotinbes- timmung in Plasma und Liquor: Ermittlung von Referenzwerten in der Padiatrie,¨ Erstellen des Biotinstatus bei Biotinidasemangel-Pa- tienten und Bestimmen der Proteinbindung in Plasma. Inauguraldis- sertation, Philosophisch-Naturwissenschaftliche Fakultat¨ der Uni- versitat¨ Basel, 1999.
[6] H.N. Bhagavan, D.B. Coursin, Depletion of biotin from brain and liver in biotin deficiency, J. Neurochem. 17 (1970) 289 –290.
[7] J.-P. Bonjour, Biotin, in: L.J. Machlin (Ed.), Handbook of Vitamin, 2nd Edition, Marcel Dekker, New York and Basel, 1991, pp. 393 –427.
[8] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein – dye binding, Anal. Biochem. 72 (1976) 248 –254.
[9] N. Chen, C.G. Trowbridge, J.B. Justice Jr., Cationic modulation of the human dopamine transporter: dopamine uptake and inhibition of uptake, J. Pharmacol. Exp. Ther. 290 (1999) 940 –949.
[10] G. Chevalier, J.M. Deniau, A. Menetrey, Evidence that biocytin is taken up by axons, Neurosci. Lett. 140 (1992) 197 –199.
[11] K. Dakshinamurti, J. Chauhan, H. Ebrahim, Intestinal absorption of biotin and biocytin in the rat, Biosci. Rep. 7 (1987) 667 –673.
[12] M. Duran, E.R. Baumgartner, T.M. Suormala, L. Bruinvis, L. Dorland, J.A.M. Smeitink, B.T. Poll-The, Cerebrospinal fluid or- ganic acids in biotinidase deficiency, J. Inher. Metab. Dis. 16 (1993) 513 –516.
[13] H. Kita, W. Armstrong, A biotin-containing compound N -(amino- methyl)biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin, J. Neurosci. Methods 37 (1991) 141 –150.
[14] J. Knappe, W. Brummer,¨ K. Biederbick, Reinigung und Eigenschaf- ten der Biotinidase aus Schweinenieren und Lactobacillus casei, Biochem. Z. 338 (1963) 599 –613.
[15] A.J. McDonald, Neuroanatomical labeling with biocytin: a review, NeuroReport 3 (1992) 821 –827.
[16] P.D. Prasad, W. Haiping, R. Kekuda, T. Fujita, Y.-J. Fei, L.D. Devoe, F.H. Leibach, V. Ganapathy, Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transpor- ter mediating the uptake of pantothenate, biotin, and lipoate, J. Biol. Chem. 273 (13) (1998) 7501 –7506.
[17] P. Rodriguez-Pombo, M. Ugarte, Biotin transport in primary culture of astrocytes: effect of biotin deficiency, J. Neurochem. 58 (1992) 1460 –1463.
[18] H.M. Said, L.P. Thuy, L. Sweetmann, B. Schatzmann, Transport of the biotin dietary derivative biocytin (N -biotinyl-L-lysine) in rat [22] T.M. Suormala, U.N. Wiesmann, F. Cruz, A. Wolf, M. Daschner, A. small intestine, Gastroenterology 104 (1993) 75 –80. Limat, B. Fowler, E.R. Baumgartner, Biotin-dependent carboxylase
[19] R. Spector, D. Mock, Biotin transport through the blood –brain activities in different CNS and skin-derived cells, and their sensitivi- barrier, J. Neurochem. 48 (1987) 400 –404. ty to biotin depletion, submitted for publication.
[20] T.M. Suormala, E.R. Baumgartner, J. Bausch, W. Holick, H. Wick, [23] W. Walz, Role of glial cells in the regulation of the brain ion Quantitative determination of biocytin in urine of patients with microenvironment, Prog. Neurobiol. 33 (1989) 309 –333. biotinidase deficiency using high-performance liquid chromatog- [24] B. Wolf, Disorders of biotin metabolism, in: C.R. Scriver, A.L. raphy (HPLC), Clin. Chim. Acta 177 (1988) 253 –270. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular
[21] T. Suormala, B. Fowler, M. Duran, A. Burtscher, A. Fuchshuber, R. Bases of Inherited Disease, 7th Edition, McGraw-Hill, New York, Tratzmuller,¨ M.J. Lenze, K. Raab, B. Baur, H. Wick, R. Baumgart- 1995, pp. 3151 –3177.
ner, Five patients with a biotin-responsive defect in holocarboxy-lase formation: Evaluation of responsiveness to biotin therapy in vivo and comparative studies in vitro, Pediatr. Res. 41 (1997) 1 –8.