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Expression of Multidrug Resistance-Associated Protein (MRP) in Brain Microvessel Endothelial Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
243, 816–820 (1998)
RC978132
Expression of Multidrug Resistance-Associated Protein (MRP) in Brain Microvessel Endothelial Cells Han Huai-Yun, David T. Secrest, Karen S. Mark, Debra Carney, Christine Brandquist, William F. Elmquist, and Donald W. Miller1 Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, Nebraska 68198-6025
Received December 24, 1997
Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell. The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control. The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines. Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC. Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB. Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents. q 1998 Academic Press
The brain microvessel endothelial cells that form the blood-brain barrier (BBB) have an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The restrictive nature of the brain microvessel endothelial cells is due in part to the formation of tight junctions
between the cells, which prevents the paracellular diffusion of most macromolecules, and a low level of pinocytic activity, which results in reduced intracellular transport (1,2). Various transport and carrier systems expressed on the brain microvessel endothelial cells also help regulate the passage of selected drugs and macromolecules across the BBB. In this regard, efflux transport systems, such as P-glycoprotein (P-gp), that actively transport agents from the brain microvessel endothelial cells back into the bloodstream, have recently been shown to have an important contribution to BBB permeability (3,4). While P-gp is clearly important in restricting the passage of many drugs and macromolecules across the blood-brain barrier, P-gp is only one member of a super family of ATP dependent efflux transport proteins (5,6). Multidrug resistance-associated protein (MRP) shares approximately 15% amino acid sequence homology with P-gp (6). Although both MRP and P-gp have broad substrate specificity, MRP is selective for organic anions, as well as glutathione and glucuronide conjugates (5,7,8). The biochemical expression of MRP in brain microvessel endothelial cells and its potential role in regulating blood-brain barrier permeability is not currently known. The objectives of the current study was to determine if MRP is expressed in the brain microvessel endothelial cells that form the blood-brain barrier. Using both functional studies with the fluorescent dye, fluorescein, and immunoblots with an MRP specific antibody, the presence of the drug efflux protein, MRP, in brain microvessel endothelial cells has been demonstrated. The coexistence of both P-gp and MRP in the BBB may provide an important mechanism for controlling the intracellular passage of selected agents in brain microvessel endothelial cells. MATERIALS AND METHODS
1
To whom correspondence should be addressed. Fax: 402-5599543. E-mail: [email protected].
0006-291X/98 $25.00
Cell isolation and culturing. Bovine brain microvessel endothelial cells (BBMEC) were used in the current study. The BBMEC
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were isolated from fresh cow brains using a combination of enzyme digestion and centrifugal separation techniques (9). The BBMEC were seeded onto collagen-coated, fibronectin-treated 6-well tissue culture plates at a density of 50,000 cells/cm2, and cultured in MEM:F12 media supplemented with 10% horse serum (platelet depleted), heparin (100 mg/ml) and amphotericin B (2.5 mg/ml). The BBMEC were used for the functional and biochemical studies upon reaching confluency (10-12 days). The human pancreatic adenocarcinoma cell line, Panc-1, was used as an MRP-positive cell line in the present studies (10). The Panc1 cells were acquired from ATCC (Rockville, MD) and cultured as described previously (10). In addition, KBv cells, a P-gp expressing cell line, was used to further delineate MRP and P-gp functional activity. The KBv cells were grown in culture media containing 1 mg/ml vinblastine to maintain drug resistance and elevated levels of P-gp expression. All functional and biochemical studies were performed after the cells reached confluency. MRP functional studies. Assessment of MRP functional activity was done by measuring the cellular accumulation of the fluorescent probe, fluorescein (FLUR). The confluent cell monolayers were exposed to FLUR (100 mM) for 60 minutes at 377C, either alone or in the presence of indomethacin (INDO; 0-100 mM), probenecid (PRB; 100 mM), valproic acid (VPA; 100 mM), or cyclosporin A (CSA; 1.6 mM). After the incubation period, the FLUR was removed, and the monolayers were washed a total of three times in ice-cold phosphate buffered saline solution (PBS). The cell monolayers were solubilized in Triton X-100 and aliquots (250 ml) were removed for determination of FLU using a fluorescence spectrophotometer (Shimadzu RF5000; Excitation wavelength 488 nm, Emission wavelength 510 nm). In separate experiments the cellular accumulation of rhodamine 123 (R123; 3.2 mM) was examined alone, and in the presence of either INDO (100 mM) or CSA (1.6 mM). The studies using R123 were performed as described for FLUR and cell samples were analyzed by fluorescence spectrophotometry (excitation wavelength 505 nm, emission wavelength 540 nm). The R123 studies were used to distinguish between MRP and P-gp related systems (10,11). Western blot experiments. Identification of MRP in the various cell lysates was done using immunoblot techniques described previously (10). Briefly, cell monolayers were solubilized in PBS containing protease inhibitors (Boehringer Mannheim; Indianapolis, IN). The cell lysates were loaded onto pre-formed 7.5% polyacrylamide gels (Bio-Rad; Hercules, CA) and the proteins separated using SDS-PAGE. The proteins were transferred onto PVDF membranes (Millipore; Bedford, MA) and immunoblots were done using the MRP specific antibody, MRPm6 (Kimaya; Seattle, WA). The transferred proteins were washed in blocking buffer and incubated overnight at 47C with MRPm6 (1:100 dilution). Following a second series of washes, a secondary horseradish peroxidase mouse Ig antibody was added (1:1500 dilution) and incubated for 30 minutes at 47C. The specific protein bands were visualized using a chemiluminescence kit (Pierce; Rockford, IL). RT-PCR studies. Total RNA was isolated from BBMEC lysates using RNA STAT-60 single-step RNA isolation reagent (Tel-Test Inc.; Friendswood, TX). The RNA in the aqueous phase was precipitated using isopropanol, and following a series of washes, was quantified by spectrophotometric analysis. Identification of the presence of RNA for MRP was determined using RT-PCR techniques described previously (12). The two primers selected for the RT-PCR experiments correlated to nucleotide sequences 4255-4273 and 4601-4617 for human MRP (GenBank Accession # L055628). The primers amplify a 362 base pair segment which is conserved in both human and rat MRP cDNA. The RT-PCR product was electrophoresed at 80 mA for approximately 30-60 minutes on a 1% agarose gel. Materials. The tissue culture plates, culture media, and rat tail collagen were purchased from Fisher (St. Louis, MO). The sera used to culture BBMEC (horse serum) and the Panc-1 and KBv cells (fetal
FIG. 1. Fluorescein accumulation in BBMEC monolayers following exposure to various concentrations of indomethacin (A) or maximally effective concentrations of valproic acid (100 mM), probenecid (100 mM), or indomethacin (10 mM) (B). Studies were performed for 60 min at 377C. Values represent the means / SEM of at least three BBMEC monolayers. *p õ 0.05 compared to control as determined by analysis of variance with Duncan’s multiple comparison of the means.
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Specificity of Indomethacin-Sensitive Efflux Transport System in MRP Positive (Panc-1) and P-gp Positive (KBv) Cell Lines Cellular accumulation (nmol/mg protein)b
Substratea
Cell line Panc-1 KBv Panc-1 KBv
Fluorescein Rhodamine 123
Indomethacin (10 mM)
Control 0.148 0.277 1.129 0.117
{ { { {
0.003 0.008 0.020 0.010
0.288 0.285 0.900 0.118
{ { { {
0.003* 0.010 0.019 0.005
Cyclosporin A (1.6 mM) 0.158 0.271 1.028 0.221
{ { { {
0.004 0.004 0.008 0.015*
a
Cells were exposed to either fluorescein (100 mM) or rhodamine 123 (3.2 mM) for a 60 minute period at 377 C. Mean { SEM of at least three monolayers per treatment group. * p õ 0.05 compared to control monolayers.
b
bovine serum) was purchased from Sigma Chemical Company (St. Louis, MO) and Gibco (Grand Island, NY), respectively. With the exception of CSA, which was purchased from Alexis Corp. (San Diego, CA), all other reagents used were obtained from Sigma Chemical Co.
RESULTS Fluorescein accumulation as an Index of MRP activity. Functional evidence for MRP in brain microvessel endothelial cell monolayers was performed by examining fluorescein accumulation in the cells in the presence and absence of various inhibitors of MRP. As shown in Fig. 1, the cellular accumulation of FLUR in primary cultured BBMEC monolayers was low and reached a maximum of approximately 0.175 nmol/mg protein within 60 minutes (Fig. 1A). However, in the presence of indomethacin, FLUR accumulation in BBMEC monolayers was enhanced more than 2-fold, reaching 0.399 nmol/mg protein within 60 minutes (Fig. 1A). The effects of indomethacin on FLUR accumulation in the BBMEC monolayers were dose-dependent with maximal effects occurring at concentrations at or above 10 mM indomethacin (Fig. 1A). While indomethacin was the most effective, PRB and VPA, agents known to interfere with either MRP or organic anion transport, were also capable of increasing FLUR accumulation in BBMEC monolayers (Fig. 1B). To determine the specificity of the indomethacin response, FLUR accumulation was examined in both MRP expressing (Panc-1) and P-gp expressing (KBv) cells (Table 1). In Panc-1 monolayers, FLUR accumulation was increased approximately 2-fold in the presence of indomethacin. In contrast, indomethacin treatment had no effect on FLUR accumulation in the KBv monolayers (Table 1). While CSA had no effect on FLUR accumulation in either cell line, significant increases in rhodamine 123 accumulation was observed in KBv monolayers following CSA treatment (Table 1). Biochemical detection of MRP in BBMEC. Western blot studies using an MRP selective antibody (MRPm6)
show an approximately 190-kD protein band in BBMEC lysates (Fig. 2A). A similar molecular weight protein band was detected in immunoblots of the MRP positive Panc-1 cell lysates using the MRPm6 antibody (Fig. 2A). In contrast, the P-gp positive KBv cell lysates had little if any detectable staining at 190-kD with the MRPm6 antibody (Fig. 2A). Several lower molecular weight protein bands were also observed with the MRPm6 antibody in both the BBMEC and Panc-1 cell lysates. Using RT-PCR techniques, RNA isolated from BBMEC was examined for the expression of MRP. As can be seen in Fig. 2B there is a single band present in the BBMEC lane corresponding to the 362 base pair sequence amplified by the selected MRP primers. As a positive control for the RT-PCR assay, a RNA template from the plasmid AW-109 was examined using probes included in the kit which amplify a 309 base pair sequence. The final lane in which no bands can be detected is a negative control which contains no RNA template (Fig. 2B). DISCUSSION The present study presents both functional and biochemical evidence supporting the expression of MRP in brain microvessel endothelial cells. The functional evidence for MRP in brain microvessel endothelial cells that form the blood-brain barrier is based on the cellular accumulation of the fluorescent marker fluorescein. While this is the first reported use of FLUR as a substrate for MRP, the fact that the compound exists as a negatively charged species under physiological conditions makes it a likely candidate for transport out of the cells by MRP (5,7,13). Furthermore, it should be noted that recent studies by Draper and colleagues (14) used a FLUR derivative, 2*,7*-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, to assess MRP activity. The observation that FLUR accumulation in BBMEC mono-
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FIG. 2. Western Blot (A) and RT-PCR (B) analysis of MRP. (A) Western blots were performed using the specific MRP antibody, MRPm6. The protein band at the top of gel (indicated by arrow) corresponds to the 190 kD MRP protein band. (B) RT-PCR was performed using probes that amplify a 360 base pair segment of MRP.
layers can be increased following treatment with known MRP inhibitors such as indomethacin (7,14) and probenecid (5,7), is consistent with the blockade of efflux transport processes out of the cell. Furthermore, the 10 mM concentration of indomethacin required for maximal effects on FLUR accumulation in the present study is similar to the concentrations of indomethacin required to reverse MRP-related drug resistance and drug efflux in cancer cells (14). The brain microvessel endothelial cells that form the blood-brain barrier are known to express the P-gp drug efflux transport system (11,15). Therefore, it was necessary to confirm that the effects of indomethacin on FLUR accumulation in the BBMEC monolayers reflects MRP and not P-gp mediated transport. This was
done by examining the specificity of the indomethacindependent increase in FLUR accumulation in Panc-1 and KBv cell monolayers. Previous studies indicate that the Panc-1 cell line expresses MRP (10) and the KBv cell line expresses P-gp (11,16) related drug efflux transport systems. The observation that FLUR accumulation was enhanced in only the Panc-1 monolayers and only following INDO treatment is indicative of an MRP drug efflux system. Furthermore, the absence of effect of INDO on rhodamine 123 accumulation in the Panc-1 cells suggests INDO is interacting in a specific manner with MRP as opposed to a nonspecific alteration in membrane permeability. The functional studies in the KBv monolayers illustrate the specificity of FLUR for MRP related drug efflux, as CSA, a potent P-gp modifying agent, had no effect on FLUR accumulation. Taken together, the functional data from the present study indicates the presence of a drug efflux transport protein in BBMEC monolayers that resembles MRP. In addition to the functional evidence supporting the presence of MRP in brain microvessel endothelial cells, conformation of the expression of MRP was also obtained using immunoblots with an MRP specific antibody. In the present study, MRPm6 reacted with a protein band of approximately 190 kD in lysates of BBMEC. This particular antibody has been used previously to identify MRP expression in various cells and tissues (10). In addition, the antibody reacted with a protein band of similar molecular weight in cell lysates from the MRP positive Panc-1 cell line. The MRPm6 antibody had little if any immunoreactivity in the KBv cells, a cell line that is drug resistant and P-gp positive. The immunoblots together with the RT-PCR studies present compelling biochemical evidence of MRP expression in BBMEC. While the present study shows unequivocally that MRP is expressed in brain microvessel endothelial cells that form the BBB, the localization within the brain microvessel endothelial cells remains to be determined. Previous studies have suggested that MRP may be located predominantly at intracellular sites within normal tissue (17). However, the enhanced cellular accumulation of FLUR following probenecid or indomethacin treatments suggest a significant amount of MRP is located on the plasma membrane of brain microvessel endothelial cells. Given the function of the BBB and the potential protective nature of the drug efflux transporters, the most likely localization of MRP in brain microvessel endothelial cells would be on the apical (lumenal) plasma membrane. Previous studies examining the distribution of MRP in normal tissue have provided conflicting information concerning the expression of MRP in the central nervous system (17,18). Immunohistochemical detection of MRP in formaldehyde-fixed tissues reported no stain-
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ing in the brain parenchyma or blood vessels (17). However, using a more sensitive detection assay, Stride and colleagues (18) reported small but detectable amounts of MRP mRNA in brain tissue. While no attempt was made to determine how much of the activity for MRP in brain tissue was attributable to the brain endothelial cells, it is possible that although the levels of mRNA for MRP in whole brain tissue are relatively low, the brain microvessel endothelial cells may be enriched in their expression of MRP. Regardless of the actual level of expression of MRP in brain microvessel endothelial cells, there is clearly a functional presence of the protein in the BBMEC. Initial studies suggest that MRP is also present in cultured human and rat brain microvessel endothelial cells as well (unpublished results). The potential influence that MRP has in BBB permeability is currently under investigation. Several in vivo studies suggests the presence of an organic anion transporter in the blood-brain barrier (19-21). In addition, other studies have reported increases in BBB permeability through altered efflux transport processes following treatment with agents such as valproic acid and probenecid (2224). Whether the transporters in these previous studies are MRP remains to be determined. Given the present studies showing MRP expression in the brain microvessel endothelial cells, and the known substrate characteristics of MRP (5,7,8), it is very likely that at least a portion of the efflux of anions out of the brain and microvessel endothelial cells is mediated by MRP. Furthermore, based on previous studies demonstrating the importance of P-gp in BBB integrity (3), it is anticipated that MRP will also have a significant contribution to BBB permeability. ACKNOWLEDGMENTS
3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17.
18.
19.
This work was supported by PHS Grant R15 NS OD35364 and a Nebraska Department of Health, Cancer and Smoking Related Diseases grant.
20. 21. 22.
REFERENCES 23. 1. Audus, K. L., Chikhale, P. J., Miller, D. W., Thompson, S., and Borchardt, R. T. (1992) Adv. Drug Res. 23, 3–66. 2. Brightman, M. W. (1989) in Implications of the Blood-Brain Bar-
24.
rier and Its Manipulation (Neuwelt, E. A., Ed.), Vol. 1, pp. 53– 84, Plenum Publishing, New York. Schinkel, A. H., Smit, J. J. M., van Tellingem, O., Beijnen, J. H., Wagenaar, E., van Deemter, L., Mol, C. A. A. M., van der Valk, M. A., Robanu-Maandag, E. C., Riele, H. P. J., Berns, A. J. M., and Borst, P. (1994) Cell 77, 491–502. Wang, Q., Yang, H., Miller, D. W., and Elmquist, W. F. (1995) Biochem. Biophys. Res. Commun. 211, 719–726. Barrand, M. A., Bagrij, T., and Neo, S. Y. (1997) Gen. Pharmacol. 28, 639–645. Loe, D. W., Deeley, R. G., and Cole, S. P. C. (1996) Eur. J. Cancer 32A, 945–957. Hollo, Z., Homolya, L., Hegedus, T., and Sarkadi, B. (1996) FEBS Lett. 383, 99–104. Jedlitschky, G., Leier, I., Buchholz, U., Barnouin, K., Kurz, G., and Keppler, D. (1996) Cancer Res. 56, 988–994. Miller, D. W., Audus, K. L., and Borchardt, R. T. (1992) J. Tiss. Cult. Meth. 14, 217–224. Miller, D. W., Fontaine, M., Kolar, C., and Lawson, T. (1996) Cancer Lett. 107, 301–306. Fontaine, M., Elmquist, W. F., and Miller, D. W. (1996) Life Sci. 59, 1521–1531. Endo, K., Maehara, Y., Kusumoto, T., Ichyoshi, Y., Kuwano, M., and Sugimachi, K. (1996) Int. J. Cancer 68, 372–377. Sarkadi, B., Muller, M., and Hollo, Z. (1996) Immunol. Lett. 54, 215–219. Draper, M. P., Martell, R. L., and Levy, S. B. (1997) Br. J. Cancer 75, 810–815. Hegmann, E. J., Bauer, H. C., and Kerbel, R. S. (1992) Cancer Res. 52, 6969–6975. Gervasoni, J. E., Fields, S. Z., Krishna, S., Baker, M. A., Rosado, M., Thuraisamy, K., Hindenburg, A. A., and Taub, R. N. (1991) Cancer Res. 51, 4955–4963. Flens, M. J., Zaman, G. J. R., van der Valk, P., Izquierdo, M. A., Schroeijers, A. B., Scheffer, G. L., van der Groep, P., de Haas, M., Meijer, C. J. L. M., and Scheper, R. J. (1996) Am. J. Pathol. 148, 1237–1247. Stride, B. D., Valdimarsson, G., Gerlach, J. H., Wilson, G. M., Cole, S. P. C., and Deeley, R. G. (1996) Mol. Pharmacol. 49, 962– 971. Adkison, K. D., Powers, K. M., Artru, A. A., and Shen, D. D. (1996) Epilepsy Res. 23, 95–104. Deguchi, Y., Inabe, K., Tomiyasu, K., Nozawa, K., Yamada, S., and Kimura, R. (1995) Pharm. Res. 12, 1838–1844. Ackley, D. C., and Yokel, R. A. (1997) Toxicology 120, 89–97. Riant, P., Bree, F., Urien, S., Hamberger, C., Albengres, E., and Tillement, J. P. (1990) Fund. Clin. Pharmacol. 4, 105–114. Deguchi, Y., Nozawa, K., Yamada, S., Yokoyama, Y., and Kimura, R. (1997) J. Pharmacol. Exp. Ther. 280, 551–560. Wong, S. L., Van Belle, K., and Sawchuk, R. J. (1993) J. Pharmacol. Exp. Ther. 264, 899–909.
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Expression of Multidrug Resistance-Associated Protein (MRP) in Brain Microvessel Endothelial Cells Han Huai-Yun, David T. Secrest, Karen S. Mark, Debra Carney, Christine Brandquist, William F. Elmquist, and Donald W. Miller1 Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, Nebraska 68198-6025
Received December 24, 1997
Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell. The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control. The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines. Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC. Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB. Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents. q 1998 Academic Press
The brain microvessel endothelial cells that form the blood-brain barrier (BBB) have an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The restrictive nature of the brain microvessel endothelial cells is due in part to the formation of tight junctions
between the cells, which prevents the paracellular diffusion of most macromolecules, and a low level of pinocytic activity, which results in reduced intracellular transport (1,2). Various transport and carrier systems expressed on the brain microvessel endothelial cells also help regulate the passage of selected drugs and macromolecules across the BBB. In this regard, efflux transport systems, such as P-glycoprotein (P-gp), that actively transport agents from the brain microvessel endothelial cells back into the bloodstream, have recently been shown to have an important contribution to BBB permeability (3,4). While P-gp is clearly important in restricting the passage of many drugs and macromolecules across the blood-brain barrier, P-gp is only one member of a super family of ATP dependent efflux transport proteins (5,6). Multidrug resistance-associated protein (MRP) shares approximately 15% amino acid sequence homology with P-gp (6). Although both MRP and P-gp have broad substrate specificity, MRP is selective for organic anions, as well as glutathione and glucuronide conjugates (5,7,8). The biochemical expression of MRP in brain microvessel endothelial cells and its potential role in regulating blood-brain barrier permeability is not currently known. The objectives of the current study was to determine if MRP is expressed in the brain microvessel endothelial cells that form the blood-brain barrier. Using both functional studies with the fluorescent dye, fluorescein, and immunoblots with an MRP specific antibody, the presence of the drug efflux protein, MRP, in brain microvessel endothelial cells has been demonstrated. The coexistence of both P-gp and MRP in the BBB may provide an important mechanism for controlling the intracellular passage of selected agents in brain microvessel endothelial cells. MATERIALS AND METHODS
1
To whom correspondence should be addressed. Fax: 402-5599543. E-mail: [email protected].
0006-291X/98 $25.00
Cell isolation and culturing. Bovine brain microvessel endothelial cells (BBMEC) were used in the current study. The BBMEC
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were isolated from fresh cow brains using a combination of enzyme digestion and centrifugal separation techniques (9). The BBMEC were seeded onto collagen-coated, fibronectin-treated 6-well tissue culture plates at a density of 50,000 cells/cm2, and cultured in MEM:F12 media supplemented with 10% horse serum (platelet depleted), heparin (100 mg/ml) and amphotericin B (2.5 mg/ml). The BBMEC were used for the functional and biochemical studies upon reaching confluency (10-12 days). The human pancreatic adenocarcinoma cell line, Panc-1, was used as an MRP-positive cell line in the present studies (10). The Panc1 cells were acquired from ATCC (Rockville, MD) and cultured as described previously (10). In addition, KBv cells, a P-gp expressing cell line, was used to further delineate MRP and P-gp functional activity. The KBv cells were grown in culture media containing 1 mg/ml vinblastine to maintain drug resistance and elevated levels of P-gp expression. All functional and biochemical studies were performed after the cells reached confluency. MRP functional studies. Assessment of MRP functional activity was done by measuring the cellular accumulation of the fluorescent probe, fluorescein (FLUR). The confluent cell monolayers were exposed to FLUR (100 mM) for 60 minutes at 377C, either alone or in the presence of indomethacin (INDO; 0-100 mM), probenecid (PRB; 100 mM), valproic acid (VPA; 100 mM), or cyclosporin A (CSA; 1.6 mM). After the incubation period, the FLUR was removed, and the monolayers were washed a total of three times in ice-cold phosphate buffered saline solution (PBS). The cell monolayers were solubilized in Triton X-100 and aliquots (250 ml) were removed for determination of FLU using a fluorescence spectrophotometer (Shimadzu RF5000; Excitation wavelength 488 nm, Emission wavelength 510 nm). In separate experiments the cellular accumulation of rhodamine 123 (R123; 3.2 mM) was examined alone, and in the presence of either INDO (100 mM) or CSA (1.6 mM). The studies using R123 were performed as described for FLUR and cell samples were analyzed by fluorescence spectrophotometry (excitation wavelength 505 nm, emission wavelength 540 nm). The R123 studies were used to distinguish between MRP and P-gp related systems (10,11). Western blot experiments. Identification of MRP in the various cell lysates was done using immunoblot techniques described previously (10). Briefly, cell monolayers were solubilized in PBS containing protease inhibitors (Boehringer Mannheim; Indianapolis, IN). The cell lysates were loaded onto pre-formed 7.5% polyacrylamide gels (Bio-Rad; Hercules, CA) and the proteins separated using SDS-PAGE. The proteins were transferred onto PVDF membranes (Millipore; Bedford, MA) and immunoblots were done using the MRP specific antibody, MRPm6 (Kimaya; Seattle, WA). The transferred proteins were washed in blocking buffer and incubated overnight at 47C with MRPm6 (1:100 dilution). Following a second series of washes, a secondary horseradish peroxidase mouse Ig antibody was added (1:1500 dilution) and incubated for 30 minutes at 47C. The specific protein bands were visualized using a chemiluminescence kit (Pierce; Rockford, IL). RT-PCR studies. Total RNA was isolated from BBMEC lysates using RNA STAT-60 single-step RNA isolation reagent (Tel-Test Inc.; Friendswood, TX). The RNA in the aqueous phase was precipitated using isopropanol, and following a series of washes, was quantified by spectrophotometric analysis. Identification of the presence of RNA for MRP was determined using RT-PCR techniques described previously (12). The two primers selected for the RT-PCR experiments correlated to nucleotide sequences 4255-4273 and 4601-4617 for human MRP (GenBank Accession # L055628). The primers amplify a 362 base pair segment which is conserved in both human and rat MRP cDNA. The RT-PCR product was electrophoresed at 80 mA for approximately 30-60 minutes on a 1% agarose gel. Materials. The tissue culture plates, culture media, and rat tail collagen were purchased from Fisher (St. Louis, MO). The sera used to culture BBMEC (horse serum) and the Panc-1 and KBv cells (fetal
FIG. 1. Fluorescein accumulation in BBMEC monolayers following exposure to various concentrations of indomethacin (A) or maximally effective concentrations of valproic acid (100 mM), probenecid (100 mM), or indomethacin (10 mM) (B). Studies were performed for 60 min at 377C. Values represent the means / SEM of at least three BBMEC monolayers. *p õ 0.05 compared to control as determined by analysis of variance with Duncan’s multiple comparison of the means.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Specificity of Indomethacin-Sensitive Efflux Transport System in MRP Positive (Panc-1) and P-gp Positive (KBv) Cell Lines Cellular accumulation (nmol/mg protein)b
Substratea
Cell line Panc-1 KBv Panc-1 KBv
Fluorescein Rhodamine 123
Indomethacin (10 mM)
Control 0.148 0.277 1.129 0.117
{ { { {
0.003 0.008 0.020 0.010
0.288 0.285 0.900 0.118
{ { { {
0.003* 0.010 0.019 0.005
Cyclosporin A (1.6 mM) 0.158 0.271 1.028 0.221
{ { { {
0.004 0.004 0.008 0.015*
a
Cells were exposed to either fluorescein (100 mM) or rhodamine 123 (3.2 mM) for a 60 minute period at 377 C. Mean { SEM of at least three monolayers per treatment group. * p õ 0.05 compared to control monolayers.
b
bovine serum) was purchased from Sigma Chemical Company (St. Louis, MO) and Gibco (Grand Island, NY), respectively. With the exception of CSA, which was purchased from Alexis Corp. (San Diego, CA), all other reagents used were obtained from Sigma Chemical Co.
RESULTS Fluorescein accumulation as an Index of MRP activity. Functional evidence for MRP in brain microvessel endothelial cell monolayers was performed by examining fluorescein accumulation in the cells in the presence and absence of various inhibitors of MRP. As shown in Fig. 1, the cellular accumulation of FLUR in primary cultured BBMEC monolayers was low and reached a maximum of approximately 0.175 nmol/mg protein within 60 minutes (Fig. 1A). However, in the presence of indomethacin, FLUR accumulation in BBMEC monolayers was enhanced more than 2-fold, reaching 0.399 nmol/mg protein within 60 minutes (Fig. 1A). The effects of indomethacin on FLUR accumulation in the BBMEC monolayers were dose-dependent with maximal effects occurring at concentrations at or above 10 mM indomethacin (Fig. 1A). While indomethacin was the most effective, PRB and VPA, agents known to interfere with either MRP or organic anion transport, were also capable of increasing FLUR accumulation in BBMEC monolayers (Fig. 1B). To determine the specificity of the indomethacin response, FLUR accumulation was examined in both MRP expressing (Panc-1) and P-gp expressing (KBv) cells (Table 1). In Panc-1 monolayers, FLUR accumulation was increased approximately 2-fold in the presence of indomethacin. In contrast, indomethacin treatment had no effect on FLUR accumulation in the KBv monolayers (Table 1). While CSA had no effect on FLUR accumulation in either cell line, significant increases in rhodamine 123 accumulation was observed in KBv monolayers following CSA treatment (Table 1). Biochemical detection of MRP in BBMEC. Western blot studies using an MRP selective antibody (MRPm6)
show an approximately 190-kD protein band in BBMEC lysates (Fig. 2A). A similar molecular weight protein band was detected in immunoblots of the MRP positive Panc-1 cell lysates using the MRPm6 antibody (Fig. 2A). In contrast, the P-gp positive KBv cell lysates had little if any detectable staining at 190-kD with the MRPm6 antibody (Fig. 2A). Several lower molecular weight protein bands were also observed with the MRPm6 antibody in both the BBMEC and Panc-1 cell lysates. Using RT-PCR techniques, RNA isolated from BBMEC was examined for the expression of MRP. As can be seen in Fig. 2B there is a single band present in the BBMEC lane corresponding to the 362 base pair sequence amplified by the selected MRP primers. As a positive control for the RT-PCR assay, a RNA template from the plasmid AW-109 was examined using probes included in the kit which amplify a 309 base pair sequence. The final lane in which no bands can be detected is a negative control which contains no RNA template (Fig. 2B). DISCUSSION The present study presents both functional and biochemical evidence supporting the expression of MRP in brain microvessel endothelial cells. The functional evidence for MRP in brain microvessel endothelial cells that form the blood-brain barrier is based on the cellular accumulation of the fluorescent marker fluorescein. While this is the first reported use of FLUR as a substrate for MRP, the fact that the compound exists as a negatively charged species under physiological conditions makes it a likely candidate for transport out of the cells by MRP (5,7,13). Furthermore, it should be noted that recent studies by Draper and colleagues (14) used a FLUR derivative, 2*,7*-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, to assess MRP activity. The observation that FLUR accumulation in BBMEC mono-
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FIG. 2. Western Blot (A) and RT-PCR (B) analysis of MRP. (A) Western blots were performed using the specific MRP antibody, MRPm6. The protein band at the top of gel (indicated by arrow) corresponds to the 190 kD MRP protein band. (B) RT-PCR was performed using probes that amplify a 360 base pair segment of MRP.
layers can be increased following treatment with known MRP inhibitors such as indomethacin (7,14) and probenecid (5,7), is consistent with the blockade of efflux transport processes out of the cell. Furthermore, the 10 mM concentration of indomethacin required for maximal effects on FLUR accumulation in the present study is similar to the concentrations of indomethacin required to reverse MRP-related drug resistance and drug efflux in cancer cells (14). The brain microvessel endothelial cells that form the blood-brain barrier are known to express the P-gp drug efflux transport system (11,15). Therefore, it was necessary to confirm that the effects of indomethacin on FLUR accumulation in the BBMEC monolayers reflects MRP and not P-gp mediated transport. This was
done by examining the specificity of the indomethacindependent increase in FLUR accumulation in Panc-1 and KBv cell monolayers. Previous studies indicate that the Panc-1 cell line expresses MRP (10) and the KBv cell line expresses P-gp (11,16) related drug efflux transport systems. The observation that FLUR accumulation was enhanced in only the Panc-1 monolayers and only following INDO treatment is indicative of an MRP drug efflux system. Furthermore, the absence of effect of INDO on rhodamine 123 accumulation in the Panc-1 cells suggests INDO is interacting in a specific manner with MRP as opposed to a nonspecific alteration in membrane permeability. The functional studies in the KBv monolayers illustrate the specificity of FLUR for MRP related drug efflux, as CSA, a potent P-gp modifying agent, had no effect on FLUR accumulation. Taken together, the functional data from the present study indicates the presence of a drug efflux transport protein in BBMEC monolayers that resembles MRP. In addition to the functional evidence supporting the presence of MRP in brain microvessel endothelial cells, conformation of the expression of MRP was also obtained using immunoblots with an MRP specific antibody. In the present study, MRPm6 reacted with a protein band of approximately 190 kD in lysates of BBMEC. This particular antibody has been used previously to identify MRP expression in various cells and tissues (10). In addition, the antibody reacted with a protein band of similar molecular weight in cell lysates from the MRP positive Panc-1 cell line. The MRPm6 antibody had little if any immunoreactivity in the KBv cells, a cell line that is drug resistant and P-gp positive. The immunoblots together with the RT-PCR studies present compelling biochemical evidence of MRP expression in BBMEC. While the present study shows unequivocally that MRP is expressed in brain microvessel endothelial cells that form the BBB, the localization within the brain microvessel endothelial cells remains to be determined. Previous studies have suggested that MRP may be located predominantly at intracellular sites within normal tissue (17). However, the enhanced cellular accumulation of FLUR following probenecid or indomethacin treatments suggest a significant amount of MRP is located on the plasma membrane of brain microvessel endothelial cells. Given the function of the BBB and the potential protective nature of the drug efflux transporters, the most likely localization of MRP in brain microvessel endothelial cells would be on the apical (lumenal) plasma membrane. Previous studies examining the distribution of MRP in normal tissue have provided conflicting information concerning the expression of MRP in the central nervous system (17,18). Immunohistochemical detection of MRP in formaldehyde-fixed tissues reported no stain-
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ing in the brain parenchyma or blood vessels (17). However, using a more sensitive detection assay, Stride and colleagues (18) reported small but detectable amounts of MRP mRNA in brain tissue. While no attempt was made to determine how much of the activity for MRP in brain tissue was attributable to the brain endothelial cells, it is possible that although the levels of mRNA for MRP in whole brain tissue are relatively low, the brain microvessel endothelial cells may be enriched in their expression of MRP. Regardless of the actual level of expression of MRP in brain microvessel endothelial cells, there is clearly a functional presence of the protein in the BBMEC. Initial studies suggest that MRP is also present in cultured human and rat brain microvessel endothelial cells as well (unpublished results). The potential influence that MRP has in BBB permeability is currently under investigation. Several in vivo studies suggests the presence of an organic anion transporter in the blood-brain barrier (19-21). In addition, other studies have reported increases in BBB permeability through altered efflux transport processes following treatment with agents such as valproic acid and probenecid (2224). Whether the transporters in these previous studies are MRP remains to be determined. Given the present studies showing MRP expression in the brain microvessel endothelial cells, and the known substrate characteristics of MRP (5,7,8), it is very likely that at least a portion of the efflux of anions out of the brain and microvessel endothelial cells is mediated by MRP. Furthermore, based on previous studies demonstrating the importance of P-gp in BBB integrity (3), it is anticipated that MRP will also have a significant contribution to BBB permeability. ACKNOWLEDGMENTS
3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17.
18.
19.
This work was supported by PHS Grant R15 NS OD35364 and a Nebraska Department of Health, Cancer and Smoking Related Diseases grant.
20. 21. 22.
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