Quantitative Atlas of Blood–Brain Barrier Transporters, Receptors, and Tight Junction Proteins in Rats and Common Marmoset
2013; Elsevier BV; Volume: 102; Issue: 9 Linguagem: Inglês
10.1002/jps.23575
ISSN1520-6017
AutoresYutaro Hoshi, Yasuo Uchida, Masanori Tachikawa, Takashi Inoue, Sumio Ohtsuki, Tetsuya Terasaki,
Tópico(s)Trace Elements in Health
ResumoThe purpose of this study was to determine the protein amounts of blood–brain barrier (BBB) permeability-related transporters, receptors, and tight junction proteins in Sprague Dawley and Wistar rats and common marmoset, and also to investigate inter-species and inter-strain differences across rodents and primates. Quantification of target proteins in isolated brain capillaries was conducted by liquid chromatography–tandem mass spectrometry-based quantitative targeted absolute proteomics, with in silico peptide selection. Most target proteins showed inter-rodent, inter-primate species, and inter-rat strain differences of less than 2-fold. Comparison of rat and human BBB showed that P-glycoprotein, multidrug resistance-associated protein 4, monocarboxylate transporter 1, l-type amino acid transporter, and organic anion transporter 3 exhibited differences of more than two-fold in protein abundance, whereas the amounts of breast cancer resistance protein, glucose transporter 1, and insulin receptor were similar in rat and human. In contrast, the differences between marmoset and human BBB were less than 2-fold for almost all measured proteins. Thus, the molecular basis of BBB functions may be similar in marmoset and human, whereas that of rats shows significant differences. The marmoset may be a good model to access in vivo human BBB permeability characteristics, as an alternative to rat and macaque monkey. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3343–3355, 2013 The purpose of this study was to determine the protein amounts of blood–brain barrier (BBB) permeability-related transporters, receptors, and tight junction proteins in Sprague Dawley and Wistar rats and common marmoset, and also to investigate inter-species and inter-strain differences across rodents and primates. Quantification of target proteins in isolated brain capillaries was conducted by liquid chromatography–tandem mass spectrometry-based quantitative targeted absolute proteomics, with in silico peptide selection. Most target proteins showed inter-rodent, inter-primate species, and inter-rat strain differences of less than 2-fold. Comparison of rat and human BBB showed that P-glycoprotein, multidrug resistance-associated protein 4, monocarboxylate transporter 1, l-type amino acid transporter, and organic anion transporter 3 exhibited differences of more than two-fold in protein abundance, whereas the amounts of breast cancer resistance protein, glucose transporter 1, and insulin receptor were similar in rat and human. In contrast, the differences between marmoset and human BBB were less than 2-fold for almost all measured proteins. Thus, the molecular basis of BBB functions may be similar in marmoset and human, whereas that of rats shows significant differences. The marmoset may be a good model to access in vivo human BBB permeability characteristics, as an alternative to rat and macaque monkey. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3343–3355, 2013 INTRODUCTIONLiquid chromatography–tandem mass spectrometry (LC–MS/MS)-based quantitative-targeted absolute proteomics (QTAP) has provided a method to understand drug pharmacokinetics in terms of the protein expression amounts of functional proteins, such as transporters, receptors, and enzymes.1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google Scholar We have found that the mRNA expression levels of CYP enzymes, except for CYP3A4, are not related to the metabolizing activity, and there is no correlation between mRNA and protein expression levels of transporters in human liver.2.Ohtsuki S. Schaefer O. Kawakami H. Inoue T. Liehner S. Saito A. Ishiguro N. Kishimoto W. Ludwig-Schwellinger E. Ebner T. Terasaki T. Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: Comparison with mRNA levels and activities.Drug Metab Dispos. 2012; 40: 83-92Crossref PubMed Scopus (344) Google Scholar Therefore, it is essential to quantify protein amounts to functionally understand drug distribution and metabolism.Successful development of central nervous system (CNS)-targeting drugs requires prediction of the blood–brain barrier (BBB) permeability of drug candidates at an early preclinical stage. One general approach to this issue is extrapolating preclinical data obtained from in vivo animal models, such as rodents and macaque monkeys, to humans.3.Hsiao P. Sasongko L. Link J.M. Mankoff D.A. Muzi M. Collier A.C. Unadkat J.D. Verapamil P-glycoprotein transport across the rat blood–brain barrier: Cyclosporine, a concentration inhibition analysis, and comparison with human data.J Pharmacol Exp Ther. 2006; 317: 704-710Crossref PubMed Scopus (85) Google Scholar,4.Lee Y.J. Maeda J. Kusuhara H. Okauchi T. Inaji M. Nagai Y. Obayashi S. Nakao R. Suzuki K. Sugiyama Y. Suhara T. In vivo evaluation of P-glycoprotein function at the blood–brain barrier in nonhuman primates using [11C]verapamil.J Pharmacol Exp Ther. 2006; 316: 647-653Crossref PubMed Scopus (92) Google Scholar The BBB permeability reflects the contributions of passive diffusion, carrier-mediated transport and receptor-mediated transcytosis to transcellular transport,5.Ohtsuki S. Terasaki T. Contribution of carrier-mediated transport systems to the blood–brain barrier as a supporting and protecting interface for the brain; importance for CNS drug discovery and development.Pharm Res. 2007; 24: 1745-1758Crossref PubMed Scopus (344) Google Scholar as well as the occurrence of paracellular transport under pathological conditions where the tight junctions are impaired.6.Rosenberg GA. Neurological diseases in relation to the blood–brain barrier.J Cereb Blood Flow Metab. 2012; 32: 1139-1151Crossref PubMed Scopus (296) Google Scholar It is thus essential to understand inter-species and inter-strain differences in the abundance and intrinsic activity of transporters, receptors, and tight junction proteins that influence BBB permeability for successful extrapolation of animal model data to humans.Two strains [Sprague Dawley (SD) and Wistar] of rats have widely been used to assess in vivo BBB permeability.7.Cannon R.E. Peart J.C. Hawkins B.T. Campos C.R. Miller D.S. Targeting blood–brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain.Proc Natl Acad Sci USA. 2012; 109: 15930-15935Crossref PubMed Scopus (112) Google Scholar,8.Tachikawa M. Kasai Y. Yokoyama R. Fujinawa J. Ganapathy V. Terasaki T. Hosoya K. The blood–brain barrier transport and cerebral distribution of guanidinoacetate in rats: Involvement of creatine and taurine transporters.J Neurochem. 2009; 111: 499-509Crossref PubMed Scopus (35) Google Scholar As the brain distribution of paclitaxel, a P-glycoprotein (P-gp) substrate, is not different between SD and Wistar rats, it has been suggested that there is no significant interrat strain difference in BBB transport function.9.Zamek-Gliszczynski M.J. Bedwell D.W. Bao J.Q. Higgins J.W. Characterization of SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats using loperamide, paclitaxel, sulfasalazine, and carboxydichlorofluorescein pharmacokinetics.Drug Metab Dispos. 2012; 40: 1825-1833Crossref PubMed Scopus (80) Google Scholar However, no data are available on differences in functional protein expression levels at the rat BBB. Direct comparison of the protein amounts is critical to clarify whether or not inter-rat strain differences exist, and is therefore important for the development of CNS-acting drugs. Positron emission tomography (PET) has revealed that P-gp substrates exhibit greater brain distribution in human and cynomolgus monkey than in rat.10.Syvanen S. Lindhe O. Palner M. Kornum B.R. Rahman O. Langstrom B. Knudsen G.M. Hammarlund-Udenaes M. Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.Drug Metab Dispos. 2009; 37: 635-643Crossref PubMed Scopus (267) Google Scholar It has thus been postulated that there may be differences in the in vivo brain distribution of drugs between rodents and primates (monkeys and humans). Because old-world primates such as rhesus and cynomolgus monkeys (macaque monkeys) are the second-nearest animals to humans in the putative evolutionary tree, they have been postulated to be good animal models for the prediction of human BBB permeability. In support of this notion, our QTAP studies have shown that the protein expression amount of P-gp at the human BBB (4.71 fmol/μg protein) is close to that in cynomolgus monkey BBB (6.06 fmol/μg protein), rather than that in mouse BBB (14.1 fmol/μg protein).1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google Scholar,11.Uchida Y. Ohtsuki S. Katsukura Y. Ikeda C. Suzuki T. Kamiie J. Terasaki T. Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (589) Google Scholar,12.Ito K. Uchida Y. Ohtsuki S. Aizawa S. Kawakami H. Katsukura Y. Kamiie J. Terasaki T. Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J Pharm Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar The common marmoset, which is a small new-world primate, has an attracted attention as a novel animal model for human CNS diseases such as Parkinson's disease, stroke, Huntington's disease, anxiety, and spinal cord injury, since transgenic techniques with tools related to embryonic stem cells and induced pluripotent stem cells have been developed for marmosets.13.Okano H. Hikishima K. Iriki A. Sasaki E. The common marmoset as a novel animal model system for biomedical and neuroscience research applications.Semin Fetal Neonatal Med. 2012; 17: 336-340Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 14.Sasaki E. Suemizu H. Shimada A. Hanazawa K. Oiwa R. Kamioka M. Tomioka I. Sotomaru Y. Hirakawa R. Eto T. Shiozawa S. Maeda T. Ito M. Ito R. Kito C. Yagihashi C. Kawai K. Miyoshi H. Tanioka Y. Tamaoki N. Habu S. Okano H. Nomura T. Generation of transgenic non-human primates with germline transmission.Nature. 2009; 459: 523-527Crossref PubMed Scopus (536) Google Scholar, 15.Sasaki E. Hanazawa K. Kurita R. Akatsuka A. Yoshizaki T. Ishii H. Tanioka Y. Ohnishi Y. Suemizu H. Sugawara A. Tamaoki N. Izawa K. Nakazaki Y. Hamada H. Suemori H. Asano S. Nakatsuji N. Okano H. Tani K. Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus).Stem Cells. 2005; 23: 1304-1313Crossref PubMed Scopus (132) Google Scholar, 16.Tomioka I. Maeda T. Shimada H. Kawai K. Okada Y. Igarashi H. Oiwa R. Iwasaki T. Aoki M. Kimura T. Shiozawa S. Shinohara H. Suemizu H. Sasaki E. Okano H. Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28.Genes Cells. 2010; 15: 959-969Crossref PubMed Scopus (102) Google Scholar The common marmoset has several advantages of comparative ease in handling because of its small size ( 200–300 g), remarkable reproductive efficiency,13.Okano H. Hikishima K. Iriki A. Sasaki E. The common marmoset as a novel animal model system for biomedical and neuroscience research applications.Semin Fetal Neonatal Med. 2012; 17: 336-340Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar and low zoonosis risk. It is thus conceivable that the common marmoset might have advantages over rats and old-world primates as an animal model. The availability of a good human-resembling animal model at the preclinical stage could drastically change the strategy for developing CNS-acting drugs.17.Orsi A. Rees D. Andreini I. Venturella S. Cinelli S. Oberto G. Overview of the marmoset as a model in nonclinical development of pharmaceutical products.Regul Toxicol Pharmacol. 2011; 59: 19-27Crossref PubMed Scopus (56) Google ScholarThe purpose of the present study was to determine the protein expression amounts of transporters, receptors, and tight junction proteins associated with BBB permeability in brain capillaries of two strains (SD and Wistar) of rats and marmoset, by means of QTAP, as well as to clarify inter-species and inter-strain similarities and differences by comparing the results with those that we have previously reported for mouse, cynomolgus monkeys, and human.1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google Scholar,11.Uchida Y. Ohtsuki S. Katsukura Y. Ikeda C. Suzuki T. Kamiie J. Terasaki T. Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (589) Google Scholar,12.Ito K. Uchida Y. Ohtsuki S. Aizawa S. Kawakami H. Katsukura Y. Kamiie J. Terasaki T. Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J Pharm Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (178) Google ScholarMATERIALS AND METHODSAnimals and ReagentsAdult SD rats (male, 8 weeks of age) and adult Wistar rats (male, 8 weeks of age) were purchased from CLEA Japan (Tokyo, Japan) (Table 1). Rats were maintained on a 12-h light/dark cycle in a temperature-controlled environment with free access to food and water; they were denied access to only food for 16 h before experiments. All experiments were approved by the Institutional Animal Care and Use Committee in Tohoku University, and were performed in accordance with the guidelines in Tohoku University.Table 1Characteristics of Rats and MarmosetsAnimalSexAgeBody Weight (g)Brain ConditionBrain RegionTime of Fasting (h)Rat-Sprague DawleyMale8 weeks230–250FreshWhole cerebrum16-WistarMale8 weeks180–200FreshWhole cerebrum16Marmoset-No. 1Female3 years286FreshRight cerebrumOver 12-No. 2Female2 years269FrozenRight cerebrumOver 12-No. 3Male4 years246FrozenRight cerebrumOver 12-No. 4Male3 years256FrozenRight cerebrumOver 12-No. 5Male3 years244FrozenRight cerebrumOver 12Fresh, the brain capillary was freshly isolated from the cerebrum immediately after the brain excision. Frozen, the excised brain was frozen and stored at -80°C for 30 days, thawed and then the brain capillary was isolated from the cerebrum. Brain region represents one used for the isolation of brain capillaries. Open table in a new tab Five adult common marmosets (Callithrix jacchus; two females and three males, 2–4 years of age, 244–286 g body weight) were bred in Central Institute for Experimental Animals (CIEA) (Kawasaki, Japan) (Table 1). The marmosets were fasted for over 12 h before brain excision, which was performed at the CIEA. All experiments were conducted after obtaining permission from the Institutional Animal Care and Use Committee of the CIEA and were performed in accordance with the CIEA guidelines.All peptides (Supplementary Tables 1 and 2) were chosen by applying our in silico selection criteria,1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google Scholar and synthesized by Thermoelectron Corporation (Sedanstrabe, Germany) with > 95% peptide purity. Other chemicals were commercial products of analytical grade. Download .doc (.25 MB) Help with doc files Supporting InformationIsolation of Brain Capillaries from Rats and MarmosetsRats were transcardially perfused with phosphate-buffered saline to remove blood under anesthesia induced with pentobarbital, brains were excised and the cerebrums were immediately used for isolation of brain capillaries (Table 1).Marmosets were euthanized by exsanguination under anesthesia induced with ketamine and xylazine. Immediately, the brains were excised without perfusion. For one marmoset (No.1), the right cerebrum was immediately used for isolation of brain capillaries (Table 1). For the other four marmosets (No. 2–5), the excised brains were frozen at −80°C, thawed, and then the right cerebrums were used for isolation of brain capillaries (Table 1).Brain capillaries were isolated by a combination of dextran density gradient separation and size filtration according to Ito et al. and Uchida et al. with minor modifications.11.Uchida Y. Ohtsuki S. Katsukura Y. Ikeda C. Suzuki T. Kamiie J. Terasaki T. Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (589) Google Scholar,12.Ito K. Uchida Y. Ohtsuki S. Aizawa S. Kawakami H. Katsukura Y. Kamiie J. Terasaki T. Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J Pharm Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Isolation of brain capillaries from rat and marmoset was performed similarly, according to the following procedure. All isolation procedures were carried out at 4°C. Two rat cerebrums (approximately 3 g) or a right cerebrum (approximately 3 g) from a marmoset were dissected into 1 mm pieces and homogenized with 20 up-and-down, unrotated strokes in five volumes of solution B (101 mM NaCl, 4.6 mM KCl, 5 mM CaCl2·2H2O2, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, 15 mM HEPES; pH 7.4) per tissue weight. The homogenate was centrifuged at 1000g for 10 min at 4°C, the pellet was suspended in solution B containing 16% dextran, and the suspension was centrifuged at 4500g for 15 min at 4°C. The supernatant was transferred to a new tube, and centrifuged again at 4500g for 15 min at 4°C. The two resulting pellets were suspended and mixed in solution A (solution B containing 25 mM NaHCO3, 10 mM glucose, 1 mM pyruvate, and 5 g/L bovine serum albumin). The suspension was loaded onto 210 μm nylon mesh, and the mesh was washed with 10 mL of solution A. The suspension passing through the 210 μm nylon mesh was loaded onto 85 μm nylon mesh, and the mesh was washed with 10 mL of solution A. The suspension passing through the 85 μm nylon mesh was loaded onto 20 μm nylon mesh, and the mesh was washed with 40 mL of solution A. The brain capillaries retained on the 20 μm nylon mesh were immediately collected using solution A, and centrifuged at 1000g for 5 min at 4°C. The supernatant was discarded and the pellet was suspended in 1 mL of solution B. The suspension was centrifuged at 1000g for 5 min at 4°C. The supernatant was discarded and the pellet was suspended in 1 mL of solution B. This suspension was centrifuged at 1000g for 5 min at 4°C after observation of brain capillaries through a microscope. The pellet was suspended in hypotonic buffer (10 mM Tris–HCl, 10 mM NaCl, 1.5 mM MgCl2, pH 7.4) and sonicated to prepare whole tissue lysates of brain capillaries. Protein concentrations of the lysates were measured by the Lowry method using DC protein assay reagent (Bio-Rad, Hercules, California), and the lysates were stored at −80°C.Quantification of Membrane Proteins by LC–MS/MSProtein expression levels of the target molecules were simultaneously determined by means of multiplexed selected/multiple reaction monitoring (SRM/MRM) analysis as described previously.1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google Scholar Whole tissue lysates of isolated brain capillaries (50 μg protein) were dissolved in denaturing buffer [7 M guanidium hydrochloride, 500 mM Tris–HCl (pH 8.5), 10 mM EDTA], and the proteins were S-carbamoylmethylated as described by Kamiie et al.1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google Scholar The alkylated proteins were precipitated with a mixture of methanol, chloroform and water. The precipitates were dissolved in 6 M urea. The dissolved samples of SD, Wistar rat, and marmoset for the comparison of the expression amounts among species were diluted fivefold with 100 mM Tris–HCl (pH 8.5) and digested with TPCK-treated trypsin (Promega, Madison, Wisconsin) at an enzyme/substrate ratio of 1:100 at 37°C for 16 h. The dissolved samples of Wistar rat for the comparison of the different enzymatic conditions were diluted fivefold with ProteaseMax surfactant (Promega; final concentration 0.05%) and 100 mM Tris–HCl (pH 8.5), and treated with lysyl endopeptidase (Wako Pure Chemical Industries, Osaka, Japan) at an enzyme/substrate ratio of 1:100 at room temperature for 3 hr. Subsequently, the lysyl endopeptidase-treated samples were digested with TPCK-treated trypsin at an enzyme/substrate ratio of 1:100 at 37°C for 16 h. The tryptic digests were mixed with internal standard peptides and formic acid, and then centrifuged at 4°C and 17,360g for 5 min.The HPLC–MS/MS analysis was performed by coupling a HPLC system (Agilent1100 system or Agilent 1200 system; Agilent Technologies, Santa Clara, Califonia) to an ESI–triple quadrupole mass spectrometer (API5000 or QTRAP5500; AB SCIEX, Framingham, Massachusetts) equipped with Turbo V ion source (AB SCIEX). Sample equivalent to 3.33 or 30 μg protein was injected onto a Waters XBridge BEH130 C18 (1.0 mm ID × 100 mm, 3.5 μm particles; Waters, Milford, Massachusetts) column together with 500 fmol of internal standard peptides. Mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Linear gradients were applied to elute and separate the peptides at a flow rate of 50 μL/min for 130 min. The gradient sequence was as follows: 99% A:1% B for 5 min after injection, then a linear gradient to 40% A:60% B at 65 min, switch to 0% A:100% B at 66 min until 68 min, then a linear gradient to 99% A:1% B at 70 min and continue to 130 min.The eluted peptides were simultaneously and selectively detected by means of electrospray ionization in a multiplexed SRM/MRM mode, which can quantify many molecules simultaneously by using up to 300 SRM/MRM transitions (Q1/Q3). The dwell time was 10 ms per SRM/MRM transition. Each molecule was monitored with four sets of SRM/MRM transitions (Q1/Q3-1, Q1/Q3-2, Q1/Q3-3, Q1/Q3-4) derived from one set of standard and internal standard peptides (Supplementary Tables 1 and 2). Chromatogram ion counts were determined by using the data acquisition procedures in Analyst software version 1.5 (AB SCIEX). Signal peaks with a peak area count of over 5000 detected at the same retention time as an internal standard peptide were defined as positive. When positive peaks were observed in three or four sets of SRM/MRM transitions, the molecules were considered to be expressed in brain capillaries, and the protein expression amounts were determined as the average of three or four quantitative values. Kamiie et al. have established that the protein expression amounts show coefficients of variation of less than 20.0% when determined from three peaks with peak area counts of over 5000.1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google ScholarIf no positive peak was observed or the positive peak(s) was only detected in one or two SRM/MRM transitions, the protein expression level in the brain capillary was defined as under the limit of quantification (LQ). The value of the LQ (fmol/μg protein) was determined as described by Uchida et al.11.Uchida Y. Ohtsuki S. Katsukura Y. Ikeda C. Suzuki T. Kamiie J. Terasaki T. Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (589) Google ScholarRESULTSQuantitative Analysis of Membrane Proteins in SD and Wistar Rat Brain CapillariesThe expression levels of 18 proteins, including 6 ATP-binding cassette (ABC) transporters, 6 solute carrier (SLC) transporters, 6 receptors, tight junction protein, and marker proteins, were analyzed in isolated brain capillaries of the two rat strains (Tables 2 and 4). Both strains showed similar protein expression amounts of 13 molecules in isolated capillaries within less than 2-fold difference (Table 2). The other five molecules were under the limit of quantification. All of the proteins detected in the isolated rat brain capillaries were also detected in isolated ddY mouse brain capillaries1.Kamiie J. Ohtsuki S. Iwase R. Ohmine K. Katsukura Y. Yanai K. Sekine Y. Uchida Y. Ito S. Terasaki T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm Res. 2008; 25: 1469-1483Crossref PubMed Scopus (412) Google Scholar with less than 2-fold difference in the protein amounts. Among ABC transporters, the protein expression amounts of Mdr1a, Mrp4 and Bcrp were determined. Mdr1a showed the most abundant expression among ABC transporters followed by Bcrp and Mrp4. Among SLC transporters, glucose transporter 1 (Glut1/Slc2a1), l-type amino-acid transporter 1 (Lat1/Slc7a5), monocarboxylate transporter 1 (Mct1/Slc16a1) and organic anion transporter 3 (Oat3/Slc22a8) were detected. Glut1 exhibits the highest expression level among all detected proteins, as is the case in marmoset brain capillaries, followed by Mct1, Lat1, and Oat3. Furthermore, insulin receptor (Insr), low-density lipoprotein receptor-related protein 1 (Lrp1), transferrin receptor 1(Tfr1), claudin-5, Na+/K+ ATPase and γ-gtp were detected. The greatest difference in expression levels between the trypsin-alone and the combination digestion conditions was 0.737-fold for γ-gtp.Table 2Protein Expression Levels of Membrane Proteins in Brain Capillaries Isolated from Sprague Dawley and Wistar Rat BrainsGene symbolProtein expression level (fmol/μg protein)Enzymatic condition SynonymSDWistarFold difference in trypsin digestion SD/WistarFold difference in Wistar rat LysC + ProteaseMax + Trypsin/TrypsinAverage of two rat strains in trypsin digestionTrypsinTrypsinLysC + ProteaseMax + TrypsinAbc transporters-Abcb1Mdr1a19.0 ± 2.019.2 ± 1.124.9 ± 1.10.9901.3019.1 ± 1.0-Abcc4Mrp41.60 ± 0.291.46 ± 0.081.23 ± 0.141.100.8421.53 ± 0.16-Abcg2Bcrp4.15 ± 0.295.74 ± 0.506.95 ± 0.730.7231.214.95 ± 0.32Slc transporters-Slc2a1Glut184.0 ± 4.198.2 ± 7.0Not measured0.855–91.1 ± 4.7-Slc7a5Lat13.41 ± 0.742.58 ± 0.84Not measured1.32–3.00 ± 0.62-Slc16a1Mct111.6 ± 0.613.5 ± 0.813.7 ± 0.70.8591.0112.6 ± 0.5-Slc22a8Oat32.13 ± 0.491.37 ± 0.181.69 ± 0.071.551.231.75 ± 0.25Receptors-InsrInsr0.785 ± 0.1111.15 ± 0.34Not measured0.683–0.968 ± 0.185-Tfr1Tfr16.74 ± 0.398.93 ± 1.168.81 ± 1.570.7550.9877.84 ± 0.53-Lrp1Lrp11.09 ± 0.141.16 ± 0.211.13 ± 0.210.9400.9741.13 ± 0.12Tight junction protein-Claudin-5Claudin-57.91 ± 0.907.00 ± 0.808.13 ± 1.741.131.167.46 ± 0.68Marker proteins-Na+/K+-- ATPaseNa+/K+ ATPase68.6 ± 4.536.2 ± 5.538.3 ± 2.51.901.0652.4 ± 5.2-γ-gtpγ-gtp3.07 ± 0.563.46 ± 0.132.55 ± 0.150.8870.7373.27 ± 0.27Brain capillaries of SD rats (adult, male, 8 weeks of age) or Wistar rats (adult, male, 8 weeks of age) were isolated from two fresh cerebrums by using a nylon mesh method (see text). Whole tissue lysate of rat brain capillaries was digested with trypsin or with a combination of lysyl endopeptidase, ProteaseMax, and trypsin under reducing and solubilizing conditions. The digests were injected into the LC–MS/MS together with internal standard peptides. The protein expression levels were calculated as an average of 6–8 quantitative values obtained from three or four SRM/MRM transitions in duplicate analyses. Protein expression levels are shown as mean ± SEM. Fold difference SD/Wistar was calculated by dividing the protein expression levels in isolated SD rat brain capillaries by the protein expression levels in isolated Wistar
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