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B, a Nitrosative Stress-Sensitive Transcription Factor, in the RegulationDepartment of Pharmaceutics, College of Pharmacy (H.-J.M., M.-H.K., H.-E.J., D.-D.K., C.-K.S., S.-J.C.), and National Research Laboratory, College of Pharmacy and Research Institute of Pharmaceutical Sciences (S.M.S., S.G.K.), Seoul National University, Seoul, Korea; and Graduate School of Pharmaceutical Science, The University of Tokyo, Tokyo, Japan (T.T.)
(Received March 21, 2007; accepted July 25, 2007)
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B (NF-B) and DNA binding activity of nuclear extracts to the NF-B consensus oligonucleotide was increased in MBEC4 cells pretreated with SNP. Taken together, these observations suggest that nitrosative stress leads to the up-regulation of the message for the efflux transporter and, ultimately, to the enhanced function, probably via a NF-B-dependent mechanism.
). Although the pathogenesis of the diabetes is not fully delineated, the literature information is quite clear on the involvement of oxidative and nitrosative stresses (Cai et al., 2005; Pacher et al., 2005) in the disease state. Nitrosative stress was defined as nitration/nitrosylation of protein, peroxidation of lipid, deoxyribonucleic acid (DNA) damage, and cell death caused by excessive production of peroxynitrite and/or other reactive nitrogen species [viz., collectively, nitrogen oxides (NOx)] (Obrosova et al., 2005) These stress conditions may be associated with the development of diabetic complications such as cardiovascular disease, nephropathy, and retinopathy (Krolewski et al., 1987). Although it is possible that these stress conditions are independently related to the complications, the mediators for these stress conditions may also be involved cooperatively in the pathogenesis of the diabetes. For example, superoxide anion is known to react chemically with NO to form peroxynitrite, a highly reactive nitrogen oxide form (Huie and Padmaja, 1993), thereby potentially exacerbating the condition. Indeed, Cai et al. (2005) reported a significant increase of 3-nitrotyrosine, a by-product of the reaction between peroxynitrite and proteins, in the serum and tissue of a streptozotocin (STZ)-induced diabetic model, which may lead to an alteration in the function of the covalently modified proteins and, ultimately, of the organ itself. Despite the aforementioned possibility, however, the pathological impact of the nitrosative stress was not fully delineated in the diabetes, especially for the relationship between the stress conditions and pharmacokinetic alteration during the disease state.
It is becoming increasingly clear that NOx is a crucial regulatory mediator for the function and the expression of the transporter (Uchiyama et al., 2005). For example, Bridges et al. (2001) have shown that taurine transporter was up-regulated after long-term exposure to NOx donors with cultured ARPE-19 cells. In addition, Uchiyama et al. (2005) reported that sodium-dependent neutral amino acid transporter was induced by the pretreatment of a NOx donor with Caco-2 cells. Because the exposure with NOx led to a regulation of more than one transport system in a number of different cell lines, a similar consequence may occur for pathological conditions that are associated with nitrosative stress, such as diabetes. Consistent with this hypothesis, the level of expression for ATP-binding cassette (ABC) transporters [e.g., Mdr2/P-glycoprotein (van Waarde et al., 2002)] was increased in the liver of rats with experimental diabetes. It is interesting that Liu et al. (2006) showed the functional impairment of P-glycoprotein in the blood-brain barrier (BBB) of rats with experimental diabetes. Since the former literature indicates the existence of functional induction for ABC transporters, including Mdr2/P-glycoprotein in the liver of diabetic rats, a regulatory mechanism specific for the blood-brain barrier may exist in the case of P-glycoprotein in diabetic conditions. Unfortunately, however, this aspect of P-glycoprotein regulation has not been extensively studied in the brain of diabetic rats.
The current study, therefore, was undertaken to investigate the underlying mechanism for the functional alteration of P-glycoprotein in the BBB of the STZ-induced diabetic rats and an in vitro model of the BBB [viz., MBEC4 cells (Tatsuta et al., 1992; Ahn et al., 2004)] pretreated with NOx donors. Since diabetes is known to produce a nitrosative stress condition and the exposure of NOx may lead to a regulation of the gene expression of efflux transporters (Heemskerk et al., 2007), the role of NOx in the functional alteration of P-glycoprotein in the BBB was of particular interest. But rather than an impairment, it was found that an STZ-induced diabetic condition led to a functional induction of P-glycoprotein in the BBB in association with the increased levels of the protein and the corresponding mRNA of P-glycoprotein. The induction was apparently mediated by the activation of NF-B, which is a putative regulatory factor for P-glycoprotein gene expression and a nuclear transcription factor that is sensitive to nitrosative stress.
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). All other chemicals were of reagent grade or better and were used without further purification.
Animals. Male Sprague-Dawley rats (Dae-Han Biolink, Eumsung, Korea), weighing 220
230 g, were used in this study. Experimental protocols involving animals in this study were reviewed by the Animal Care and Use Committee of the College of Pharmacy, Seoul National University, according to National Institutes of Health guidelines (National Institutes of Health publication number 85-23, revised 1985) in Principles of Laboratory Animal Care.
Induction of Experimental Diabetes in Rats by STZ Administration. The rats were randomly divided into two groups: control and diabetes. Freshly prepared STZ solution (STZ powder dissolved in a citrate buffer of pH 4.5, the final concentration for STZ of 60 mg/ml) at a dose of 60 mg/kg was administered once via intraperitoneal injection to overnight-fasted rats. Control rats received an intraperitoneal injection of the same volume of citrate buffer (i.e., the vehicle). On day 3, blood samples (50 µl, via tail vein blood sampling) were obtained from the rats for the determination of blood glucose level. The rats having a blood glucose level exceeding 300 mg/dl (determined by a OneTouch glucometer; LifeScan Inc., Milpitas, CA) were considered to be experimental diabetic and used in subsequent studies. All rats were maintained for up to 28 days with free access to food and tap water in temperature- and humidity-controlled quarters. Blood glucose concentrations and weights were monitored weekly and subsequent doses were provided as necessary.
Determination of NOx. NOx level of plasma or NOx donors under our cell culture conditions was measured spectrophometrically by the use of the Griess reaction (Green et al., 1982). Approximately 400 µl of blood samples were obtained from control or diabetic rats, and the plasma was obtained by centrifugation (14,000 rpm, for 15 min). Nitrate contained in the sample was first reduced by nitrate reductase to nitrite, and the Griess reagent, a mixture (1:1) of 0.2% naphthylethylene-diamine and 2% sulfonamide in 5% phosphoric acid, was then added. After 5 min of standing at room temperature, the concentration of total nitrite was determined spectrophotometrically at 550 nm using NaNO3 solution as the standard. Total nitrite level was regarded as the sample NOx concentration.
In Vivo Brain Uptake Study. Under light ether anesthesia, the rats received implantations in their femoral artery and vein, using catheters made of polyethylene tubing (PE-50; Clay Adams, Parsippany, NJ) filled with heparinized saline (25 U/ml). After recovery (approximately 120 min after the completion of the surgery) from anesthesia, CsA solution was bolus-injected to the rat via the venous catheter at a dose of 1 mg/kg. Each dosing solution contained 1 mg of CsA in 1 ml of the vehicle (PEG400/ethanol = 9:1). The blood samples were collected at 30, 60, 120, and 180 s. Immediately after the last blood collection, the rat was decapitated, and the brain tissue was collected and weighed. The blood and brain samples were then solubilized in 1 ml of Soluene-350 (PerkinElmer Life and Analytical Sciences, Boston, MA) and transferred to scintillation vials for the determination of radioactivity. The brain uptake clearance was estimated by standard integration plot analysis (Kusuhara et al., 1997), in which the uptake clearance was obtained by dividing the amount of CsA in the brain at time t by the area under the plasma concentration-time curve up to the time t for CsA.
Real-Time PCR Analysis. When it was necessary to determine the mRNA levels for P-glycoprotein in the brain of control/diabetic rats and MBEC4 cells pretreated with SNP, real-time monitoring of the PCR was performed using the LightCycler 1.5 system (Roche Applied Science, Indianapolis, IN). Pairs of forward/reverse primers specific for Mdr1a, Mdr1b, Mrp1, Mrp2, and β-actin were synthesized as described by van Vliet et al. (2004) and Serrano et al. (2003). The FastStart DNA Master SYBR Green Kit (Roche Applied Science) was used for the quantitative PCR analysis (van Vliet et al., 2004). The cycling conditions were carried out as follows: Mdr1a, Mrp1, and Mrp2, initial denaturation at 95°C for 6 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 59°C for 5 s, and extension at 72°C for 10 s; Mdr1b, initial denaturation at 95°C for 6 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 5 s, and extension at 72°C for 20 s. The temperature transition rate was set at 20°Cs-1. To distinguish the specific amplification product from nonspecific products or primer dimmers, a melting curve was constructed from the amplification reaction obtained by maintaining the temperature at 65°C for 15 s, followed with a gradual temperature increase rate of 0.1°C/s to 95°C. For this study, the signal acquisition mode was set at "continuous".
The relative quantification of the amount of the target message in the tested brain tissue samples was obtained by measuring Ct. The following equations were used to quantify the amount of the message:
 | (1) |
 | (2) |
where CtMdr1 represents the Ct value of the target gene and Ctβ-actin the Ct value of the endogenous control reference gene. Ctlinear represents the -fold change in the mRNA expression between the control and the treated groups, assuming a doubling of the target sequence with each PCR cycle.
MBEC4 Cell Culture. For this study, MBEC4 cells, immortalized mouse brain capillary endothelial cells (Tatsuta et al., 1992), were used as an in vitro model of the BBB. Standard protocols were used for subculturing in a T-flask and seeding in 12-well culture plates (Ahn et al., 2004). Cells were maintained at 37°C in Dulbecco's modified Eagle's medium (low glucose), 10% fetal bovine serum, 1% nonessential amino acid solution, 100 units/ml penicillin, and 0.1 mg/ml streptomycin in an atmosphere of 5% CO2 and 90% relative humidity.
Determination of CsA Uptake in MBEC4 Cells. When it was necessary to determine the uptake kinetics, the culture medium was removed from MBEC4 cells and the cells were subsequently washed three times with uptake buffer. The uptake buffer contained Hanks' balanced salt solution with 25 mM HEPES and 25 mM glucose (pH 7.4). Uptake was initiated by addition of 1 ml of uptake buffer containing 1 µM CsA with trace levels (i.e., 1% of total CsA concentration) of the radiolabeled drug. Fifteen-minute uptake at 37°C was measured for this study. Upon completion of the incubation, the uptake was terminated by the aspiration of medium followed by washing (three times) with ice-cold uptake buffer with unlabeled CsA. The cells were then solubilized in 1 ml of 0.1 N NaOH and transferred to scintillation vials for the determination of radioactivity. The amount of uptake of [3H]CsA was adjusted to pmol/mg protein in each sample.
CsA Uptake in the Presence of Various NOx Donors in MBEC4 Cells. To determine the uptake of CsA in MBEC4 cells in the presence of various NOx donors, confluent cultures of MBEC4 cells were incubated at 37°C with serum-free medium in either the absence or the presence (viz., 1 mM for all donors, final concentration) of NOx donors, SNAP, DETA, SIN-1, or SNP, for 24 h. Upon completion of the pretreatment, the pretreatment medium was aspirated, the cells were washed three times with uptake buffer, and the uptake of CsA was determined, similar to the procedure described in the previous section. When it was necessary to study the temporal dependence of the effect of SNP, the cells were pretreated with the donor for various periods of exposure (from 0.5 to 24 h) and the uptake was determined in a similar manner. Since the impact of SNP may be mediated by a number of different NOx, SNP-mediated stimulation on the efflux of CsA was studied in MBEC4 cells by the pretreatment of SNP in the presence (all in final concentration) of either ascorbate [10 mM; an antioxidant (Bridges et al., 2001)], methylene blue [1 mM; an NO scavenger (Bridges et al., 2001)], or SOD [750 unit/ml; superoxide scavenger (Uchiyama et al., 2005)]. The potential cytotoxic effects of the NOx donors (1 mM) were also evaluated by protein (Lowry et al., 1951) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays.
Total RNA Isolation and cDNA Synthesis. Total RNA was isolated from the brain tissues and the MBEC4 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The concentration and purity of the RNA were determined spectrophotometrically at 260/280 nm. Approximately 1 µg of total RNA was used in each 20-µl reaction mixture containing 5 mM MgCl2, 1 mM deoxynucleoside-5'-triphosphate, 5 U RNase inhibitor, 5 U avian myeloblastosis virus reverse transcriptase, and 0.125 µM oligo-dT-adaptor primer (Takara Shuzo Co., Ltd., Shiga, Japan). The reactions were carried out by incubating the mixture at 42°C for 30 min, heating to 99°C for 5 min, and subsequent cooling to 4°C.
Semiquantitative RT-PCR Analysis. When it was necessary to determine the mRNA levels for P-glycoprotein in MBEC4 cells, RT-PCR was carried out using primer pairs specific for mice Mdr1b [5'-TGCTTATGGATCCCAGAGTGAC-3' (sense) and 5'-TTGGTGAGGATCTCTCCGGCT-3' (antisense)]. The primers specific for mice GAPDH were 5'-CTCATgACCACAgTCCATgC-3' (sense) and 5'-CACATTGGGGGTAggAACAC-3' (antisense). The fragments of specific genes were amplified through 30 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min and terminated by a final step of 72°C for 15 min. The PCR products were separated by gel electrophoresis using 1.5% (w/v) agarose, then inspected under UV light after ethidium bromide staining and photographed using a digital camera.
Preparation of Nuclear and Cytosolic Fractions. Nuclear extracts were prepared essentially according to the method of Schreiber et al. (1990). In brief, cells in dishes were washed with ice-cold PBS. Cells were then scraped and allowed to swell after addition of 100 µl of lysis buffer containing 10 mM HEPES (pH 7.9), 0.5% Nonidet P-40, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. Cell membranes were lysed by vortexing the mixture; the lysate was then incubated for 10 min on ice and centrifuged at 7200g for 5 min. The supernatant (containing cytosolic fraction) was stored at -70°C until use. The pellet (containing crude nuclei) was resuspended in 50 µl of a buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride; then, it was incubated for 30 min on ice. The samples were subsequently centrifuged at 15,800g for 10 min to obtain the nuclear extract (i.e., the supernatant). The nuclear extracts were stored at -70°C until use.
Western Blot Analysis. Western blot analysis was performed to determine levels of P-glycoprotein of brain homogenate in rats or the lysates of MBEC4 cells pretreated with SNP. Western blot analysis was also performed to determine levels of NF-B in the nuclear extract of MBEC4 cells exposed to SNP or by SNP exposure in the presence of ascorbate, methylene blue, or SOD. The samples (20 µg for P-glycoprotein of brain homogenate in rats or NF-B in the nuclear extract of MBEC4 cells, or 100 µg for P-glycoprotein of the lysates of MBEC4 cells pretreated with SNP) were loaded on 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate and electrophoretic separation was carried out. The gel was then transferred onto a nitrocellulose membrane. The membrane was probed with either a primary monoclonal mouse antibody, C219 (Signet Laboratories, Dedham, MA), for the determination of P-glycoprotein level, or with a rabbit polyclonal anti-NF-B p65 (Santa Cruz Biotechnology, Santa Cruz, CA) for the determination of NF-B level. Either peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (Zymed Laboratories, South San Francisco, CA) was used as a secondary antibody. The bound antibody was detected using an enhanced chemiluminescence detection system (GE Healthcare).
Electrophoretic Mobility Shift Assay. A double-stranded DNA probe for the consensus sequence of NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') was used for gel shift analysis after end-labeling of the probe with [
-32P]ATP and T4 polynucleotide kinase. The reaction mixture contained 2 µl of 5x binding buffer with 20% glycerol, 5 mM MgCl2, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM dithiothreitol, 0.25 mg/ml poly(dI-dC), and 50 mM Tris·Cl (pH 7.5), 30 µg of nuclear extracts, and sterile water in a total volume of 10 µl. Incubations were initiated by addition of a 1-µl probe (106 cpm) and continued for 20 min at room temperature. The specificity of the DNA/protein binding was confirmed by competition reactions, in which a 20-fold molar excess of unlabeled NF-B oligonucleotide was added to each reaction mixture before the addition of radiolabeled probe. For supershift assay, the antibody (2 µg) was added to the reaction mixture and additionally incubated for 1 h at 25°C. Samples were loaded onto 4% polyacrylamide gels at 100 V. The gels were removed, fixed, and dried, followed by autoradiography.
Data Analysis. When it was necessary to determine the brain uptake clearance by an integration plot method, the rate of the compound in the brain was calculated by eq. 3 (Kusuhara et al., 1997),
 | (3) |
where Xbrain represents the amount of drug in the brain, CLuptake represents the uptake clearance to the brain, and 
represents the area under the concentration time curve from time 0 to the time of brain sampling.
When it was necessary to compare the means between the treatments, the unpaired t test, or one-way ANOVA, followed by Duncan's test, was typically used. For this study, p < 0.05 was accepted as denoting statistical significance. Data were expressed as the mean ± S.D.
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). In addition, the enhanced level of NOx in the plasma was consistent with other experimental diabetic models (Bank and Aynedjian, 1993). These observations suggest, therefore, that the STZ administration to the rats led to experimental diabetes in this study.
Kinetics of CsA Transport to the Brain in Control Versus STZ-Induced Diabetic Rats. Since CsA is known to partition to red blood cells in a time-dependent manner, the blood concentration, rather than the plasma concentration, was measured to determine the pharmacokinetics in this study. After an intravenous administration of CsA, a substrate of P-glycoprotein, at a dose of 1 mg/kg, the temporal profiles of mean arterial blood concentration for the immunosuppressive drug were not significantly different between control and STZ-induced diabetic rats, suggesting that the systemic pharmacokinetics was not affected by the diabetic condition (data not shown), consistent with the study of Ogata et al. (1996). In contrast, however, the transport of CsA to the brain was significantly different. Table 2 summarizes the kinetic parameters for CsA transport to the brain. Whereas the brain-to-plasma concentration ratio for mannitol, a substance that is not permeable to the BBB (Ahn et al., 2004), was not affected (i.e., 40.6 ± 1.84 versus 39.8 ± 1.21 µl/g brain for control versus diabetic rats, respectively), the concentration ratio for CsA was significantly reduced in the diabetic rats (37.5 ± 7.43 µl/g brain), compared with the control rats (56.9 ± 5.66 µl/g brain). In addition, the apparent brain uptake clearance by integration plot analysis (Kusuhara et al., 1997) was also reduced by the induction of diabetes (Table 2). When the brain uptake kinetics was monitored with the progression of the hyperglycemic condition (Fig. 1), the reduced penetration was readily apparent at 1 week after STZ administration, and the reduced penetration continued throughout the experimental period ( 4 weeks).
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Comparison of the Levels of mRNA and Protein for P-glycoprotein in Control Versus STZ-Induced Diabetic Rats. Since the reduced penetration of CsA may be related to a functional induction of the efflux transporter (e.g., P-glycoprotein) for the drug, the levels of the mRNA and the protein for P-glycoprotein were examined in both the control and the STZ-induced diabetic rats. When mRNA levels of Mdr1a and Mdr1b were determined in brain homogenates by real-time RT-PCR, as indicated by the 
Ct value, both were significantly increased compared with levels in the control rats (p < 0.001) (for Mdr1a, 4.01- ± 1.09-fold; for Mdr1b, 2.91- ± 1.15-fold; Fig. 2). In addition, the Mdr1a form was expressed higher than was the Mdr1b form, the isoform of Mdr1a, in the brain homogenate of the control rats, which is consistent with the literature (Mei et al., 2004). Similar to the change in Mdr1 isoforms, the Mrp2 mRNA expression was significantly increased in the brain of diabetic rats (2.28-fold of controls), whereas the Mrp1 mRNA level was not affected by the induction of experimental diabetes (Fig. 2).
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). In addition to the comparable functions between the rat and the mouse Mdr1 protein, significant amino acid sequence homology was noted between the species (94.7% between the rat and mouse form for Mdr1a; 93% between the rat and mouse form for Mdr1b). Based on the literature evidence, P-glycoproteins from the rat and mouse were regarded as functionally comparable in subsequent studies. To study the underlying mechanism for the enhanced expression and the function of P-glycoprotein, MBEC4 cells, an in vitro model of the BBB (Ahn et al., 2004), was used. The level of NOx generated from 1 mM NOx donors in the cell culture condition is summarized in Table 3. The NOx level (in order from the highest to the lowest) was DETA > SIN-1 > SNAP > SNP. The NOx concentration in the plasma obtained from the diabetic rats was 117 ± 12.3 µM compared with a concentration of 158 ± 20.0 µM in incubation media that contained 1 mM SNP. Therefore, the level of NOx exposure in the in vitro and in vivo experiments was relatively comparable. When the cell was exposed to NOx donors, SNAP, DETA, SIN-1, or SNP, for 24 h at a concentration of 1 mM, the functional alteration on the uptake of CsA in the MBEC4 cells was variable (Fig. 4). Among the NOx donors, the functional enhancement (i.e., reduced uptake) was apparent for SNP and SIN-1, whereas the stimulatory effect was absent for SNAP and DETA. In the cases of the NOx donors that exhibited the stimulatory effect, SNP was the most pronounced. Figure 4B shows the concentration-stimulation relationship for SNP in MBEC4 cells. The concentration-dependent stimulatory effect was observed for the treatments at SNP concentrations higher than 100 µM. Temporal dependence of the stimulation by SNP showed that, after a transient impairment (0.51 h of the SNP treatment), the functional induction was readily apparent after 24-h of the treatment (Fig. 4C). In subsequent studies, the treatment with SNP was set at 1 mM/24 h for the exposure time. To further test the functional induction of P-glycoprotein in MBEC4 cells exposed to SNP, intracellular accumulation of CsA (initial concentration of 10 nM) in the presence and absence of verapamil (10 µM, the final concentration in the transport medium), a known inhibitor for P-glycoprotein function (Matsuzaki et al., 1999), was compared in control and SNP-pretreated MBEC4 cells. The accumulation of CsA was 3.51 ± 0.168 and 2.48 ± 0.147 pmol/mg protein in control and SNP-pretreated cells, respectively. In contrast, in the presence of verapamil (10 µM), there was greater accumulation of CsA, 4.94 ± 0.278 and 4.81 ± 0.684 pmol/mg protein in control and SNP-pretreated cells, respectively. This observation is consistent with the hypothesis that the functional induction by SNP-pretreated MBEC4 cells is mediated by the induction of P-glycoprotein. Collectively, these results indicate that SNP and SIN-1, but not all NOx donors, had a stimulatory effect on the function of P-glycoprotein.
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Comparison of the Levels of mRNA and Protein for P-glycoprotein in MBEC4 Cells with or without SNP Pretreatment. In a preliminary study, we attempted to measure the level of Mdr1a in MBEC4 cells with quantitative RT-PCR. However, amplification curves for Mdr1a could not be constructed up to 45 cycles with a number of annealing conditions, probably because of limited Mdr1a expression in MBEC4 cells (Matsuzaki et al., 1999). Therefore, we measured only Mdr1b in subsequent studies of MBEC4 cells. RT-PCR analysis was used to study the effect of SNP treatment on the level of P-glycoprotein mRNA. The level of Mdr1b mRNA was markedly increased in SNP-treated MBEC4 cells, compared with untreated cells (Fig. 5A). The level of a housekeeping gene, GAPDH, was not affected by the SNP treatment with MBEC4 cells (Fig. 5A). When mRNA levels of Mdr1b were determined in SNP-pretreated MBEC4 cells by real-time RT-PCR, as indicated by the Ct value, the level was significantly increased compared with untreated MBEC4 cells (p < 0.001) (i.e., for Mdr1b, 3.09- ± 0.28-fold; Fig. 5B). Consistent with the mRNA level, the level of P-glycoprotein protein was markedly increased approximately 1.74-fold in SNP-pretreated MBEC4 cells (Fig. 5C).
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Ct value, the addition of ascorbate, methylene blue, or SOD attenuated the increase in P-glycoprotein mRNA levels by SNP (p < 0.001) (Fig. 6B).
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Potential Involvement of NF-B in the SNP-Mediated Enhanced Expression of P-glycoprotein in MBEC4 Cells. To study the underlying mechanism for the SNP-mediated induction in the expression and the function of P-glycoprotein, we hypothesized that the exposure of an MBEC4 cell with the NOx donor may lead to an activation of NF-B, a putative regulator for P-glycoprotein expression (Thevenod et al., 2000; Kuo et al., 2002), thereby elevating the level of the P-glycoprotein expression and, thus, its function. Since NF-B activation is involved in the translocation of the protein to the nucleus, the protein level was quantified in the nuclear fraction by Western blot analysis. Based on the gel electrophoresis data (Fig. 7A) and the software analysis of the band density, the nuclear translocation of NF-B was readily apparent after 1 h of the SNP treatment in MBEC4 cells, and the activation was still apparent up to 6 h (the last sampling time) after SNP treatment (e.g., at 3 h of SNP treatment, 155 ± 12.4% of the band density of the untreated MBEC4 cells). In the case of the reference protein (β-actin), the density of the band was not affected by the SNP treatment. In addition, pretreatment with ascorbate (an antioxidant, 10 mM), methylene blue (an NO scavenger, 1 mM), or SOD (superoxide scavenger, 750 U/ml) attenuated nuclear translocation of NF-B in MBEC4 cells exposed to SNP (1 mM, at 3 h of treatment) (Fig. 7B). To test this hypothesis further, electrophoretic mobility shift for NF-B DNA binding activity was performed with the nuclear extracts of MBEC4 cells exposed to SNP using a radiolabeled NF-B consensus oligonucleotide. SNP (1 mM, after 3 h of treatment) markedly increased the binding activity of nuclear extracts to the NF-B consensus oligonucleotide (Fig. 8A). The addition of 20-fold excess unlabeled NF-B abolished the NF-B DNA binding complex. Also, anti-p65 antibody supershifted the retarded band (Fig. 8B).
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) demonstrated that the resting cerebral blood flow values were reduced by 40% compared with the control value, suggesting that the physiological alteration may be related to the alteration in the transport of CsA across the BBB. However, since CLapp,br of CsA was found to be 6.90 µl/min/g brain in diabetic rats, the value is much lower than the cerebral blood flow rate of the rat (i.e., 920 µl/min/g brain) (Kusuhara et al., 1997), suggesting that the change in the blood flow to the brain is not likely to be the primary factor in the CsA transport. In addition, an in vitro observation of MBEC4 cells was consistent with the in vivo results. Furthermore, the brain uptake kinetics for mannitol, an impermeable substance to the BBB, was not affected by the induction of the diabetic condition, suggesting that the physical integrity of the barrier is not altered by the experimental diabetic condition.
In this study, mRNA levels of rMdr1a (4.01-fold), rMdr1b (2.91-fold), and rMrp2 (2.28-fold) were significantly elevated in STZ-induced diabetic rats compared with the control (Fig. 2) when normalized to mRNA level for β-actin (a reference marker). Consistent with the enhanced level of rMrp2 in diabetic rats, it was recently demonstrated that the permeability of the BBB for fluorescein, a substrate for Mrp2, was decreased in STZ-induced diabetic rats (Hawkins et al., 2007). it is interesting to note, however, that rMrp1 mRNA levels were apparently unaffected in the diabetic condition, suggesting that certain specificity exists for the regulation of transporters in the BBB. In the case of rMdr1, the increase of protein level (1.92-fold) was somewhat comparable to that of the mRNA (2.91- to 4.01-fold).
In our study, we measured mRNA and protein levels of P-glycoprotein in whole brain homogenates rather than in brain microvessel endothelial cells in control and STZ-induced diabetic rats. The literature suggests that brain microvessel endothelial cells (viz., the BBB), not astrocytes and neurons, account for the majority of P-glycoprotein found in the brain tissue (Lester and Potschka, 2005). In addition, higher P-glycoprotein expression was associated with higher function of the transporter in the BBB compared with the transporter in astrocytes (Declèves et al., 2000). Therefore, despite possible expression of P-glycoprotein in astrocytes and neurons and/or enhanced expression of the transporter in these cells under pathophysiological condition(s) (e.g., epilepsy) (Volk et al., 2004), the transporter in astrocytes and neurons may play a secondary role in the transport of CsA from the systemic circulation to the brain tissue. The function of P-glycoprotein in multiple brain cells warrants further investigation.
Because NO, a radical, is relatively reactive, the oxide form may produce a number of NOx (e.g., inorganic nitrite and peroxynitrite), depending on the rate of NO release. In the literature, SIN-1 and SNP were reported to produce predominantly peroxynitrite (viz., slow NO-releasing donors), whereas SNAP and DETA may preferentially generate NO and other nitrogen oxides (viz., rapid NO-releasing donors) (Walia et al., 2003). The results of this study indicate that the exposure of MBEC4 cells with slow NO-releasing donors led to a functional induction of P-glycoprotein, whereas no effect on the function was noted in the case of the pretreatment of MBEC4 cells with rapid NO-releasing donors. Consistent with this observation, the stimulatory effect of SNP on the function and expression of P-glycoprotein was partially reversed by the presence of ascorbate (antioxidant), methylene blue (NO scavenger), or SOD (superoxide scavenger) (Fig. 6). Taken together, these observations indicate that peroxynitrite, rather than NO itself or other nitrogen oxide forms, is involved in the functional induction of the efflux transporter. Since an increased formation of 3-nitrotyrosine (a by-product of the reaction between peroxynitrite and tyrosine residue of target protein) of proteins was found in the heart of mice with experimental diabetes (Cai et al., 2005), peroxynitrite may be endogenously overproduced in experimental diabetes. Consistent with the literature, we found that NOx concentration was 117 ± 12.3 µM in the plasma of the diabetic rats. Therefore, the functional induction found in the present in vivo study may also be mediated by the overproduction of peroxynitrite. In addition, since NOx level in vivo was comparable to that measured in the in vitro culture condition (Table 3), the in vitro experimental condition appears relevant to the in vivo condition in terms of NOx exposure.
In this study, it was suggested that the functional induction via the increased level of mRNA and protein for P-glycoprotein was mediated by the presence of SNP that potentially produce peroxynitrite in vitro (Fig. 5). In the literature, various factors that are involved in the transcription process are activated under nitrosative conditions. Among these, NF-B is reported to be activated by peroxynitrite in various cell types. For example, the exposure to peroxynitrite donor SIN-1 increased a NF-B-dependent luciferase reporter vector in rat lung epithelial cells (Janssen-Heininger et al., 1999), as well as inducible nitric-oxide synthase protein levels via the activation of NF-B in the bovine microvessel endothelium (Cooke and Davidge, 2002). Consistent with the literature, the data from the present study indicate that the exposure of SNP to MBEC4 cells, an in vitro model of the BBB, leads to nuclear translocation of NF-B (Fig. 7) and increased DNA binding activity of nuclear extracts to the NF-B consensus oligonucleotide (Fig. 8). It is believed that this represents the first indication in the literature wherein NF-B activation has been observed in mouse brain microvessel endothelial cells via SNP exposure. Our data collectively suggest that NOx (e.g., peroxynitrite) alters DNA binding activity of NF-B, which, in turn, may enhance transcription of the P-glycoprotein, thereby inducing the expression and function of P-glycoprotein. Consistent with this hypothesis, a number of reports indicate that NF-B is involved in the increased gene expression of P-glycoprotein (Thevenod et al., 2000; Kuo et al., 2002), probably by binding of NF-B in the promoter region (Kuo et al., 2002).
Recently, Liu et al. (2006) reported that the induction of experimental diabetes led to impaired function and expression of P-glycoprotein in the rat BBB, a result that directly contradicts the data obtained in the current study. The underlying reasons for the discrepancy are unknown. However, differences in the experimental design (e.g., difference in the STZ dosage, duration of diabetes, P-glycoprotein substrate used, and/or the time of brain/blood sampling) may have contributed to the discrepancy. In addition, it is possible that the nitrosative stress may have a complex impact on P-glycoprotein function during the time course of the stress progression, thus leading to contradictory results. For example, we demonstrated that in vitro nitrosative stress induced by SNP exposure led first to a transient impairment and, then, eventually to the induction of the transporter (Fig. 4C). Consistent with this observation, Bauer et al. (2007) recently reported that exposure of rat brain capillaries to tumor necrosis factor-
, an inflammatory mediator, caused a rapid decrease in P-glycoprotein transport activity in the short term (after 2-3 h) and then a rapid increase in both function and expression via NF-B activation in the long term (i.e., after 6 h). Furthermore, the complex nature of nitrosative stress was documented in the regulation of the expression of transporters. For example, in another nitrosative model using lipopolysaccharide treatment, function and/or expression of ABC transporters (i.e., P-glycoprotein) were differentially regulated in a tissue-specific manner. Namely, lipopolysaccharide-induced acute inflammation caused down-regulation of P-glycoprotein in the liver, the intestine, and the brain, but up-regulation of P-glycoprotein in the kidney (Kalitsky-Szirtes et al., 2004; Hartmann et al., 2005; Wang et al., 2005; Heemskerk et al., 2007).
In this study, we investigated the role of nitrosative stress and alterations in the function/expression of P-glycoprotein both in vivo and in vitro. Zhou and Kuo (1997) found that addition of insulin independently induced the Mdr1 gene in rat hepatocytes without nitrosative stress. Therefore, administration of insulin may induce function and expression of P-glycoprotein independent of nitrosative stress.
In conclusion, the induction of experimental diabetes led to a decrease in CLapp,br for CsA, a well known substrate for P-glycoprotein, which was associated with an increased level of mRNA and protein for the transporter. The induction of the function and expression of P-glycoprotein was also noted in MBEC4 cells exposed to certain NOx donors (e.g., SNP). The nuclear translocation of NF-B and the increased DNA binding activity of nuclear extracts to the NF-B consensus sequence were detected in MBEC4 cells pretreated with SNP. Since a variety of substrates are subjected to the efflux process via P-glycoprotein, the regulation may have a broad implication in understanding the altered pharmacokinetics of the substrates in experimental as well as clinical diabetes. Therefore, the regulatory mechanism of the efflux transporter may be clinically relevant.
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ABBREVIATIONS: NOx, nitrogen oxide(s); NO, nitric oxide; STZ, streptozotocin; SNP, sodium nitroprusside; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetyl-dl-penicillamine; DETA, diethylenetriamine; MBEC4, mouse brain capillary endothelial; BBB, blood-brain barrier; RT-PCR, reverse transcription-polymerase chain reaction; Mdr, multidrug resistance; Mrp, multidrug resistance protein; ABC, ATP-binding cassette; NF-B, nuclear factor-B; CsA, cyclosporin A; SOD, superoxide dismutase; ANOVA, analysis of variance; CL, clearance; CLapp,br, apparent brain clearance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Address correspondence to: Dr. Suk-Jae Chung, Department of Pharmaceutics, College of Pharmacy, Seoul National University, San 56-1 Shinlim-dong, Kwanak-gu, Seoul 151-742, Korea. E-mail: sukjae{at}plaza.snu.ac.kr
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