Functional Induction of P-glycoprotein in the Blood-Brain Barrier of Streptozotocin-Induced Diabetic Rats: Evidence for the Involvement of Nuclear Factor- B, a Nitrosative Stress-Sensitive Transcription Factor, in the Regulation

The objective of this study was to investigate the transport kinetics of cyclosporin A, a well known substrate for P-glycoprotein (P-gp), across the blood-brain barrier (BBB), and the expression of the transporter in the brain of streptozotocin-induced diabetic rats. The in vivo transport clearance of cyclosporin A was significantly reduced in diabetic rats compared with that in the control. The decreased transport was associated with the increased level of mRNA and the protein for P-glycoprotein in the rat brain. The functional activity of the efflux transporter in mouse brain capillary endothelial (MBEC4) cells, an in vitro model of the BBB, was also stimulated when slow nitric oxide (NO)-releasing donors were present, whereas the stimulation was absent in the case of rapid NO-releasing donors (e.g., S-nitroso-N-acetyl-dl-penicillamine and diethylenetriamine). The stimulatory effect was highest for sodium nitroprusside (SNP) and the functional induction associated with the increased mRNA and protein level of the transporter. The pretreatment of the cell with SNP along with ascorbate, methylene blue, or superoxide dismutase attenuated the induction of function and expression for P-glycoprotein, suggesting that the reaction product between superoxide and NO is involved in the induction of function and expression. The level of nuclear translocation of nuclear factorB (NFB) and DNA binding activity of nuclear extracts to the NFB 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 NFB-dependent mechanism. Diabetes mellitus, an endocrine metabolic disorder, represents one of the most common geriatric diseases in developed countries (Rodriguez et al., 1996). 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. This study was supported by a grant (A03-0001-A71005-06M4-13010A) from the Ministry of Health & Welfare, Republic of Korea, and a grant from the health fellowship foundation. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.015800. 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; NFB, nuclear factorB; CsA, cyclosporin A; SOD, superoxide dismutase; ANOVA, analysis of variance; CL, clearance; CLapp,br, apparent brain clearance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 0090-9556/07/3511-1996–2005$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 11 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 15800/3260347 DMD 35:1996–2005, 2007 Printed in U.S.A. 1996 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from 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 NFB, which is a putative regulatory factor for P-glycoprotein gene expression and a nuclear transcription factor that is sensitive to nitrosative stress. Materials and Methods Materials. [H]Cyclosporin A (CsA; specific activity 9 Ci/mmol) and [C]mannitol (specific activity 53.7 mCi/mmol) were purchased from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). STZ, sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), S-nitroso-N-acetyl-dlpenicillamine (SNAP), diethylenetriamine (DETA), methylene blue, ascorbate, superoxide dismutase (SOD), Dulbecco’s modified Eagle’s medium, nonessential amino acid solution, penicillin-streptomycin, Hanks’ balanced salt solution, and HEPES were obtained from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA). MBEC4 cells were previously generated by our research group (Tatsuta et al., 1992). 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 l, 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 temperatureand 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 l 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 -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 . 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