Drug Metabolism and Disposition Fast Forward
First published on July 20, 2005; DOI: 10.1124/dmd.105.005231
0090-9556/05/3310-1547-1554$20.00
DMD 33:1547-1554, 2005
THE BLOOD-BRAIN BARRIER SODIUM-DEPENDENT MULTIVITAMIN TRANSPORTER: A MOLECULAR FUNCTIONAL IN VITRO-IN SITU CORRELATION
Seonghee Park, and
Patrick J. Sinko
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers-The State University of New Jersey, Piscataway, New Jersey
(Received April 18, 2005;
accepted July 18, 2005)
 |
Abstract
|
|---|
The molecular mechanism of biotin brain uptake was investigated using an in vitro bovine blood-brain barrier (BBB) cell model and an in situ mouse brain perfusion technique. A functional uptake/transport correlation of the in vitro and in situ characteristics of biotin uptake was investigated. Morphological and immunochemical characteristics (e.g., factor VIII expression) of the primary culture of brain microvessel endothelial cells (BMECs) were confirmed. Gene expression of the multidrug resistance (Mdr1) and sodium-dependent multivitamin (SMVT) transporters was also determined in BMECs. Biotin transport was saturable and Na+-dependent at the luminal side of the BBB. The estimated half-saturation concentrations (Km) of biotin uptake in vitro and in situ were 49.1 and 35.5 µM, respectively, supporting the presence of a carrier-mediated biotin transport system. Inhibition studies using various biotin derivatives and structural analogs demonstrated the structural requirements for biotin-SMVT interaction. Desthiobiotin and pantothenic acid significantly inhibited the uptake of biotin, whereas 2-iminobiotin and diaminobiotin were very weak inhibitors. Based on our results, there was a good correlation between the in vitro and in situ BBB models, suggesting that when a single membrane transporter is involved in substrate uptake, flexibility in choosing the experimental model can be afforded. The current results are also consistent with the suggestion that the properties of the BBB are likely to be organ-specific rather than species-specific. Further mechanistic and comparative studies are needed to validate these results. In conclusion, the in vitro transporter-based mechanism studies produced valuable molecular functional transport results that correlated well with in situ results.
The BBB serves as a regulatory interface for drug entry into the central nervous system (Goldstein and Betz, 1986
). In vitro cell culture and in situ or in vivo techniques for studying drug transport into the brain, including perfusion, microdialysis, and autoradiography, have been developed (Feng, 2002). The selection of the appropriate methodology is an important consideration for obtaining mechanistic biopharmaceutical information that will allow for a meaningful interpretation of the results, since there are numerous advantages and disadvantages associated with each method.
The in vitro BBB models have been used to study drug transport and metabolism at the cellular level. Cultures of microvessel endothelial cells have been derived from human, canine, bovine, murine, porcine, and rat brain (Pardridge, 1999). The in vitro BBB cell culture model offers several advantages over whole-animal studies: 1) the cellular environment such as the composition of the buffers and temperature can be easily controlled, 2) the potentially confounding effects of surrounding cells (e.g., neurons, astrocytes, and pericytes) on the transport properties of the BBB are controlled, 3) any influence from blood pressure and enzymes present in the blood is eliminated, and 4) in vitro studies are time- and cost-efficient since they are amenable to higher throughput (Audus et al., 1990). However, differences or changes in the expression or function of drug transporters compared with in vivo models may limit the usefulness of in vitro models. For example, some nutrient transporter systems have been reported to be down-regulated by as much as 100-fold in vitro (Pardridge, 1995). However, transporter expression is not always down-regulated. For example, although multidrug resistance-associated protein transporters are functionally active in cultured brain endothelial cells, P-glycoprotein expression in human brain endothelial cell culture models is lower than in isolated microvessels, and multidrug resistance protein expression increased in the cell culture model (Seetharaman et al., 1998). Therefore, it is important to understand the differences in transporter expression and function between these models. In addition, cell-culturing conditions can dramatically affect transporter expression and function (Williams et al., 2003). Some of the factors that affect the properties of cultured cells include 1) the cell culture medium, 2) the cell seeding density, 3) the supporting matrix (e.g., collagen), and 4) the presence or absence of essential nutrients and growth factors. All of these factors should be carefully considered when attempting to use preclinical models to study the impact of drug transporters on brain uptake.
The in vitro BBB model is much more permeable than what is typically observed in vivo, resulting in, on average, 100 times greater permeability values for compounds where passive diffusion is thought to be the primary route of brain uptake (Chikhale et al., 1994). Although the results of these models can be correlated, their utility for prediction of in vivo brain uptake is limited since it will nearly always be overestimated. Drug uptake measured in the intact brain using the in situ perfusion method, unlike in vitro models of the BBB, provides a much closer approximation of in vivo conditions. Permeability values for various reference compounds obtained by the perfusion method agree with values obtained in animal studies using the intravenous injection method (Smith, 1996). Besides enhanced accuracy and sensitivity, the perfusion method has an advantage over other in vivo methods in that the perfusate composition can be controlled. Traditional in vivo brain distribution studies using brain tissue homogenates measure "total" drug concentration in the brain, providing no information regarding intra- or extracellular drug concentration (Cisternino et al., 2001). Therefore, in situ brain perfusion can offer mechanistic insight into the in vivo situation and is complementary to the in vitro and in vivo methods.
Biotin, a water-soluble vitamin, is essential for normal cellular functions, growth, and development and is involved in various metabolic reactions, including fatty acid biosynthesis, gluconeogenesis, and catabolism of amino acids (Said et al., 1987). Biotin is a substrate for the SMVT (Prasad et al., 1999), and its transport into various mammalian tissues has been shown to occur by a specific saturable process (Said et al., 1987; Baur and Baumgartner, 1993; Ma et al., 1994; Prasad et al., 1997). In the present study, biotin was used as a model substrate to assess differences in SMVT function between the in vitro and in situ models of the BBB, since transport characteristics of cultured cells do not always reflect those in vivo.
In the current report, a mechanistic comparison of the molecular kinetic parameters and transport properties of biotin-SMVT interactions across the BBB is investigated in vitro and in situ. Hence, the aims of the present study were: 1) to validate an in vitro BBB model using a primary culture of bovine BMECs, 2) to investigate detailed mechanisms and various factors affecting biotin uptake into the brain using the in vitro BBB model as well as an in situ mouse brain perfusion technique, and 3) to examine the kinetic correlation of the in vitro and in situ characteristics of biotin uptake and to assess the feasibility of using in vitro results.
|
Materials and Methods
|
|---|
Materials. Amphotericin B (cell-culture tested), dextran (average mol. wt. 78,000), dimethyl sulfoxide, heparin, HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), HEPES sodium salt, penicillin G (benzyl penicillin; cell-culture tested), Percoll (colloidal polyvinylpyrrolidone-coated silica for cell separation), polymyxin B sulfate (cell-culture tested), sodium bicarbonate (NaHCO3; cell-culture tested), streptomycin sulfate (cell-culture tested), endothelial cell growth supplement (from bovine neural tissue), and plasma-derived horse serum were purchased from Sigma-Aldrich (St. Louis, MO) and used as obtained. Collagenase/dispase and dispase were purchased from Roche Diagnostics (Indianapolis, IN) and used as obtained. Collagen (rat tail, type I) was purchased from Collaborative Biomedical Products (Bedford, MA). Ham's F-12 nutrient mixture (F-12) and minimum essential medium (MEM) were purchased from Mediatech (Herndon, VA) and used as obtained. D-[8,9-3H]Biotin (42–46 Ci/mmol; radiochemical purity of 98%) and [14C]sucrose (495 mCi/mmol; radiochemical purity greater than 98%) were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK) and Moravek Biochemicals, Inc. (Brea, CA), respectively. Unlabeled biotin, desthiobiotin, diaminobiotin, 2-iminobiotin, biocytin, pantothenic acid, and D-glucose were purchased from Sigma-Aldrich. Solvable was purchased from Packard Instruments (Downers Grove, IL). All other chemicals and reagents were purchased from Fischer Scientific (Houston, TX) and were of analytical grade.
Animals. Male FVB mice (26–30g) were purchased from Taconic (Germantown, NY) and maintained under standard conditions of temperature and lighting with ad libitum access to food and water. All experimental procedures complied with the protocol (02-029) approved by the Institutional Review Board Use and Care of Animal Committee and housed in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities at Rutgers University.
Isolation and Cultivation of BMECs. BMECs were isolated from the gray matter of fresh bovine brains using a two-step enzymatic method and cultured as described previously (Bowman et al., 1981, 1983; Goldstein et al., 1984; Audus and Borchardt, 1987). The frozen BMECs were rapidly thawed in a water incubator at 37°C and washed two times in MEM-F-12 containing 10% horse serum. The cells were centrifuged for 10 min at 2000 rpm. The final pellet was resuspended in an appropriate volume of the BMEC culture medium (45% MEM, 45% F-12, and 10% plasma-derived horse serum with 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, 125 µg/ml heparin, and 50 µg/ml endothelial cell growth supplement) and counted using a hemacytometer. The cell culture plates were coated with 50 µg/ml rat-tail collagen, type I, 2 h prior to seeding. The BMECs were seeded at a density of 50,000 cells/cm2 in the 24-well dishes (Corning Life Sciences, Acton, MA) for uptake studies and six-well dishes for morphological studies. The BMECs were incubated at 37°C and 5% CO2 and 95% humidity. The BMEC culture medium was changed on the third day after plating and then changed every other day thereafter. The BMECs reached confluency on day 6 or 7 after seeding.
Morphological Characterization of BMEC Monolayers. BMECs were seeded at 50,000 cells/cm2 and grown in a six-well cell culture plate (Corning Life Sciences). After the cultured BMECs formed a monolayer (6–7 days), they were fixed with 70% ethanol and examined using a Nikon TMS-F phase-contrast microscope (Nikon USA, Melville, NY).
For electron microscopic examination, BMECs were seeded at 150,000 cells/cm2 and grown in a Permanox dish (Electron Microscopy Sciences, Fort Washington, PA). The dish was not treated with collagen. Upon confluency, the monolayers were fixed with 2.5% glutaraldehyde and 4% paraformaldehyde in cacodylate buffer (Karnowsky's modified method), postfixed in 1% osmium tetroxide in the same buffer for 1 h, progressively dehydrated in acetone, and embedded in epon/spur resin. Thin sections (80 nm) were stained with uranyl acetate and lead citrate. They were then examined using a JEOL 1200 transmission electron microscope (JEOL, Tokyo, Japan) (Orlowski and Meister, 1970; Faulkner et al., 1979; Baron-Van Evercooren et al., 1986).
Immunohistochemical Characterization of BMEC Monolayers. Using immunohistochemical techniques, BMECs were shown to express specific markers found only in endothelial cells, such as factor VIII antigen. They were assayed at six-well plate histochemically with anti-factor VIII antibody and immunohistochemical kit for von Willebrand antigen (Zymed Laboratories, Inc., South San Francisco, CA). 3,3'-Diaminobenzidine (Sigma-Aldrich) was used as a chromogen. Immunostaining using glial fibrillary acidic protein (GFAP) antibody was also performed to inspect contamination by glial cells surrounding the brain microvessels.
Determination of Mdr1 and SMVT Gene Expression. The presence of multidrug-resistant (Mdr1) and SMVT in BMECs were determined using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from BMECs was isolated with TRIzol reagent (Promega, Madison, WI). The first strand of cDNA was synthesized using 3 µg of RNA, 2 pmol of reverse primer, 10 mM dithiothreitol, 0.5 mM concentration each of dATP, dCTP, dGTP, and dTTP, and 200 units of Superscript II reverse transcriptase as described by the manufacturer (Invitrogen, Carlsbad, CA). For determination of Mdr1 gene expression, two specific primers were synthesized based on human Mdr1. The sequences for forward and reverse Mdr1 primers were 5'-ATC GTG TCC CAG GAG CCC ATC CTG-3' and 5'-AGA CAC GGT CGT CGA CGA CTA CGC-3', respectively. For determination of SMVT gene expression, two specific primers were synthesized based on human SMVT. The sequences for forward and reverse hSMVT primers were 5'-CTG TCC GTG CTG GCC CTG GGC-3' and 5'-GAC CSG GCC AAT GAG GCA GCC-3', respectively. PCR was performed using 50 µl of reaction volume containing 10 ng of cDNA, 0.2 mM MgCl2, 0.5 µM primers, and 2.5 units of TaqDNA polymerase. The reaction was run for 30 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The PCR products were electrophoresed through 1.5% agarose gel containing ethidium bromide, and images were captured using NucleoTech imaging system (NucleoTech, San Mateo, CA).
Uptake Measurements Using BMEC Monolayers. All uptake studies were performed according to the methods described by Baur and Baumgartner (2000). Briefly, uptake studies were performed at 37°C or 4°C with confluent BMEC monolayers. Cells were rinsed twice with Hanks' buffer salt solution (HBSS; Invitrogen) buffered with HEPES (10 mM, pH 7.4). Subsequently, the cells were incubated (1 ml) with radiolabeled biotin and/or other test compounds. After the desired incubation period, cells were washed three times with ice-cold buffer. Finally, the cells were solubilized by 0.5 ml of 1 N NaOH at room temperature overnight and neutralized by adding 0.5 ml of 1 N HCl. The uptake was measured by liquid scintillation counting. All uptake results were normalized by protein amount, determined using the reagent from Bio-Rad (Hercules, CA). Uptake was corrected for nonspecific adsorption as measured by [14C]sucrose association and was expressed as moles per milligram of protein according to the formula (Baur and Baumgartner, 2000):
 | (1) |
where 3H dpmcells and 14C dpmcells are dpm of 3H test compound and [14C]sucrose, respectively, associated with cell solubilisates; 3H dpm inc.sol and 14C dpm inc.sol are the total dpm of 3H test compound and [14C]sucrose, respectively, in the incubation solution; V is the incubation volume; c is the concentration of test compound in the incubation solution; and mg is the protein content per well. The concentration dependence of biotin uptake was studied at pH 7.4 with an uptake buffer prepared using HBSS-buffered HEPES (10 mM). In general, uptake studies were carried out as described previously. Different concentrations of biotin (1–200 µM) spiked with trace amounts of [3H]biotin at 37°C for 1 min were used for uptake studies. The concentration of biotin was determined by liquid scintillation counting. Inhibition studies were performed by coincubating biotin (10 µM) with biotin derivatives such as biocytin, desthiobiotin, 2-iminobiotin, and diaminobiotin, or structural analogs such as pantothenic acid at 200 µM concentration. To find the effect of Na+, NaCl in HBSS-HEPES was replaced iso-osmotically with choline-Cl. The stoichiometry of the biotin-Na+ coupling was examined by increasing the concentration of Na+ (i.e., the activation method). To determine the number of Na+ ions interacting with the carrier, the data were fit to the Hill equation. After the cells were solubilized in 1 N NaOH and neutralized by adding 1 N HCl, 100 µl of solution was taken from each well for protein concentration determination. Protein concentration was determined by Bradford method (Bradford, 1976) using the Bio-Rad reagent with bovine serum albumin used as the standard. The absorbance was read at 595 nm, and the amount of protein/well was calculated from the standard curves. The cells collected from two separate isolations were used for each experiment, and each experiment was performed in quadruplicate.
In Situ Brain Perfusion. The in situ brain perfusion technique used in the experiments was similar to that described elsewhere (Takasato et al., 1984; Dagenais et al., 2000) with modifications. Briefly, mice were anesthetized with ketamine/xylazine (140/8 mg/kg i.p.). After exposure of the left common carotid artery, the left external carotid artery was ligated at the bifurcation of the common carotid artery with the internal carotid artery. The left common carotid artery was ligated caudally. A polyethylene tube (0.30 x 0.70 mm o.d.) filled with heparin (20 units/ml) was catheterized into the left common carotid artery under the microscope. For perfusion experiments, the left hemisphere of the mouse brain was perfused with perfusion buffer containing test compounds at a selected flow rate through the catheter that was connected to the perfusion pump. The perfusion buffer consisted of bicarbonate buffered physiological saline [128 mM NaCl, 4.2 mM KCl, 24 mM NaHCO3, 2.4 mM NaH2PO4, 1.5 mM CaCl2, and 0.9 mM MgSO4 (Momma et al., 1987)]. D-Glucose (9 mM) was added prior to an experiment. The perfusate was bubbled with a mixture of 95% O2 and 5% CO2 for pH control (7.4) and maintained at 37°C. Immediately prior to initiation of the perfusion, the cardiac ventricles were severed to eliminate the contribution of contralateral blood flow. Multiple time point experiments (20, 40, 60, and 90 s) were performed at a calibrated flow rate (Harvard pump PHD2000; Harvard Apparatus, Holliston, MA). To determine concentration dependence of biotin uptake, various concentrations of unlabeled biotin were added into the perfusate that contained [3H]biotin and [14C]sucrose as a vascular marker. For inhibition studies, the perfusate contained [3H]biotin and [14C]sucrose with or without biotin derivatives or structural analogs to produce an appropriate drug concentration. The perfusion was terminated by decapitation. The brain was removed from the skull and dissected, and each hemisphere was weighed and placed in a preweighed scintillation vial. Brain and perfusion fluid samples were digested in 0.7 ml of Solvable at 37°C for 24 h. Scintillation cocktail (5 ml) was added to each vial, and radioactivities of 3H and 14C were determined simultaneously by dual liquid scintillation counting. All data are reported for the left hemisphere.
Calculations and Data Analysis. The kinetic parameters for the concentration dependence studies were calculated by performing nonlinear regression using the Michaelis-Menten equation:
 | (1) |
where 
is the rate of substrate uptake, [C] is the substrate concentration, Vmax is the maximal uptake rate, and Km is the half-saturating concentration. Results from sodium dependence studies were plotted using the Hill equation (Coval, 1970; Heck, 1971). The Hill coefficient (n) was determined by fitting the data using the following equation:
 | (2) |
where is the uptake rate, C is the concentration of Na+, Kn is the concentration of Na+ necessary for half-maximal activation, and n is the Hill coefficient. Data are expressed as mean ± standard deviation (S.D.).
Brain perfusion results were analyzed as previously described (Park and Sinko, 2005). Briefly, brain vascular volume (Vvasc, ml · 100g-1), defined as the ratio of the vascular marker concentration in brain to that in the perfusate, was determined using the following equation:
 | (3) |
where X* is the amount of radiolabeled inulin in the brain (dpm · 100 g-1), and C* is the perfusate concentration (dpm · ml-1). The unidirectional transfer coefficient Kin (ml · min-1 · 100 g-1) was calculated using the following relationship:
 | (4) |
where Xbrain is the amount of radiotracer in the brain (dpm · 100 g-1) corrected for vascular contamination (Xtotal - Vvasc · Cpf), Xtotal (dpm · 100 g-1) is the total quantity of tracer measured in the tissue sample (vascular + extravascular), and Cpf is the tracer concentration in the perfusate (dpm · ml-1). In a single time point experiment, Xbrain/T replaced dXbrain/dt, where T was the perfusion time (minutes). Apparent brain distributional volumes (Vbrain, ml · 100 g-1) were calculated from:
 | (5) |
Apparent cerebrovascular PS (product of permeability and surface area) for biotin was estimated as:
 | (6) |
where Fpf is the regional cerebral perfusion fluid flow. Fpf was determined using brain uptake data of [14C]diazepam in separate experiments was 39.2 µl/g brain/s (Park and Sinko, 2005); P is the apparent permeability coefficient for biotin at the BBB, and S is the mouse brain capillary surface area. The concentration dependence of cerebrovascular PS for biotin during perfusion can be described as:
 | (7) |
where Vmax is the maximal uptake rate for the saturable component, Km is the half-saturation concentration of biotin, and Kd is the first order constant for the nonsaturable transport. Values for the kinetic parameters were obtained by fitting eqs. 1, 2, and 7 to the data using nonlinear least-squares regression analysis program (GraphPad Prism 4; Graphpad Software Inc., San Diego, CA). Data are presented as mean ± S.E.M. for three to four animals.
When appropriate, two-sided Student t tests and analysis of variance followed by Dunnett's test were used to determine the significance of differences between experimental groups for in vitro and in situ studies, respectively. GraphPad (GraphPad Software Inc.) was used for statistical analysis. Statistical significance was determined at the levels of significance as indicated.
View larger version (102K):
[in this window]
[in a new window]
|
FIG. 1. A, immunohistochemical staining of factor VIII antigen in primary cultures of bovine BMECs. The cells retain factor VIII as shown by typical perinuclear staining. B, immunohistochemical staining of GFAP antigen in primary cultures of bovine BMECs. Response of BMECs to GFAP immunostaining is negative, indicating that the purity of the endothelial cells is high.
|
|
|
Results
|
|---|
Identification and Immunohistochemical Characterization of BMEC Monolayers. Structure of confluent BMECs was examined using light and electron microscopy. Typical spindle-shaped endothelial cells with dimensions approximately 5 µm across the broadest part and around 50 µm long were observed. The BMECs showed a uniform phase-bright appearance with dark granular inclusions. The cells grew out approximately radially with some swirling patterns and whirls. The cells became confluent 6 to 7 days after plating. A similar pattern to that seen in the tight junctions of brain capillaries in vivo (Shivers, 1979) was observed in confluent BMEC monolayers using electron microscopy. The region between endothelial cells was characterized by a continuous network of complex anastomosing membrane particle arrays.
Positive response of factor VIII as a red granular population was found in a confluent monolayer (Fig. 1A). The endothelial origin of the cells is supported by the immunohistochemical finding of factor VIII (von Willebrand antigen). GFAP immunostaining was negative (Fig. 1B), indicating the absence of any contaminating cells.
SMVT and Mdr1 Gene Expression in BMECs. A single amplified DNA band was detected in BMECs (Fig. 2). For Mdr1, the DNA was 
480 base pairs (bp) as expected from the size of the fragment between the forward (3767 to 3790) and reverse (4214 to 4191) primer positions. For SMVT, the DNA was 400 bp as expected from the size of the fragment between the forward (923–953) and reverse (1333–1313) primer positions.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 2. Expression of SMVT and Mdr1 gene in BMECs. Images of the PCR products after electrophoresis through a 1.5% agarose gel indicating the presence and expression of the SMVT and Mdr1 gene in BMECs are shown. Primers and PCR conditions used are described under Materials and Methods. Lanes A and B represent SMVT (ca. 400 bp) and Mdr1 (ca. 480 bp), respectively.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3. Concentration dependence of biotin uptake into BMECs. Cells were incubated for 1 min at 37°C in HBSS-HEPES with various biotin concentrations and [3H]biotin (15 nM). Values are means ± S.D. (n = 4).
|
|
In Vitro Biotin Uptake Studies. BMECs were grown in monolayers, and upon reaching confluency, the concentration (1–200 µM) and sodium dependence (0–130 mM) of biotin uptake was determined at 37°C. The SMVT-mediated transport of biotin in BMECs was concentration-dependent and saturable (Fig. 3). Kinetic parameters were determined by fitting the experimental values to the Michaelis-Menten equation and were found to be 49.1 ± 8.1 µM and 313.2 ± 14.3 pmol/mg protein/min for Km and Vmax, respectively. The influence of sodium concentration on biotin transport was studied by increasing the Na+ concentration in the incubation medium and measuring initial uptake rates during 1-min incubation. NaCl was replaced iso-osmotically by choline-Cl. The hyperbolic dependence of the uptake of biotin on extracellular Na+ concentration is shown in Fig. 4. The data were fit to the Hill equation, and the value for n was found to be 1.03 ± 0.06. The value for n was confirmed from the slope of the Hill plot (Fig. 4, inset). This result suggests that the Na+-biotin coupling ratio for the transporter is 1, i.e., about one Na+-ion is cotransported per biotin molecule across the cell membrane. Furthermore, inhibition studies were performed by coincubating biotin with various derivatives and structural analogs of biotin such as pantothenic acid, desthiobiotin, 2-iminobiotin, diaminobiotin, and biocytin. Table 1 summarizes the inhibitory effect of several compounds on the uptake of 10 µM of biotin with 1-min incubation. Among the biotin derivatives and structural analogs, desthiobiotin and pantothenic acid significantly inhibited biotin uptake, whereas 2-iminobiotin and diaminobiotin were very weak inhibitors. When biotin was incubated in HBSS-HEPES where the NaCl was replaced by choline-Cl, intracellular accumulation of biotin was almost abolished.
View this table:
[in this window]
[in a new window]
|
TABLE 1 Inhibition of biotin uptake into BMECs BMECs were incubated for 1 min in HBSS-HEPES containing biotin (control) and inhibitor as indicated. Values are means ± S.D. (n = 4). Values were compared with control using Student's paired two-tailed t test.
|
|
Time Course of Biotin Uptake. Unidirectional uptake kinetics were required to obtain transfer coefficients (Kin) from single time point experiments. Brain uptake of biotin was evaluated for various perfusion times (20, 40, 60, and 90 s). Biotin uptake was expressed as apparent brain distributional volume, Vbrain, in the left hemisphere during perfusion (Fig. 5). Biotin uptake into the brain was appreciable, and the relationship between brain uptake of biotin and perfusion time for up to 90 s was linear (r2 = 0.99), extrapolating to 0 at 0 s during the perfusion while no BBB disruption was observed. In this study, [14C]sucrose was used as a vascular marker for correcting substrate concentration in the intravascular space. The estimated intravascular volumes using [14C]sucrose were not significantly different from those measured using [3H]inulin, another vascular marker. The distributional volume of [14C]sucrose was constant (1.2 ± 0.1 ml x 100/g brain, mean ± S.E.M; n = 12) during the perfusion (40–90 s). A 60-s perfusion time was chosen for all subsequent experiments using a 2.1-ml/min perfusion flow rate.
View larger version (10K):
[in this window]
[in a new window]
|
FIG. 5. Time course of biotin transport across the BBB using an in situ mouse brain perfusion technique. Brain uptake of [3H]biotin (12 nM) was evaluated for various perfusion times (20, 40, 60, and 90 s). Biotin uptake was expressed as apparent brain distributional volume, Vbrain, in the left hemisphere during perfusion (flow rate, 2.1 ml/min). The relationship between biotin brain uptake and perfusion time for up to 90 s was linear (r2 = 0.99), while no BBB disruption was observed. The distributional volume of [14C]sucrose was constant (1.2 ± 0.1 ml x 100/g brain, means ± S.E.M., n = 12) during perfusion time (40–90 s). Data are means ± S.E.M. (n = 34 mice).
|
|
Concentration Dependence of Biotin Uptake in Situ. The concentration dependence of unidirectional transport into mice brains was determined for biotin using the in situ brain perfusion technique. Figure 6 shows that the relationship between cerebrovascular PS for [3H]biotin in the left hemisphere and biotin concentration of perfusate. Cerebrovascular PS decreased as biotin concentration in the perfusate increased, suggesting that carrier-mediated transport is involved in biotin uptake into the brain. The concentration dependence of cerebrovascular PS to [3H]biotin was described adequately by a model with saturable and nonsaturable components. The curve represents the least-squares fit of eq. 7 to the data (r2 = 0.99), where Km = 35.4 ± 4.9 µM, Vmax = 7.26 ± 1.08 pmol/g brain/s, and Kd = 0.13 ± 0.01 µl/g brain/s.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 6. Cerebrovascular PS for [3H]biotin in the left hemisphere as a function of biotin concentration. Initial brain uptake of [3H]biotin was measured at various concentrations (1–400 µM) of biotin for 60 s. The result shows the concentration dependence of unidirectional influx of [3H]biotin. Line represents the fitting by a modified Michaelis-Menten equation (PS = [Vmax/(Km + Cpf) + Kd] and estimated values for kinetic parameters such as Km, Vmax, and Kd were 35.4 ± 4.9 µM, 7.26 ± 1.08 pmol/g brain/s, and 0.13 ± 0.01 µl/g brain/s, respectively. Data are means ± S.E.M. (n = 3 4). The brain vascular volume estimated using [14C]sucrose was almost constant (1.27 ± 0.03 ml x 100/g brain, n = 26).
|
|
Effect of Various Inhibitors and Na+ Influence on Biotin Uptake. Biotin uptake (10 µM) was measured for 60 s in the absence (control; open column) or presence (solid column) of various putative inhibitors (200 µM). Figure 7 illustrates that pantothenic acid and desthiobiotin significantly inhibited the transport of biotin across the BBB, whereas biocytin, 2-iminobiotin, and diaminobiotin had no significant effect. To study the influence of Na+ on biotin transport, perfusion was performed using a Na+-free buffer in which NaCl was iso-osmotically replaced by KCl. Biotin uptake was significantly reduced in the presence of K+ as compared with Na+.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 7. Effect of various inhibitors and ionic composition on biotin uptake into the mice brains. Uptake of biotin (10 µM) was measured for 60 s in the absence (control; open column) or presence (solid column) of inhibitors (200 µM), except for Na+ influence on biotin uptake. Inhibition effect of pantothenic acid and desthiobiotin on biotin uptake was statistically significant, whereas biocytin, 2-iminobiotin, and diaminobiotin were very weak inhibitors. To study an influence of Na+ on biotin uptake, perfusion was performed using a Na+-free buffer in which NaCl was iso-osmotically replaced with KCl. Biotin uptake was significantly decreased by the replacement with K+. Each result represents the mean ± S.E.M. (n = 34). The statistical significance of differences from the control was determined by analysis of variance followed by Dunnett's test (*, P < 0.05; **, P < 0.01).
|
|
|
Discussion
|
|---|
Bovine BMECs were isolated and successfully cultured. It was observed that all morphological and immunohistochemical properties of the in vitro bovine BMEC culture such as the expression of the endothelial cell-specific marker factor VIII are consistent with those of the BBB in vivo. Excellent attachment and growth were achieved when BMECs were cultured on rat-tail collagen-coated wells with an optimized seeding density. BMECs grew to confluent monolayers within 7 days after seeding and were used for cellular uptake studies. The transport properties of BMECs were characterized by investigating the mechanism of biotin uptake through a series of detailed studies.
In the present study, a saturable biotin transport mechanism on the luminal side of the BBB was identified, yielding a Km of 49.1 ± 8.1 µM in BMECs. Results of kinetic and inhibition studies show good agreement with those previously published in a calf brain cell culture model of the BBB (e.g., Km = 49 µM) (Baur and Baumgartner, 2000). Spector and Mock reported that biotin was transported into the brain by a saturable system with a Km of roughly 100 µM for rats (Spector and Mock, 1987), using the perfusion method and 20 µM for rabbits (Spector and Mock, 1988) by injecting intravenously and intraventricularly. In the current study, kinetic properties of biotin transport into mice brains were investigated using the in situ brain perfusion technique. To test for the existence of a biotin carrier-mediated system in mice, the initial rate of biotin uptake as a function of concentration was evaluated. The results indicated that biotin uptake across the BBB involved both saturable and nonsaturable processes. It is expected that more than 60% of biotin will be transported into the brain via a carrier-mediated process under normal physiological conditions. However, since blood biotin concentrations under physiological conditions are in the nanomolar range in humans and other mammals (Horsburgh and Gompertz, 1978), biotin uptake across the BBB may not show saturable behavior. Therefore, the existence of a carrier system for biotin brain uptake was confirmed by investigating the inhibitory effect of unlabeled biotin, biotin structural analogs, and biotin derivatives on biotin uptake.
Transmembrane ion gradients play a vital role in substrate transport via a membrane-linked transport system. Two ions, Na+ and H+, have been identified as the main coupling ions in such transport processes (Ma et al., 1994). Biotin uptake into BMECs was Na+-dependent with a coupling coefficient of 1 for biotin uptake as a function of increasing NaCl concentration. At pH 7.3, biotin is charged, since its pKa value is 4.5. Thus, the biotin carrier in the BMEC membrane must be electrically uncharged to maintain electroneutrality of the Na+/biotin transport complex, since biotin is deprotonated at physiological pH. The effect of Na+ on the uptake of biotin across the BBB using an in situ mouse brain perfusion technique was also examined after replacing the Na+ gradient iso-osmotically with a gradient of other monovalent cations such as K+. The in situ result showed significant inhibition of biotin uptake upon replacing the Na+ gradient, as observed in our in vitro BBB model.
Inhibition studies using structural analogs may provide valuable information regarding structure-transporter relationships. However, the involvement of multiple transporters in the translocation of a single drug or nutrient may complicate the interpretation of mechanism study results. Biotin and its analogs were selected for study since, at this point in time, SMVT appears to be the only transporter involved in the translocation of biotin and its analogs. This gave us a unique opportunity to probe in vitro-in situ transport properties of a single uptake/influx transporter in the brain. Biocytin, with a blocked carboxyl group on the valeric acid moiety, did not significantly inhibit biotin uptake, compared with unlabeled biotin and desthiobiotin in vitro. This result suggests that a free carboxyl group may be important for efficient interactions with the transporter. Diaminobiotin, which lacks an intact carbamide moiety, and 2-iminobiotin, which has amino group in the 5-position of imidazoline ring instead of carbonyl group, did not efficiently inhibit biotin uptake either. Therefore, it appears that a free carboxyl group and an intact carbamide moiety in the imidazolidone ring of the biotin molecule may be important for efficient transporter interactions. Similar structural requirements for the biotin transporter were reported in several cell lines (Ma et al., 1994; Prasad et al., 1997; Said et al., 1998) and other tissues such as the intestine (Said et al., 1987), kidney (Baur and Baumgartner, 1993), or placenta (Grassl, 1992). Biotin uptake in the presence of pantothenic acid was significantly inhibited. The transporter for biotin uptake is also involved in mediating the transport of pantothenate and lipoate (Prasad et al., 1997). SMVT transcripts have been identified in various rat tissues including brain. The recent finding of an SMVT cloned from rat placenta (Prasad et al., 1998) and rabbit intestine (Prasad et al., 1999) showed that this transporter is responsible for the uptake of pantothenate, biotin, and lipoate. Similar inhibition patterns were observed in situ and the results showed good agreement.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 8. Comparison of inhibitory effects and Na+ influence on biotin uptake between in vitro and in situ. Influence of Na+ ion and inhibitory effects of structural analogs and biotin derivatives on biotin (15 nM) uptake were determined in an in vitro uptake experiment or by the in situ mice brain perfusion technique; (1) unlabeled biotin (200 µM), (2) unlabeled biotin (100 µM), (3) influence of Na+ ion, (4) unlabeled biotin (50 µM), (5) pantothenic acid, (6) unlabeled biotin (10 µM), (7) desthiobiotin, (8) diaminobiotin, (9) biocytin, and (10) 2-iminobiotin. Each point is the mean ± S.E.M. for four (in vitro) or three four (in situ) experiments. The solid line represents a linear regression line (r2 = 0.93), and the dotted lines are 95% confidence limits.
|
|
Figure 8 demonstrates a linear relationship of Na+ dependence and other inhibitory effects between the in vitro and in situ uptake of biotin under various conditions. A slope of 0.83 (r2 = 0.93) was obtained when in situ and in vitro data were correlated, indicating a good correspondence between in vitro and in vivo properties. When kinetic parameters such as Km were compared, estimated Km values using the in vitro BBB model and in situ brain perfusion technique were 49.1 ± 8.1 and 35.4 ± 4.9 µM, respectively. The order of affinity (1/Km) for the cerebrovascular biotin transport system was comparable with that obtained in the in vitro BBB model. Kinetic parameters, functional properties, and effects of inhibitors were well preserved between intact mice brains and BMECs in vitro. Interestingly, the present results are consistent with that of DeBoer, who suggested that other properties of the BBB are likely to be organ-specific rather than species-specific (De Boer and Sutanto, 1997). Therefore, a primary culture of BMECs may be useful as an in vitro BBB model to investigate the mechanism and properties of biotin transport across the BBB. The major limitation is that, due to the unusually high passive permeability, the in vitro results may not be directly useful for predicting the extent of brain exposure. On the other hand, the model has the advantage that mechanism studies can be performed in a much higher throughput than in situ studies.
In summary, we have confirmed the existence of a biotin transporter in both in vitro and in vivo BBB models and investigated the characteristics of a biotin transporter using in vitro BBB model and an in situ mice brain perfusion technique. In our studies, biotin was transported into the brain by a saturable but a low-capacity transport system and Na+-dependent process with biotin-Na+ coupling. The present study results also suggest some structural requirements for efficient substrate interactions with SMVT. The in vitro and in vivo BBB models show good agreement and suggest that in vitro transporter-based mechanism studies yield valuable results but are limited because of the inability to predict total brain exposure.
|
Footnotes
|
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.005231.
ABBREVIATIONS: BBB, blood-brain barrier; SMVT, sodium-dependent multivitamin transporter; F-12, Ham's F-12 nutrient mixture; MEM, minimum essential medium; BMEC, brain microvessel endothelial cells; GFAP, glial fibrillary acidic protein; Mdr, multidrug-resistant; PCR, polymerase chain reaction; HBSS, Hanks' buffer salt solution; bp, base pair(s).
Address correspondence to: Dr. Patrick J. Sinko, Rutgers University, Ernest Mario School of Pharmacy, 160 Frelinghuysen Road, Piscataway, NJ 08854. E-mail: sinko{at}rci.rutgers.edu
|
References
|
|---|
Audus KL, Bartel RL, Hidalgo IJ, and Borchardt RT (1990) The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm Res 7: 435-451.[CrossRef][Medline]
Audus KL and Borchardt RT (1987) Bovine brain microvessel endothelial cell monolayers as a model system for the blood-brain barrier. Ann NY Acad Sci 507: 9-18.[Abstract]
Baron-Van Evercooren A, Gansmuller A, Gumpel M, Baumann N, and Kleinman HK (1986) Schwann cell differentiation in vitro: extracellular matrix deposition and interaction. Dev Neurosci 8: 182-196.[Medline]
Baur B and Baumgartner ER (1993) Na(+)-dependent biotin transport into brush-border membrane vesicles from human kidney cortex. Pflugers Arch 422: 499-505.[CrossRef][Medline]
Baur B and Baumgartner ER (2000) Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood-brain barrier. Brain Res 858: 348-355.[CrossRef][Medline]
Bowman PD, Betz AL, Ar D, Wolinsky JS, Penney JB, Shivers RR, and Goldstein GW (1981) Primary culture of capillary endothelium from rat brain. In Vitro 17: 353-362.[Medline]
Bowman PD, Ennis SR, Rarey KE, Betz AL, and Goldstein GW (1983) Brain microvessel endothelial cells in tissue culture: a model for study of blood-brain barrier permeability. Ann Neurol 14: 396-402.[CrossRef][Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Chikhale EG, Ng KY, Burton PS, and Borchardt RT (1994) Hydrogen bonding potential as a determinant of the in vitro and in situ blood-brain barrier permeability of peptides. Pharm Res 11: 412-419.[CrossRef][Medline]
Cisternino S, Rousselle C, Dagenais C, and Scherrmann JM (2001) Screening of multidrug-resistance sensitive drugs by in situ brain perfusion in P-glycoprotein-deficient mice. Pharm Res 18: 183-190.[CrossRef][Medline]
Coval ML (1970) Analysis of Hill interaction coefficients and the invalidity of the Kwon and Brown equation. J Biol Chem 245: 6335-6336.[Abstract/Free Full Text]
Dagenais C, Rousselle C, Pollack GM, and Scherrmann JM (2000) Development of an in situ mouse brain perfusion model and its application to mdr1a P-glycoprotein-deficient mice. J Cereb Blood Flow Metab 20: 381-386.[Medline]
De Boer AG and Sutanto W (1997) Drug Transport across the Blood-Brain Barrier: In Vitro and in Vivo Techniques, CRC Press, Boca Raton, FL.
Faulkner G, Dubois-Dalcq M, Hooghe-Peters E, McFarland HF, and Lazzarini RA (1979) Defective interfering particles modulate VSV infection of dissociated neuron cultures. Cell 17: 979-991.[CrossRef][Medline]
Feng MR (2002) Assessment of blood-brain barrier penetration: in silico, in vitro and in vivo. Curr Drug Metab 3: 647-657.[CrossRef][Medline]
Goldstein GW and Betz AL (1986) The blood-brain barrier. Sci Am 255: 74-83.[Medline]
Goldstein GW, Betz AL, and Bowman PD (1984) Use of isolated brain capillaries and cultured endothelial cells to study the blood-brain barrier. Fed Proc 43: 191-195.[Medline]
Grassl SM (1992) Human placental brush-border membrane Na(+)-biotin cotransport. J Biol Chem 267: 17760-17765.[Abstract/Free Full Text]
Heck HD (1971) Statistical theory of cooperative binding to proteins. The Hill equation and the binding potential. J Am Chem Soc 93: 23-29.[CrossRef][Medline]
Horsburgh T and Gompertz D (1978) A protein-binding assay for measurement of biotin in physiological fluids. Clin Chim Acta 82: 215-223.[CrossRef][Medline]
Ma TY, Dyer DL, and Said HM (1994) Human intestinal cell line Caco-2: a useful model for studying cellular and molecular regulation of biotin uptake. Biochim Biophys Acta 1189: 81-88.[Medline]
Momma S, Aoyagi M, Rapoport SI, and Smith QR (1987) Phenylalanine transport across the blood-brain barrier as studied with the in situ brain perfusion technique. J Neurochem 48: 1291-1300.[CrossRef][Medline]
Orlowski M and Meister A (1970) The gamma-glutamyl cycle: a possible transport system for amino acids. Proc Natl Acad Sci USA 67: 1248-1255.[Abstract/Free Full Text]
Pardridge WM (1995) Transport of small molecules through the blood-brain barrier: biology and methodology. Adv Drug Deliv Rev 15: 5-36.[CrossRef]
Pardridge WM (1999) Blood-brain barrier biology and methodology. J Neurovirol 5: 556-569.[Medline]
Park S and Sinko PJ (2005) P-Glycoprotein and multidrug resistance-associated proteins limit the brain uptake of saquinavir in mice. J Pharmacol Exp Ther 312: 1249-1256.[Abstract/Free Full Text]
Prasad PD, Ramamoorthy S, Leibach FH, and Ganapathy V (1997) Characterization of a sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate in human placental choriocarcinoma cells. Placenta 18: 527-533.[CrossRef][Medline]
Prasad PD, Wang H, Huang W, Fei YJ, Leibach FH, Devoe LD, and Ganapathy V (1999) Molecular and functional characterization of the intestinal Na+-dependent multivitamin transporter. Arch Biochem Biophys 366: 95-106.[CrossRef][Medline]
Prasad PD, Wang H, Kekuda R, Fujita T, Fei YJ, Devoe LD, Leibach FH, and Ganapathy V (1998) Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate. J Biol Chem 273: 7501-7506.[Abstract/Free Full Text]
Said HM, Ortiz A, McCloud E, Dyer D, Moyer MP, and Rubin S (1998) Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid. Am J Physiol 275: C1365-C1371.
Said HM, Redha R, and Nylander W (1987) A carrier-mediated, Na+ gradient-dependent transport for biotin in human intestinal brush-border membrane vesicles. Am J Physiol 253: G631-G636.
Seetharaman S, Maskell L, Scheper RJ, and Barrand MA (1998) Changes in multidrug transporter protein expression in endothelial cells cultured from isolated human brain microvessels. Int J Clin Pharmacol Ther 36: 81-83.[Medline]
Shivers RR (1979) The blood-brain barrier of a reptile, Anolis carolinensis. A freeze-fracture study. Brain Res 169: 221-230.[CrossRef][Medline]
Smith QR (1996) Brain perfusion systems for studies of drug uptake and metabolism in the central nervous system, in Models for Assessing Drug Absorption and Metabolism (Borchardt RT ed) pp 285-307, Plenum Press, New York.
Spector R and Mock D (1987) Biotin transport through the blood-brain barrier. J Neurochem 48: 400-404.[CrossRef][Medline]
Spector R and Mock DM (1988) Biotin transport and metabolism in the central nervous system. Neurochem Res 13: 213-219.[CrossRef][Medline]
Takasato Y, Rapoport SI, and Smith QR (1984) An in situ brain perfusion technique to study cerebrovascular transport in the rat. Am J Physiol 247: H484-H493.
Williams GC, Knipp GT, and Sinko PJ (2003) The effect of cell culture conditions on saquinavir transport through and interactions with, MDCKII cells overexpressing hMDR1. J Pharm Sci 92: 1957-1967.[CrossRef][Medline]
This article has been cited by other articles:
|
|
|
|
 
V. S. Subramanian, J. S. Marchant, and H. M. Said
Biotin-responsive basal ganglia disease-linked mutations inhibit thiamine transport via hTHTR2: biotin is not a substrate for hTHTR2
Am J Physiol Cell Physiol,
November 1, 2006;
291(5):
C851 - C859.
[Abstract]
[Full Text]
[PDF]
|
|
|
![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Top
Abstract
Materials and Methods
