The octapeptide angiotensin II produces a number of physiological actions, the most common of these being its peripheral role as a potent vasoconstrictor (1). The renin-angiotensin system also is known to exist in the brain (1-3). Brain angiotensin II appears to function in the central regulation of blood pressure, the regulation of body fluid volume, and the control of hormone release (4). Angiotensin II does not readily penetrate the blood-brain barrier (BBB), and the effects of angiotensin II are generally considered to be mediated by receptors in the circumventricular regions of the brain, which lack the BBB.
Speth and Harik (5) and Grammas et al. (6) observed that there were angiotensin II-receptor binding sites on the brain microvessel endothelial cells (BMECs) lining the BBB and that these binding sites have biologic activity (7,8). Subsequently, studies by Guillot and Audus (9) reported that there were specific angiotensin II binding sites on BMECs in primary culture and that the cells internalized the peptide. Angiotensin II receptors also are found on peripheral vascular endothelial cells and mediate transendothelial transport of the peptide (10). The subtyping of these binding sites, however, has not been reported.
Two major subtypes of angiotensin II receptors are known to exist, designated AT1 and AT2. Both receptor subtypes have been found in brain tissue of rats, monkeys, and humans. Most of the known actions of angiotensin II are mediated through the AT1 receptor (11). The development of nonpeptide antagonists has made it possible to distinguish between the two receptor subtypes. Losartan is a highly specific nonpeptide AT1 antagonist, and PD 123,319 is a highly specific nonpeptide AT2 antagonist. These antagonists lack the partial agonist activities of peptide angiotensin II analogs, allowing study of angiotensin II processing without interfering side effects (11).
The objective of this study was to use primary cultures of BMECs to reveal the receptor-subtype populations that regulate angiotensin II uptake and distribution across the BBB. The nonpeptide antagonists losartan and PD 123,319 were used in this study to distinguish the AT1- and AT2-receptor subtypes on BMECs. The definition of receptor subtypes providing for the clearance of peptides at the BBB contributes to our knowledge of fundamental physiological mechanisms regulating cerebrovascular levels of vasoeffectors. In addition, characterization of BBB carrier mechanisms potentially can provide physiological strategies to circumvent this barrier beneficially by covalently linking pharmacophores to receptor ligands known to be transported across the barrier (12,13).
MATERIALS AND METHODS
[3H]-angiotensin II (specific activity, 45 Ci/mmol) was purchased from Amersham (Arlington Heights, IL, U.S.A.). Losartan was donated by Merck & Co. (Rahway, NJ, U.S.A.), and PD 123,319 ditrifluoroacetate was purchased from Research Biochemicals International (Natick, MA, U.S.A.). Cyclosporin A was purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). The AT1 (N-10) rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). All other compounds used were of the highest commercially available quality.
Bovine brain microvessel endothelial cells (BMECs) were isolated from gray matter of cerebral cortices by enzymatic digestion and subsequent centrifugation, and seeded into primary culture as detailed by Audus and Borchardt (14-16). This preparation has been extensively characterized, as reported in the literature (14-18). Isolated BMECs were seeded at a density of 50,000 cells/cm2 into 12-well plates and 100-mm tissue culture dishes (Corning Costar Corporation, Cambridge, MA, U.S.A.), pretreated with rat-tail collagen and bovine fibronectin (Sigma Chemical Co.) in a culture medium consisting of 45% minimum essential medium, 45% F-12 Ham nutrient mixture (Gibco Life Technologies, Grand Island, NY, U.S.A.), 10 mM HEPES, pH 7.4, 13 mM sodium bicarbonate, 10% plasma-derived equine serum, 100 μg/ml heparin, 100 μg/ml streptomycin, 100 μg/ml penicillin G, 50 μg/ml polymyxin B, and 2.5 μg/ml amphotericin B (Sigma Chemical Co.). The cells were cultured at 37°C with 95% humidity and 5% CO2. Cells were fed on the third day after seeding and then every 2 days until confluent monolayers were formed (10-14 days).
Binding and internalization studies
The effect of the nonpeptide antagonists on [3H]angiotensin II binding and internalization was studied in 12-well plates at 37°C. Aliquots of specified concentrations of losartan and PD 123,319 dissolved in culture medium, containing equine serum, were added to each well with a pulse of [3H]angiotensin II. The plates were incubated for 30 min at 37°C. At the conclusion of the experiment, the medium was aspirated from each well, and the wells were rinsed 3 times with ice-cold culture medium. The cell monolayers were then solubilized by the addition of 1.0 ml of 0.2N NaOH/0.5% Triton X-100. An aliquot of 100 μl was removed to analyze protein content by using the BCA protein assay kit (Pierce, Rockford, IL, U.S.A.). The remaining lysate from each well was collected, placed in a scintillation vial with 10 ml of scintillation cocktail, and assayed with a Beckman LS 6800 scintillation counter (Irvine, CA, U.S.A.).
Rhodamine 123 uptake studies were also performed on confluent monolayers in 12-well plates. Angiotensin II, losartan, PD 123,319, or cyclosporin A dissolved in culture medium was added to each well, and the plate was preincubated for 1 h at 37°C. After the 1-h preincubation, rhodamine 123 was added to each well for a final concentration of 5 μM, and the plate was incubated at 37°C for an additional hour. After the second incubation, the plate was rinsed, and the monolayers were solubilized as described earlier. The contents of each well were collected into cuvettes and assayed by fluorescence using an SLM-Aminco 4800 fluorometer (Urbana, IL, U.S.A.) (ex, 500 nm; em, 550 nm). After we measured relative fluorescence, 100 μl was removed from each cuvette and assayed for protein content with the BCA protein assay kit (Pierce).
Transendothelial permeability studies
Polycarbonate membranes (pore size, 0.4 μm) were placed into 100-mm tissue culture dishes and coated with rat-tail collagen and bovine fibronectin. BMECs were grown to confluent monolayers, as determined by inspection of the areas around the membranes by using an inverted microscope. The basolateral side of the cells was defined as the side facing the collagen matrix. Once confluent monolayers were formed, the membranes were carefully placed into a horizontal Side-bi-Side diffusion apparatus (Crown Glass, Inc., Somerville, NJ, U.S.A.) for transendothelial permeability studies. The area of the diffusion membrane was 0.636 cm2. The donor and receiver chambers were filled with 3.0 ml culture medium, containing equine serum, and the temperature was maintained at 37°C with an external circulating water bath. The contents of each chamber were stirred with Teflon-coated magnetic stir bars at a speed of 600 r/min driven by an external-drive console (Crown Glass). The apical to basolateral transport of a pulse of [3H]angiotensin II was studied for 30 min. An aliquot of 25 μl was removed from the donor chamber to be assayed for initial donor concentration. Sample aliquots of 100 μl were removed from the receiver chamber at various intervals. After the 30-min receiver sampling time, different concentrations of losartan or PD 123,319 were added to the donor chamber. Receiver samples were then taken for an additional 60 min. After each receiver sample, the volume was replaced with fresh medium. The samples were placed in a scintillation vial with 10 ml of scintillation cocktail and assayed by scintillation spectrometry. The flux was determined by plotting pmoles versus minutes for each sampling interval (0-30 min and 30-90 min). The apparent permeability coefficient was calculated by using the equation Equation (1) where flux is the slope of each line, A is the area of the membrane, and CDo is the initial donor concentration. The donor concentration did not change by >10% during the period of these experiments.
Total cellular extract was prepared by using confluent monolayers of BMECs. The cells were lysed with a 45-min incubation in ice-cold phosphate-buffered saline containing 3% sodium dodecylsulfate (SDS) and protease inhibitors [0.1 mM leupeptin, 73 μM pepstatin A, and 100 μg/ml phenylmethylsulfonyl fluoride (PMSF)]. The cell lysate was then centrifuged at 12,000 g for 15 min, and the supernatant containing solubilized membrane proteins was stored at −20°C and used for further analysis. Total cell protein was measured by the Micro BCA protein assay (Pierce) by using bovine serum albumin as a standard. Cell plasma membranes were prepared by using confluent monolayers of BMECs. The cells were removed from the tissue-culture plates with a trypsin/EDTA solution. The cell suspension was then diluted in 5 volumes of ice-cold 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 (TE buffer) and homogenized. The homogenate was passed through a fine-gauge syringe needle and then centrifuged at 500 g for 15 min to pellet unbroken cells and nuclei, leaving a membrane containing supernatant. The plasma membranes were collected by centrifugation at 40,000 g for 60 min. The pellets were washed in 10 volumes of TE buffer and recentrifuged at 40,000 g for 60 min. The pellets were resuspended in TE buffer and stored at −70°C (19). The protein concentration was measured by the Micro BCA protein assay (Pierce) by using bovine serum albumin as a standard. Cell proteins were electrophoresed on a 10% Tris-Glycine gel (NOVEX, San Diego, CA, U.S.A.) and transferred to a PVDF membrane. Immunoreactive protein was detected with the AT1 polyclonal antibody by using the enhanced chemiluminescence method (ECL) as per the manufacturer's protocol (Amersham, Downers Grove, IL, U.S.A.). A positive control peptide (Santa Cruz Biotechnology) and a negative control (serum albumin) were used to establish and confirm blot conditions.
The comparison between mean values within each experimental series was performed by one-way analysis of variance by using Dunnett's test to compare several treatments against a control.
The temperature and concentration dependence of the binding and internalization kinetics for angiotensin II were described in an earlier report (9). In this study, the uptake of [3H]angiotensin II alone and with different concentrations of losartan is shown in Fig. 1. When coincubated for 30 min at 37°C, micromolar concentrations of losartan were observed significantly to inhibit the binding and uptake of [3H]angiotensin II. In Fig. 2, the effect of PD 123,319 ditrifluoroacetate on angiotensin II uptake is shown. When different concentrations of PID 123,319 were coincubated with [3H]angiotensin II for 30 min, there was no significant change in the amount of angiotensin II taken up by the cells.
Because the concentrations of losartan required to inhibit angiotensin II binding and internalization were higher than expected and earlier studies suggested a role for P-glycoprotein in mediating losartan distribution across the BBB (20), we performed rhodamine 123 uptake studies. The uptake of rhodamine 123 alone and with angiotensin II, losartan, PD 123,319, and cyclosporin A is shown in Fig. 3. The cells were exposed for 1 h with 100 μM angiotensin II, 750 μM losartan, 500 μM PD 123,319, or 1 μg/ml cyclosporin A. After the 1-h incubation, 5 μM rhodamine 123 was added to each well, and the cells were incubated another hour. As shown in Fig. 3, losartan, PD 123,319, and cyclosporin A significantly increased the uptake of rhodamine 123 in BMECs, indicating that only the nonpeptide antagonists interacted with P-glycoprotein.
The permeation of angiotensin II across BMECs was found to increase linearly over time, as shown in Fig. 4 at 37°C. The transport of a pulse of [3H]angiotensin II was monitored alone for 30 min before addition of the antagonists. The effect of losartan on angiotensin II transport is shown in Fig. 5, where again, micromolar concentrations of losartan were found significantly to decrease the transport of [3H]angiotensin II. There was no decrease in angiotensin II transport seen with PD 123,319, as shown in Fig. 6.
Western blot studies, shown in Fig. 7A and B, were generated by using an AT1-receptor polyclonal antibody and show that the AT1 receptor is present in BMECs. Figure 7A shows the Western analysis of total cell lysate, and Fig. 7B shows the blot of BMEC membranes compared with total cell lysate. The blots indicated that there was a greater concentration of AT1 receptors in the membrane portion of the cells.
Speth and Harik (5) and Grammas et al. (6) originally described the presence of specific angiotensin II-binding sites on brain microvessels. A hypothesis developed that angiotensin binding sites in the cerebrovasculature regulated water and ion transport (7,8). Guillot and Audus (9,21) subsequently described angiotensin II binding sites on primary cultures of bovine BMECs, a well-characterized in vitro system representing BBB endothelium. Scatchard analysis of the concentration-dependent binding of angiotensin II to BMECs revealed a single population of binding sites with an apparent Kd of 3.1 nM. In addition, angiotensin II was observed to be internalized intact by BMECs, and apical application of the peptide regulated permeability properties of the BMEC monolayers at concentrations similar to the binding site affinity. The latter findings were recently confirmed by other researchers in human BMECs (22).
In this study, we showed that the binding and uptake of angiotensin II by BMECs was inhibited by losartan, the AT1-receptor antagonist and not the AT2 antagonist, PD 123,319. This observation was consistent with the presence of a single population of angiotensin II binding sites that were described by Guillot and Audus (9). Therefore it appears that an AT1 receptor mediates clearance of angiotensin II by BMECs. Although our data include both binding and internalization, the concentrations at which an antagonist was inhibitory were higher than expected based on other receptor studies (11) and our observation that specific binding of angiotensin II to bovine BMECs could be inhibited by nanomolar concentrations of peptide analogs, saralasin and sarathrin (9).
Our earlier studies on the uptake and transport of losartan itself indicated that the compound was an apparent substrate for P-glycoprotein at submicromolar concentrations (20). Therefore P-glycoprotein may be a competing mechanism for losartan interactions with the BMEC membrane. To determine whether the antagonists and angiotensin II were potential substrates for P-glycoprotein, the uptake studies were performed with rhodamine 123, a specific substrate for the P-glycoprotein efflux pump (23-26). The uptake of rhodamine 123 alone and with pretreatment of the monolayers by using angiotensin II, losartan, PD 123,319, or a positive control, cyclosporin A (26), suggested that both antagonists were inhibitors of P-glycoprotein. Angiotensin II does not affect rhodamine 123 uptake; therefore it appears that the nonpeptide antagonists may not be as effective in blocking BMEC uptake and binding because of their added affinity for P-glycoprotein. On the other hand, the micromolar concentrations used here remain within the concentration range of losartan antagonist activity across a variety of different species, tissue types, and angiotensin II-mediated biologic functions (27).
Angiotensin II was previously shown to be transported across arterial endothelial cell monolayers by a specific, saturable mechanism that could be inhibited by unlabeled peptide. Mineo et al. (10) required micromolar concentrations of angiotensin to inhibit arterial endothelial cell transport of the radiolabeled angiotensin II. We observed that angiotensin II crosses monolayers of BMECs intact (28,29) at a rate slightly higher than expected based on its molecular weight. The transport was also a saturable process (Km, 1.7 nM), and angiotensin II transport from the apical to the basolateral side was greater than the reverse direction (28,30). In our study, the differential effects of the two angiotensin II-receptor antagonists on the passage of the peptide through monolayers of BMECs affirmed the role of an AT1-mediated mechanism with losartan inhibiting the transport of angiotensin II. As observed in the uptake studies, the AT2-antagonist effect on angiotensin II transport also was absent. Consistent with the uptake studies, micromolar concentrations of losartan were required to inhibit angiotensin transport.
The AT1 receptor belongs to the class of G protein-coupled seven-transmembrane receptors and is made up of 359 amino acids with an unmodified molecular weight of 41,000 (11). By using Western blot analysis, we confirmed the expression of the AT1 receptor in BMECs. Further, we demonstrated that the AT1 receptor resides in the membrane portion of BMECs. The AT1 receptor is responsible for most of the known biologic effects of angiotensin II, including those of the central nervous system (11,31). Therefore, expression in the BMECs was consistent with most other angiotensin II-mediated functions. At the time of this study, antibodies for the AT2 receptor were not available potentially to rule out expression of this class of receptors in BMECs.
In addition to peripheral endothelia (10), other cell types have been shown to internalize angiotensin II, including adrenocortical cells (32) and vascular smooth-muscle cells (33,34). Our results suggest that the presence of the AT1-receptor populations mediating BMEC uptake and transport of angiotensin II may serve as a local physiological mechanism for controlling angiotensin II distribution in the cerebrovasculature. This observation appears consistent with the local control of the renin-angiotensin systems associated with specific tissues (1-3). Alternatively, the AT1 receptors might provide a mechanism that could be exploited for facilitating delivery of some substances via chimeric constructs into the central nervous system like other endogenous peptides, insulin, and transferrin (35,36). However, like insulin, it is also apparent that angiotensin II has significant biologic activity at the BBB, regulating the BMEC secretion of other vasoeffectors (9,21,22,37) and the permeability properties of the cells (7-9) that would preclude its use in chimeric constructs. The nonpeptide antagonists such as losartan could provide alternative targeting ligands in a chimeric construct; unfortunately, losartan appears to be a substrate for BMEC efflux mechanisms (20). Therefore, future works in our laboratory will focus on the transport of alternative ligands for the carrier, including metabolically stable angiotensin II peptide analogs.
In summary, we have shown that the uptake and transport of angiotensin II by BMECs appears to be mediated by the AT1 receptor. These results form a better understanding of the regulation of cerebrovascular levels of angiotensin II by the BBB and may lead to possible delivery strategies for therapeutic chimeric peptides.
Acknowledgment: This work was supported by the American Heart Association's Kansas Affiliate, a PhRMA predoctoral fellowship (J.M.R.), an AFPE predoctoral fellowship (J.M.R.), and a Higuchi graduate fellowship (J.M.R.). We gratefully acknowledge the support of Corning Costar for use of the Cellular and Molecular Biopharmaceutics Handling Laboratory.
1. Reid JL, Rubin PC. Peptides and central neural regulation of the circulation. Physiol Rev
2. Paul M, Bader M, Steckelings UM, Voigtlander T, Ganten D. The renin-angiotensin system in the brain: localization and functional significance. Drug Res
3. Wright JW, Harding JW. Regulatory role of brain angiotensins in the control of physiological and behavioral responses. Brain Res Rev
4. Phillips MI. Functions of angiotensin in the central nervous system. Annu Rev Physiol
5. Speth RC, Harik SI. Angiotensin II
receptor binding sites in brain microvessels. Proc Natl Acad Sci U S A
6. Grammas P, Diglio C, Giacomelli F, Weiner J. Cerebrovascular angiotensin II
receptors in spontaneously hypertensive rats. J Cardiovasc Pharmacol
7. Grubb RLJ, Raichle ME. Intraventricular angiotensin II
increases brain vascular permeability. Brain Res
8. Oztas B, Sandalci U. Reversibility of blood-brain barrier
dysfunction in acute hypertension induced by angiotensin. Exp Neurol
9. Guillot FL, Audus KL. Some characteristics of specific angiotensin II
binding sites on bovine brain microvessel endothelial cell monolayers. Peptides
10. Mineo C, Yagyu Y, Imanaka T, Takano T. Transcellular transport of angiotensin II
through a cultured arterial endothelial monolayer. Exp Cell Res
11. Timmermans PBMWM, Wong PC, Chiu AT, et al. Angiotensin II
receptors and angiotensin II
receptor antagonists. Pharmacol Rev
12. Pardridge WM. Peptide drug delivery to the brain.
New York: Raven Press, 1991.
13. Friden PM. Receptor-mediated transport of peptides and proteins across the blood-brain barrier
. In: Pardridge WM, ed. The blood-brain barrier.
New York: Raven Press, 1993:229-47.
14. Audus KL, Borchardt RT. Characterization of an in vitro blood-brain barrier
model system for studying drug transport and metabolism. Pharm Res
15. Audus KL, Borchardt RT. Characteristics of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers. J Neurochem
16. Audus KL, Borchardt RT. Bovine brain microvessel endothelial cell monolayers as a model system for the blood-brain barrier
. Ann NY Acad Sci
17. Baranczyk-Kuzma A, Audus KL, Borchardt RT. Catecholamine-metabolizing enzymes of bovine brain microvessel endothelial cell monolayers. J Neurochem
18. Bowman PD, Ennis SR, Rarey KE, Betz AL, Goldstein GW. Brain microvessel endothelial cells
in tissue culture: a model for study of blood-brain barrier
permeability. Ann Neurol
19. McKenzie FR. Basic techniques to study G-protein function. In: Milligan G, ed. Signal transduction: a practical approach.
Oxford: IRL Press, 1992:33-4.
20. Rose JM, Audus KL. Cellular mechanisms regulating losartan
transfer across brain capillary endothelial cells in primary culture. S T P Pharma Sci
21. Guillot FL, Audus KL. Angiotensin peptide regulation of fluid-phase endocytosis in brain microvessel endothelial cell monolayers. J Cerebr Blood Flow Metab
22. Stanimirovic D, Morley P, Ball R, Hamel E, Mealing G, Durkin JP. Angiotensin II
-induced fluid phase endocytosis in human cerebrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway. J Cell Physiol
23. Hegmann EJ, Bauer HC, Kerbel RS. Expression and functional activity of P-glycoprotein in cultured cerebral capillary endothelial cells. Cancer Res
24. Jette L, Murphy GF, LeClerc J, Beliveau R. Interaction of drugs with P-glycoprotein in brain capillaries. Biochem Pharmacol
25. Lautier D, Canitrot Y, Salmon JM. Effects of vinblastine, colchicine, and verapamil on rhodamine 123 accumulation in human P-glycoprotein-positive leukemia cells. Anticancer Res
26. Wang Q, Yang H, Miller DW, Elmquist WF. Effect of the P-glycoprotein inhibitor, cyclosporin A, on the distribution of rhodamine-123 to the brain: an in vivo microdialysis study in freely moving rats. Biochem Biophys Res Commun
27. Smith RD, Chiu AT, Wong PC, Herblin WF, Timmermans PBMWM. Pharmacology of nonpeptide angiotensin II
receptor antagonists. Annu Rev Pharmacol Toxicol
28. Anderson JM, Wimalasena R, Foster K, Stobaugh JF, Audus KL. Internalization and transport of angiotensin II
by bovine brain microvessel endothelial cells
(BBMECs) [Abstract]. Pharm Res
29. Rose MJ, Lunte SM, Carlson RG, Stobaugh JF. Development of hydroquinone-based derivatization reagents for the quantitation of amines using electrochemical detection [Abstract]. Pharm Res
30. Rose JM, Audus KL. Angiotensin II
transport in bovine brain microvessel endothelial cells
31. Barnes JM, Steward LJ, Barber PC, Barnes NM. Identification and characterisation of angiotensin II
receptor subtypes in human brain. Eur J Pharmacol
32. Crozat AA, Penhoat A, Saez JM. Processing of angiotensin II
)AII by cultured bovine adrenocortical cells. Endocrinology
33. Griendling KK, Delafontaine P, Rittenhouse SE, Gimbrone MA, Alexander RW. Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II
-stimulated cultured vascular smooth muscle cells. J Biol Chem
34. Ullian ME, Linas SL. Role of receptor cycling in the regulation of angiotensin II
surface receptor number and angiotensin II
uptake in rat vascular smooth muscle cells. J Clin Invest
35. Fukuta M, Okada K, Iinuma S, Yanai S, Toguchi H. Insulin fragments as a carrier for peptide delivery across the blood-brain barrier
. Pharm Res
36. Pardridge WM, Buciak JL, Friden PM. Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier
in vivo. J Pharmacol Exp Ther
37. Gimbrone MA, Alexander RW. Angiotensin II
stimulation of prostaglandin production in cultured human vascular endothelium. Science