1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
YEXER6426_proof ■ 16 May 2014 ■ 1/7
Experimental Eye Research xxx (2014) 1e7
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier Q12 Q1
Yoshiyuki Kubo 1, Ayaka Tomise 1, Ai Tsuchiyama, Shin-ichi Akanuma, Ken-ichi Hosoya* Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2013 Accepted in revised form 4 May 2014 Available online xxx
The elimination of spermine, the end product of cellular polyamine, from the retina to the blood across the blood-retinal barrier (BRB) was investigated. The in vivo microdialysis study revealed that the elimination of [3H]spermine from vitreous humor after vitreous bolus injection was in a biexponential manner. The rate constant for the elimination of [3H]spermine during the terminal phase was estimated to be 1.67-fold greater than that of [14C]D-mannitol, a bulk flow marker, and the difference in the terminal elimination rate constant between [3H]spermine and [14C]D-mannitol was reduced in the presence of 50 mM spermine, suggesting a retina-to-blood transport system for [3H]spermine across the BRB. The retina-to-blood transport of [3H]spermine was also supported by a study of the retinal uptake index (RUI). The in vitro transport study with TR-iBRB2 cells, a model cell line of the inner BRB, revealed time-, concentration- and temperature-dependent transport of [3H]spermine, suggesting the involvement of carrier-mediated processes in spermine transport across the inner BRB. The in vitro study also suggested that the transport of spermine at the inner BRB is pH-, membrane potential- and Cl-sensitive and Naþinsensitive, and these functional properties of spermine transport suggest only a minor contribution of spermine transporters, such as CCC9 (SLC12A8), the expression of which was suggested at the inner BRB. In the inhibition study, [3H]spermine transport was markedly inhibited by putrescine, spermidine, spermine and agmatine while substrates of a well-characterized organic cation transporter (OCTs/ SLC22A) and a cationic amino acid transporter (CATs/SLC7A) had no effect, suggesting the involvement of unknown transporters in spermine elimination from the retina. Ó 2014 Published by Elsevier Ltd.
Keywords: polyamine spermine transport blood-retinal barrier polyamine transporter
1. Introduction Polyamines, such as putrescine, spermidine and spermine, are known as bioactive amines that are ubiquitously distributed in the animal tissues (de Vera et al., 1995; Moinard et al., 2005). Polyamines have been suggested to play an important role in cellular proliferation and differentiation (Tabor and Tabor, 1976; Pegg and McCann, 1982), and alterations of polyamine concentration, in cells, blood and urine, have been reported in patients with diseases including cancer, psoriasis, multiple sclerosis, Duchenne muscular dystrophy and chronic renal failure (Wallace and Caslake, 2001; Noga et al., 2012; Russell and Stern, 1981; Swendseid et al., 1980; Saito et al., 1983). In cancer cells, the cellular concentration of polyamine was reported to be elevated by the activation of
* Corresponding author. Tel.: þ81 76 434 7505; fax: þ81 76 434 5172. E-mail address: [email protected] (K.-i. Hosoya). 1 Kubo Y and Tomise A contributed equally to this work.
ornithine decarboxylase that catalyzes the biosynthesis of putrescine (LaMuraglia et al., 1986; Cañizares et al., 1999), and the proliferation of cancer cells was reported to be suppressed in the presence of an ornithine decarboxylase inhibitor, such as adifluoromethylornithine (Quemener et al., 1992; Meyskens et al., 2008). In the retina associated with vision sense, the physiological significance of polyamines has been reported, and the loss of cone cells, photoreceptor cells, is known to be caused by polyamine deficiency in the developing rabbit retina (Withrow et al., 2002). In a study with multiple sclerosis model mice, the cell loss in the retinal ganglion cell layer has been shown to be inhibited by the oral administration of spermidine, suggesting the antioxidative effect of polyamines (Guo et al., 2011). In addition, the involvement of spermine was suggested in the retinal degeneration observed in gyrate atrophy of the choroid and retina since the excess amount of spermine induced cell apoptosis in a study involving the bovine retina (Kaneko et al., 2007). These reports suggest that the regulation of polyamine concentration is important in the retina. In the
http://dx.doi.org/10.1016/j.exer.2014.05.002 0014-4835/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Q2 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 2/7
2
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
cellular biosynthesis of polyamines, it is known that putrescine is produced from ornithine by ornithine decarboxylase (ODC), and putrescine is converted into spermidine by spermidine synthase (Sturman et al., 1976; Pegg and McCann, 1982; Seiler, 2004). ODC is one of the rate-limiting enzymes in biosynthesis of polyamine, and its cellular expression and activity were detectable in the retina (Yanagihara et al., 1996; Biedermann et al., 1998). In addition, the localization of ODC and spermine/spermidine in Müller cells was reported to suggest the role of spermine/spermidine in regulation of Kþ channels (Biedermann et al., 1998). Spermine synthase catalyzes the conversion of spermidine into spermine that is known to be the end product, suggesting the involvement of a spermine elimination system from the retina in the regulation of polyamine concentration (Sturman et al., 1976; Pegg and McCann, 1982; Seiler, 2004). In the retina, the blood-retinal barrier (BRB) separates the retinal tissue and the circulating blood, and the BRB includes two barrier structures, the inner and outer BRB, that are formed by the retinal capillary epithelial cells and retinal pigment epithelial (RPE) cells, respectively. In order to maintain the retinal function, membrane transporters expressed by the BRB contribute to the nutrient supply and endobiotic removal across the BRB since the retinal capillary epithelial cells and RPE cells form the tight junction to restrict the paracellular solute transport at the inner and outer BRB, respectively (Stewart and Tuor, 1994; Cunha-Vaz, 2004; Hosoya et al., 2011, 2012). In particular, it is known that the inner BRB is involved in nourishing two-thirds of the retina (Tomi and Hosoya, 2010), and previous reports about retinal capillary endothelial cells revealed the expression of various membrane transporters, such as cationic amino acid transporter 1 (CAT1/SLC7A1), neutral and basic amino acid transporter (yþLAT2/ SLC7A6), monocarboxylate transporter 1 (MCT1/Slc16a1), equilibrative nucleoside transporter 2 (ENT2/SLC29A2), L (leucinereferring)-type amino acid transporter (LAT1/SLC7A5), taurine transporter (TAUT/SLC6A6) and glucose transporter (GLUT1/ SLC2A1) (Tomi et al., 2009; Hosoya et al., 2001a; Nagase et al., 2006; Tomi et al., 2005, 2007a, 2007b; Usui et al., 2013; Takata et al., 1992; Kumagai et al., 1996). Regarding polyamine transport, polyamine transporters have been identified in various organisms, including prokaryotes and eukaryotes (Igarashi and Kashiwagi, 2010). In mammals, several transport systems for polyamines have been reported in tissues, such as lymphocytes, intestine, kidney and brain (Kakinuma et al., 1988; Iseki et al., 1991; Kobayashi et al., 1999; Masuko et al., 2003), and organic cation transporter 1 (OCT1/SLC22A1), L-carnitine transporter 2 (CT2/SLC22A16) and the splice variant of cation-chloride cotransporter 9 (CCC9/SLC12A8) have been reported to recognize spermine as a substrate (Busch et al., 1996; Aouida et al., 2010; Daigle et al., 2009). However, little is known about the mechanism for the elimination of spermine from the retina to the circulating blood, and any clarification of spermine elimination will be beneficial in maintenance of the retina and the clinical treatment of retinal diseases, such as diabetic retinopathy and gyrate atrophy of choroid and retina (Matsushita et al., 2010; Nicoletti et al., 2003; Kaneko et al., 2007). In this study, in vivo and in vitro studies were performed to investigate the elimination of spermine from the retina to the circulating blood. In the in vivo study, the blood-to-retina and retina-to-blood transports of spermine were analyzed by means of the retinal uptake index (RUI) and microdialysis, respectively. In the in vitro study, spermine transport was analyzed in a conditionally immortalized cell line of retinal capillary endothelial cells (TRiBRB2 cells), an in vitro model cell line of the inner BRB (Hosoya et al., 2001a, 2001b).
2. Materials and methods 2.1. Animals Male Wistar rats (160e300 g) were purchased from Japan SLC (Hamamatsu, Japan). The use of experimental animals followed the guidelines instituted by the Animal Care Committee in the University of Toyama and by the Association for Research in vision and Ophthalmology (ARVO) Statement. 2.2. Reagents Chemicals were of reagent grade and available commercially, and [1-14C]D-mannitol ([14C]D-mannitol, 55 mCi/mmol) and [1-14C] n-butanol ([14C]n-butanol, 2 mCi/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). [Terminal methylene-3H]spermine ([3H]spermine, 50 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). 2.3. Microdialysis study A microdialysis study was performed as described elsewhere (Katayama et al., 2006; Hosoya et al., 2009; Yoneyama et al., 2010; Akanuma et al., 2013). In brief, after anesthetizing rats (250e300 g) with sodium pentobarbital (60 mg/kg), their heads were placed on a stereotaxic frame (Narishige, Tokyo, Japan). To prevent eye blinking, 2% xylocaine was instilled to locally anesthetize their eyelids, and a 25G needle was inserted through the pars plana at a depth of 3.0 mm. After removal of the needle, 1 mL Ringer-HEPES solution (141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 10 mM HEPES, pH 7.4) containing [3H]spermine (2.0 mCi) and [14C]D-mannitol (0.2 mCi) were administered by means of a microsyringe (Hamilton, Reno, NE) at a depth of 3.0 mm from the eye surface. Immediately after, a microdialysis probe (TEP-50; Eicom, Kyoto, Japan) was implanted into the vitreous chamber, and the probe was fixed on the eye surface by means of surgical glue (Daiichi-Sankyo, Tokyo, Japan). To continuously supply Ringer-HEPES solution to the probe (2 mL/min, 37 C), an infusion pump (Harvard, Holliston, MA) and polyethylene tubing (Natsume, Tokyo, Japan) were used. The radioactivity in the dialyzate collected at designated times was determined with a liquid scintillation counter (LSC-5200, Aloka, Tokyo, Japan). Data analysis was performed as described previously (Yoneyama et al., 2010; Akanuma et al., 2013), and the data obtained were used in the calculation of CP, the vitreous concentrations normalized by the injected dose (% of dose/mL), by means of Eq. (1), where Dosetracer and CT are the total radioactivity in the solution after injection (dpm) and the concentration in the dialyzate (dpm/mL), respectively.
CP ¼ CT =Dosetracer 100
(1)
CP(t) defined as the CP at time t, was subjected to the nonlinear least-square regression analysis program, MULTI (Yamaoka et al., 1981), and was fitted to Eq. (2), where a and b are the apparent first-order rate constants for the initial and terminal phase, respectively, and A and B are intercepts on the y-axis for each exponential segment.
CP ðtÞ ¼ A eat þ B ebt
(2)
The probe recovery was estimated from Eq. (3), and CV (dpm/ mL) is the concentration in the test solution. In the present study, the recovery values were constant over 180 min, and the value for [3H]spermine and [14C]D-mannitol were 5.22% and 8.69%, respectively.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 3/7
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
Recovery ð%Þ ¼ CT =CV 100
(3)
In the inhibition study, Ringer-HEPES solution containing unlabeled compounds was supplied to the probe. The inhibitory effect on [3H]spermine elimination from the vitreous humor was evaluated from the difference in b values between [3H]spermine and [14C]D-mannitol (Db), and the effect was expressed as a percentage of the control estimated from Eq. (4).
ð% of controlÞ ¼ ðDb in the presence of inhibitorÞ= ðDb in the absence of inhibitorÞ 100
(4)
2.4. In vivo study of blood-to-retina transport of [3H]spermine The carotid artery injection method in rats was adopted to study the permeability of spermine to the retina as described previously (Hosoya et al., 2010; Alm and Törnquist, 1981; Pardridge and Fierer, 1985). In brief, a test solution was prepared by adding [3H]spermine (5.0 mCi, 0.5 mM) and a diffusible internal reference [14C]n-butanol (0.5 mCi) to 200 mL Ringer-HEPES buffer (pH 7.4). The test solution was injected into the common carotid artery of anesthetized rats (160e180 g), and decapitation was performed 15 s after injection. The retinas were collected to be lysed and neutralized with 2 N sodium hydroxide and hydrochloric acid, and the radioactivity in samples was measured by liquid scintillation counting. As described previously (Hosoya et al., 2010; Kubo et al., 2013), to express the distribution characteristics of compounds, the retinal uptake index (RUI) was used and was estimated from Eq. (5).
RUI ð%Þ ¼ ð½3 H=½14 Cðdpm in the retinaÞÞ=ð½3 H=½14 C ðdpm in the injectate solutionÞÞ 100
(5)
2.5. Study of [3H]spermine transport in TR-iBRB2 cells The [3H]spermine uptake by TR-iBRB2 cells, a conditionally immortalized rat capillary endothelial cell line, was investigated as described previously (Tomi et al., 2009; Usui et al., 2013; Kubo et al., 2013). In brief, TR-iBRB2 cells (passage number 23e35) were maintained in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceuticals, Tokyo, Japan) with 10% fetal bovine serum, 20 mM NaHCO3, and antibiotics at 33 C (Hosoya et al., 2001a, 2001b). An uptake study was performed by means of extracellular fluid (ECF)buffer (122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, 10 mM D-glucose and 10 mM HEPES (pH 7.4)). The cells to be cultured were rinsed with ECFbuffer, and incubated with 10 nM [3H]spermine (0.1 mCi) in 200 mL ECF-buffer at 37 C. The ion-dependence of [3H]spermine uptake by TR-iBRB2 cells was examined in Naþ-free ECF buffer as described previously (Ohkura et al., 2010; Kubo et al., 2013). Uptake was terminated at the designated time, and the cells were lysed and neutralized in 1 N sodium hydroxide and hydrochloric acid. The cellular radioactivity and protein content were determined by means of liquid scintillation counting and a DC protein assay kit (Bio-Rad, Hercules, CA), respectively. As described elsewhere (Hosoya et al., 2001b; Usui et al., 2013), the cellular uptake of [3H] spermine was expressed as the cell-to-medium (cell/medium) ratio calculated from Eq. (6).
Cell=medium ratio ¼ ð½3 H dpm per cell protein ðmgÞÞ= ð½3 H dpm per mL mediumÞ
(6)
In the estimation of the kinetic parameters for cellular spermine uptake by means of MULTI (Yamaoka et al., 1981), the data obtained were fitted to a two saturable processes model (Eq. (7)), where V,
3
[S], Vmax1, Vmax2, Km1 and Km2 are the uptake rate of spermine, the concentration of spermine in ECF-buffer, the maximum uptake rate for the high-affinity process, the maximum uptake rate for the lowaffinity process, the MichaeliseMenten constant for the highaffinity process and the MichaeliseMenten constant for the lowaffinity process, respectively.
V ¼ Vmax1 ½S=ðKm1 þ ½SÞ þ Vmax2 ½S=ðKm2 þ ½SÞ
(7)
2.6. Expression analysis by reverse transcription polymerase chain reaction (RT-PCR) The investigation of the transcript expression of CT2 and CCC9 was performed as described previously (Tomi et al., 2007a, 2007b; Yoneyama et al., 2010). In brief, TRIzol reagent (Invitrogen, Carlsbad, CA) and an RNeasy Mini kit (Qiagen, Hilden, Germany) were used in the preparation of total RNA from TR-iBRB2 cells and primary-cultured RPE cells (Babu et al., 2011; Usui et al., 2013), and 1 mg total RNA was subjected to single-strand cDNA synthesis by means of reverse transcription (RT) and oligo dT primer. GeneAmp PCR system 9700 (Applied Biosystems, Foster, CA) was used in the polymerase chain reaction for rat CT2 through 35 cycles at 94 C for 30 s, 47 C for 30 s, and 72 C for 30 s, and for rat CCC9 through 35 cycles at 94 C for 30 s, 61 C for 30 s, and 72 C for 30 s. Primers for rat CT2 were designed by referring to mouse CT2 (GenBank ID: NM_027572), and the sense and antisense sequences were 50 AGTCCTTGCTTCGATGGCTA-30 and 50 -AGCGTGTGATCATGAAGCTG30 , respectively. Primers for rat CCC9 were designed by referring to rat CCC9 (SLC12A8, GenBank ID: NM_153625), and the sense and antisense sequences were 50 -CGAGAGGCCCTCCGCTCTGA-30 and 50 TCCGGGCCTTGTCTGGGCAT-30 , respectively. Agarose gel electrophoresis with ethidium bromide was performed to separate and visualize the PCR products under ultraviolet light, and the resultant products were confirmed by sequence analysis using a DNA sequencer (ABI PRISM 3130; Applied Biosystems). 2.7. Statistical analysis The kinetic parameters are represented as the mean S.D., and others are shown as the mean S.E.M. An unpaired Student’s t-test was performed in the statistical analysis of two groups, and oneway analysis of variance followed by Dunnett’s test was performed in the analysis of several groups. 3. Results 3.1. Retina-to-blood transport of [3H]spermine from the vitreous humor The time-profile of the [3H]spermine remaining in the vitreous humor after bolus injection was examined (Fig. 1A), and [3H]spermine exhibited a biexponential elimination from the vitreous humor with an apparent elimination constant (b) during terminal phase of 15.3 103 1.03 103 min1 (Fig. 1B). [14C]D-Mannitol, co-injected as a bulk flow marker, also exhibited a biexponential elimination from the vitreous humor with an apparent elimination constant (b) during terminal phase of 9.14 103 0.70 103 min1, and the b value of [3H]spermine was 1.67-fold greater than that of [14C]D-mannitol (Fig. 1B), showing that the decline in [3H] spermine was significantly greater than that of [14C]D-mannitol. In the inhibition study, unlabeled spermine showed a significant reduction in the b value difference between [3H]spermine and [14C] D-mannitol while no significant effect was observed in the presence of tetraethylammonium (TEA) (Table 1). These results suggest the
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 4/7
4
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
Fig. 1. Time-profile of [3H]spermine and [14C]D-mannitol in the vitreous humor after vitreous bolus injection (A) and elimination rate constants (b) of [3H]spermine and [14C]Dmannitol in the terminal phase (B). Open circles and triangles represent the concentration in the dialyzate of [14C]D-mannitol and [3H]spermine, respectively. Each point and column represents the mean S.E.M. (n ¼ 3). *p < 0.01, significantly different from [14C]D-mannitol.
involvement of the retina-to-blood carrier-mediated transport process at the BRB in spermine elimination from the retina. 3.2. Blood-to-retina transport of [3H]spermine To examine the in vivo blood-to-retina transport of spermine at the BRB, the retinal uptake index (RUI) was estimated using a carotid artery single injection in rats. In Table 2, the RUI value of [3H] spermine was shown to be 42.5%, and no significant effect on the value (43.2%) was observed in the presence of 50 mM spermine, suggesting that the blood-to-retina carrier-mediated transport of spermine is minor at the BRB. 3
3.3. Uptake of [ H]spermine by TR-iBRB2 cells In order to investigate the transport of spermine at the inner BRB, the uptake study with TR-iBRB2 cells, an in vitro model cell line, was performed. As seen from the results, [3H]spermine uptake by TR-iBRB2 cells exhibited a time-dependent increase for 10 min at least, with an initial uptake rate of 24.9 4.2 mL/(min mg protein) (Fig. 2A). TR-iBRB2 cells showed a temperature-dependent uptake of [3H]spermine, exhibiting a [3H]spermine uptake significantly reduced by 90.0% at 4 C, (Fig. 2A). In the study of pH-dependence, [3H]spermine transport in TR-iBRB2 cells was significantly increased and decreased at pH 6.4 and 8.4, respectively, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a proton uncoupler, canceled the increased uptake at pH 6.4 (Fig. 2B). In the study of ion-dependence, the transport of [3H]spermine was significantly reduced in Cl-free buffer and Kþ-replacement buffer by 20.5% and 64.8%, respectively, and no alteration was observed in Naþ-free buffer (Fig. 2C). In the study of spermine transport over Table 1 Inhibitory effect on the elimination rate constant (b) difference between [3H]spermine and [14C]D-mannitol during the terminal phase. % of control Control Spermine TEA
100 18 26.6 6.4* 91.7 23.2
Each inhibitor was perfused in the microdialysis probe. Inhibitory effect was examined in the presence of spermine (50 mM) and TEA (50 mM). Each value represents the mean S.E.M. (n ¼ 4e5). *p < 0.01, significantly different from the control. TEA, tetraethylammonium.
the concentration range of 1e200 mM (Fig. 3), the transport of [3H] spermine was shown to be concentration-dependent, exhibiting a Km1 of 0.968 0.369 mM, a Vmax1 of 42.7 7.3 pmol/ (min mg protein), a Km2 of 158 67 mM and a Vmax2 of 382 88 pmol/(min mg protein). These results suggest the involvement of pH-, Cl- and a membrane potential-dependent carrier-mediated transport systems in the [3H]spermine transport in TR-iBRB2 cells. 3.4. Expression of spermine transport molecules To investigate the expression of rat CT2 (SLC22A16) and rat CCC9 (SLC12A8) at the BRB, RT-PCR analysis was performed. The results obtained showed that the PCR product for CT2 (271 bp) was not detected in TR-iBRB2 cells and RPE cells, suggesting that the expression of CT2 is minor at the inner and outer BRB (Fig. 4A). On the other hand, the PCR product for CCC9 (401 bp) was detected in TR-iBRB2 cells and RPE cells (Fig. 4B), suggesting the expression of CCC9 at the inner and outer BRB. 3.5. Inhibitory effect on the transport of [3H]spermine in TR-iBRB2 cells The inhibition study of [3H]spermine transport in TR-iBRB2 cells was performed to examine the transport characteristics of spermine at the inner BRB. As the results, in the presence of spermine, spermidine, putrescine and agmatine, [3H]spermine transport was strongly reduced by more than 87% (Table 3). Tetraethylammonium (TEA) exhibited a significant but weak inhibitory effect on [3H] spermine transport by 18% while no significant inhibition was observed in the presence of 1-methyl-4-phenylpyridinium (MPPþ), serotonin, L-carnitine, L-histidine, choline, L-arginine, cimetidine and L-ornithine (Table 3).
Table 2 In vivo [3H]spermine uptake by rat retina. RUI (%) Control Spermine
42.5 1.7 43.2 2.6
[3H]Spermine (5.0 mCi/rat) and [14C]n-butanol (0.5 mCi/rat) were injected into the common carotid artery in the absence (control) or presence of spermine (50 mM). Each value represents the mean S.E.M. (n ¼ 3e4).
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 5/7
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
5
Fig. 3. Concentration-dependent uptake of spermine. The uptake of [3H]spermine (0.1 mCi, 10 nM) was examined at 37 C for 5 min, over the concentration range 1e 200 mM. The data obtained were analyzed by means of MichaeliseMenten (main graph) and EadieeScatchard (inset graph) analyses. Each point represents the mean S.E.M (n ¼ 3).
the elimination system for spermine across the BRB is thought to have a significant influence in the polyamine concentration in the retina. Therefore, in the present study, in vivo and in vitro analyses were performed to investigate the mechanism for the retina-toblood transport of spermine at the inner BRB that nourishes twothirds of the retina (Tomi and Hosoya, 2010). In the in vivo analysis, microdialysis was performed to study the retina-to-blood transport of [3H]spermine at the BRB using [14C]Dmannitol as a bulk flow marker for passage from the vitreous humor to Schlemm’s canal and/or the uveoscleral outflow route. The concentrationetime profile for [3H]spermine and [14C]D-mannitol in the dialyzate showed that their concentration in the vitreous humor was reduced in a biexponetial manner, and [3H]spermine and [14C]D-mannitol exhibited a steeper initial slope (a) than the later slope (b), supporting the hypothesis that the initial and later declines reflect diffusion into the vitreous humor, including the microdialysis tube, after vitreous bolus injection and elimination from the vitreous humor of both [3H]spermine and [14C]D-mannitol (Fig. 1A). In the comparison of the b values, [3H]spermine exhibited a 1.67-fold greater b value than that of [14C]D-mannitol (Fig. 1B), suggesting that [3H]spermine was subjected to retina-to-blood transport at the BRB in addition to its elimination from the vitreous humor via bulk flow and passive diffusion. In the inhibition study, the results suggest the involvement of a spermine-sensitive and carrier-mediated transport system in the retina-to-blood transport of spermine at the BRB since unlabeled spermine significantly reduced the difference in the b value between [3H]spermine
Fig. 2. Uptake of [3H]spermine by TR-iBRB2 cells. (A) Time-dependent uptake of [3H] spermine (0.1 mCi, 10 nM) was investigated at 37 C (closed circles), and temperaturedependence was examined at 4 C (open circle). Extracellular pH-dependence (B) and Naþ-, Cl- and membrane potential-dependence (C) of [3H]spermine uptake was performed at 37 C for 5 min. Extracellular pH-dependence was examined with or without 50 mM FCCP. Each point and column represents the mean S.E.M (n ¼ 3). *p < 0.01, significantly different from the control.
4. Discussion Polyamines are cationic compounds that are ubiquitously distributed in tissues, and their involvement has been reported in the maintenance of retinal function and the pathogenesis of severe retinal diseases, such as gyrate atrophy of choroid and retina (Withrow et al., 2002; Kaneko et al., 2007; Guo et al., 2011), showing that the regulation of polyamine concentration is important in the maintenance of a healthy retina. In particular, spermine is the end product in the cellular biosynthesis of polyamines, and
Fig. 4. Expression of rat CT2 (SLC22A16) and rat CCC9 (SLC12A8). Expression of CT2 (A) and CCC9 (B) was analyzed by RT-PCR. (þ) and () represent PCR, with and without reverse transcription, respectively.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 6/7
6
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7 Table 3 Inhibitory effect on [3H]spermine uptake by TR-iBRB2 cells. % of control Control Putrescine Spermidine Spermine Agmatine TEA MPPþ Serotonin L-Carnitine Choline (5 mM) Cimetidine L-Arginine L-Ornithine L-Histidine
100 11.8 3.61 3.27 12.3 81.2 91.0 93.7 101 109 119 115 119 103
2 0.5* 0.19* 0.18* 0.36* 3.7* 1.2 4.1 3 3 0.6 5 6 4
The uptake of [3H]spermine (10 nM, 0.1 mCi) by TR-iBRB2 cells was examined in the absence (control) or presence of 1 mM compounds at 37 C for 5 min. Choline was used at a concentration of 5 mM. Each value represents the means S.E.M. (n ¼ 3e15). *p < 0.01, significantly different from the control. TEA, tetraethylammonium; MPPþ, 1methyl-1-phynylpyridinium.
and [14C]D-mannitol in spite of no significant effect being exhibited by TEA (Table 1). The study with a carotid single injection suggested the minor contribution of the blood-to-retina carrier-mediated transport of spermine at the BRB because unlabeled spermine had no significant effect on the RUI value of [3H]spermine (Table 2). The in vivo studies suggest the involvement of the retina-toblood carrier-mediated transport process in the elimination of spermine from the retina, and the in vitro transport study with TRiBRB2 cells, an in vitro model of the inner BRB (Hosoya et al., 2001a, 2001b), was used in order to investigate the functional properties of spermine transport. From the results, the involvement of carriermediate transport processes were suggested in spermine transport at the inner BRB since the transport of [3H]spermine occurred in a time-, concentration-, and temperature-dependent manner, with high-affinity (Km1 ¼ 0.968 mM) and low-affinity (Km2 ¼ 158 mM) processes (Figs. 2A and 3). In the study of ion coupling, the pH-sensitivity of spermine transport was suggested at the inner BRB since the transport of [3H]spermine in TR-iBRB2 cells was significantly altered under acidic and alkaline extracellular pH conditions and a proton uncoupler showed the cancelation of the enhanced transport observed at acidic extracellular pH (Fig. 2B). The significant decrease in [3H]spermine transport, in the Kþ- and Cl-free buffers, suggested the membrane potential- and Cl-sensitivities of spermine transport at the inner BRB (Fig. 2C). The study also suggests that spermine transport is Naþ-insensitive since the transport of [3H]spermine in TR-iBRB2 cells exhibited no significant change in the Naþ-free buffer (Fig. 2C). These results suggest the involvement of pH-, membrane potential- and Cl-sensitive carrier-mediated processes in the retina-to-blood transport of spermine at the inner BRB. Recent research on transporters suggests that OCT1 (SLC22A1), CT2 (SLC22A16) and the splice variant of CCC9 (SLC12A8) recognize spermine (Busch et al., 1996; Aouida et al., 2010; Daigle et al., 2009), and the undetectable expression of OCT1 at the inner BRB has been reported previously (Tomi et al., 2007a, 2007b). The detectable and undetectable expressions of CCC9 and CT2 in TR-iBRB2 cells suggest the possible contribution of CCC9 at the inner BRB (Fig. 4). However, TR-iBRB2 cells exhibited the Cl-sensitity of [3H]spermine transport to suggest that CCC9 is not responsible for spermine transport at the inner BRB because the Cl-insensitivity of CCC9 was previously reported although CCC9 is a member of the cation-coupled chloride cotransporters family (Daigle et al., 2009).
In the inhibition study, the results obtained suggest the involvement of a polyamine transporter in the retina-to-blood transport of spermine since the transport of [3H]spermine in TRiBRB2 cells was markedly inhibited by polyamines, such as putrescine, spermidine, spermine and agmatine (Table 3). Although TEA exhibited a slight effect on the transport of [3H]spermine in TRiBRB2 cells (Table 3), the contribution of organic cation transporters is thought to be minor at the inner BRB because no significant decrease in the [3H]spermine transport was shown by MPPþ, serotonin, L-carnitine, choline and cimetidine (Table 3), which are typical substrates of identified organic cation transporters (Otsuka et al., 2005; Engel and Wang, 2005; Ohta et al., 2006; Koepsell, 2013). In addition, no significant effect on the transport of [3H] spermine was exhibited by L-arginine, L-ornithine and L-histidine (Table 3), which are known as substrates of SLC7A family members, such as LAT1 (SLC7A5), CATs (SLC7A1-4) and yþLAT2 (SLC7A6) (Kanai et al., 1998; Segawa et al., 1999; Pineda et al., 1999; Tomi et al., 2009), and this suggests that the well-characterized members of SLC7A family are not responsible for the retina-to-blood transport of spermine, implying the involvement of polyaminespecific but unknown transporters at the inner BRB. In conclusion, the present analyses suggest the involvement of carrier-mediated transport processes in the elimination of spermine, the end product of polyamine biosynthesis, across the inner BRB. The inhibition study suggests the minor contribution of wellidentified transporters to the retina-to-blood transport of spermine, and show that unknown transporters contribute to spermine elimination from the retina. The present findings will be helpful to improve our understanding of the regulation of spermine concentration in the retina, and for the maintenance of a healthy retina. Conflict of interest The authors declare that they have no conflict of interest. Uncited references Hayasaka et al., 2011; Hosoya and Tomi, 2005.
Q11
Acknowledgments This study was supported, in part, by a Grant-in Aid for Scientific Q3,4 Research from the Ministry of Education, Science, Sports, and Cul- Q5,6 ture, the Japan Society for the Promotion of Science, and The Nakatomi Foundation, Japan. Q7 References Akanuma, S., Soutome, T., Hisada, E., Tachikawa, M., Kubo, Y., Hosoya, K., 2013. Naþindependent nucleoside transporters regulate adenosine and hypoxanthine levels in Müller cells and the inner blood-retinal barrier. Investig. Ophthalmol. Vis. Sci. 54, 1469e1477. Alm, A., Törnquist, P., 1981. The uptake index method applied to studies on the blood-retinal barrier. I. A methodological study. Acta Physiol. Scand. 113, 73e79. Aouida, M., Poulin, R., Ramotar, D., 2010. The human carnitine transporter SLC22A16 mediates high affinity uptake of the anticancer polyamine analogue bleomycinA5. J. Biol. Chem. 285, 6275e6284. Babu, E., Ananth, S., Veeranan-Karmegam, R., Coothankandaswamy, V., Smith, S.B., Boettger, T., Ganapathy, V., Martin, P.M., 2011. Transport via SLC5A8 (SMCT1) is obligatory for 2-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells. Investig. Ophthalmol. Vis. Sci. 52, 5749e5757. Biedermann, B., Skatchkov, S.N., Brunk, I., Bringmann, A., Pannicke, T., Bernstein, H.G., Faude, F., Germer, A., Veh, R., Reichenbach, A., 1998. Spermine/ spermidine is expressed by retinal glial (Müller) cells and controls distinct Kþ channels of their membrane. Glia 23, 209e220. Busch, A.E., Quester, S., Ulzheimer, J.C., Waldegger, S., Gorboulev, V., Arndt, P., Lang, F., Koepsell, H., 1996. Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1. J. Biol. Chem. 271, 32599e32604.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 7/7
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
Q8
Cañizares, F., Salinas, J., de las Heras, M., Diaz, J., Tovar, I., Martinez, P., Peñafiel, R., 1999. Prognostic value of ornithine decarboxylase and polyamines in human breast cancer: correlation with clinicopathologic parameters. Clin. Cancer Res. 5, 2035e2041. Cunha-Vaz, J.G., 2004. The blood-retinal barriers system. Basic concepts and clinical evaluation. Exp. Eye Res. 78, 715e721. Daigle, N.D., Carpentier, G.A., Frenette-Cotton, R., Simard, M.G., Lefoll, M.H., Noël, M., Caron, L., Noël, J., Isenring, P., 2009. Molecular characterization of a human cation-Cl- cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport. J. Cell Physiol. 220, 680e689. de Vera, N., Camón, L., Martínez, E., 1995. Blood polyamines in the rat. J. Pharm. Pharmacol. 47, 204e205. Engel, K., Wang, J., 2005. Interaction of organic cations with a newly identified plasma membrane monoamine transporter. Mol. Pharmacol. 68, 1397e1407. Guo, X., Harada, C., Namekata, K., Kimura, A., Mitamura, Y., Yoshida, H., Matsumoto, Y., Harada, T., 2011. Spermidine alleviates severity of murine experimental autoimmune encephalomyelitis. Investig. Ophthalmol. Vis. Sci. 52, 2696e2703. Hayasaka, S., Kodama, T., Ohira, A., 2011. Retinal risks of high-dose ornithine supplements: a review. Br. J. Nutr. 106, 801e811. Hosoya, K., Tachikawa, M., 2012. The inner blood-retinal barrier: molecular structure and transport biology. Adv. Exp. Med. Biol. 763, 85e104. Hosoya, K., Tomi, M., 2005. Advances in the cell biology of transport via the inner blood-retinal barrier: establishment of cell lines and transport functions. Biol. Pharm. Bull. 28, 1e8. Hosoya, K., Kondo, T., Tomi, M., Takanaga, H., Ohtsuki, S., Terasaki, T., 2001a. MCT1mediated transport of L-lactic acid at the inner blood-retinal barrier: a possible route for delivery of monocarboxylic acid drugs to the retina. Pharm. Res. 18, 1669e1676. Hosoya, K., Tomi, M., Ohtsuki, S., Takanaga, H., Ueda, M., Yanai, N., Obinata, M., Terasaki, T., 2001b. Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat. Exp. Eye Res. 72, 163e172. Hosoya, K., Makihara, A., Tsujikawa, Y., Yoneyama, D., Mori, S., Terasaki, T., Akanuma, S., Tomi, M., Tachikawa, M., 2009. Roles of inner blood-retinal barrier organic anion transporter 3 in the vitreous/retina-to-blood efflux transport of p-aminohippuric acid, benzylpenicillin, and 6-mercaptopurine. J. Pharmacol. Exp. Ther. 329, 87e93. Hosoya, K., Yamamoto, A., Akanuma, S., Tachikawa, M., 2010. Lipophilicity and transporter influence on blood-retinal barrier permeability: a comparison with blood-brain barrier permeability. Pharm. Res. 27, 2715e2724. Hosoya, K., Tomi, M., Tachikawa, M., 2011. Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier. Expert Opin. Drug Deliv. 8, 1571e1587. Igarashi, K., Kashiwagi, K., 2010. Characteristics of cellular polyamine transport in prokaryotes and eukaryotes. Plant Physiol. Biochem. 48, 506e512. Iseki, K., Kobayashi, M., Miyazaki, K., 1991. Spermine uptake by rat intestinal brushborder membrane vesicles. Biochim. Biophys. Acta 1068, 105e110. Kakinuma, Y., Hoshino, K., Igarashi, K., 1988. Characterization of the inducible polyamine transporter in bovine lymphocytes. Eur. J. Biochem. 176, 409e414. Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., Endou, H., 1998. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem. 273, 23629e23632. Kaneko, S., Ueda-Yamada, M., Ando, A., Matsumura, S., Okuda-Ashitaka, E., Matsumura, M., Uyama, M., Ito, S., 2007. Cytotoxic effect of spermine on retinal pigment epithelial cells. Investig. Ophthalmol. Vis. Sci. 48, 455e463. Katayama, K., Ohshima, Y., Tomi, M., Hosoya, K., 2006. Application of microdialysis to evaluate the efflux transport of estradiol 17-beta glucuronide across the rat blood-retinal barrier. J. Neurosci. Methods 156, 249e256. Kobayashi, M., Fujisaki, H., Sugawara, M., Iseki, K., Miyazaki, K., 1999. The presence of an Naþ/spermine antiporter in the rat renal brush-border membrane. J. Pharm. Pharmacol. 51, 279e284. Koepsell, H., 2013. The SLC22 family with transporters of organic cations, anions and zwitterions. Mol. Aspects Med. 34, 413e435. Kubo, Y., Kusagawa, Y., Tachikawa, M., Akanuma, S., Hosoya, K., 2013. Involvement of a novel organic cation transporter in verapamil transport across the inner blood-retinal barrier. Pharm. Res. 30, 847e856. Kumagai, A.K., Vinores, S.A., Pardridge, W.M., 1996. Pathological upregulation of inner blood-retinal barrier Glut1 glucose transporter expression in diabetes mellitus. Brain Res. 706, 313e317. LaMuraglia, G.M., Lacaine, F., Malt, R.A., 1986. High ornithine decarboxylase activity and polyamine levels in human colorectal neoplasia. Ann. Surg. 204, 89e93. Masuko, T., Kusama-Eguchi, K., Sakata, K., Kusama, T., Chaki, S., Okuyama, S., Williams, K., Kashiwagi, K., Igarashi, K., 2003. Polyamine transport, accumulation, and release in brain. J. Neurochem. 84, 610e617. Matsushita, K., Fukumoto, M., Kobayashi, T., Kobayashi, M., Ishizaki, E., Minami, M., Katsumura, K., Liao, S.D., Wu, D.M., Zhang, T., Puro, D.G., 2010. Diabetesinduced inhibition of voltage-dependent calcium channels in the retinal microvasculature: role of spermine. Investig. Ophthalmol. Vis. Sci. 51, 5979e 5990. Meyskens Jr., F.L., McLaren, C.E., Pelot, D., Fujikawa-Brooks, S., Carpenter, P.M., Hawk, E., Kelloff, G., Lawson, M.J., Kidao, J., McCracken, J., Albers, C.G., Ahnen, D.J., Turgeon, D.K., Goldschmid, S., Lance, P., Hagedorn, C.H., Gillen, D.L., Gerner, E.W., 2008. Difluoromethylornithine plus sulindac for the prevention of
7
sporadic colorectal adenomas: a randomized placebo-controlled, double-blind trial. Cancer Prev. Res. (Phila) 1, 32e38. Moinard, C., Cynober, L., de Bandt, J.P., 2005. Polyamines: metabolism and implications in human diseases. Clin. Nutr. 24, 184e197. Nagase, K., Tomi, M., Tachikawa, M., Hosoya, K., 2006. Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier. Biochim. Biophys. Acta 1758, 13e19. Nicoletti, R., Venza, I., Ceci, G., Visalli, M., Teti, D., Reibaldi, A., 2003. Vitreous polyamines spermidine, putrescine, and spermine in human proliferative disorders of the retina. Br. J. Ophthalmol. 87, 1038e1042. Noga, M.J., Dane, A., Shi, S., Attali, A., van Aken, H., Suidgeest, E., Tuinstra, T., Muilwijk, B., Coulier, L., Luider, T., Reijmers, T.H., Vreeken, R.J., Hankemeier, T., 2012. Metabolomics of cerebrospinal fluid reveals changes in the central nervous system metabolism in a rat model of multiple sclerosis. Metabolomics 8, 253e263. Ohkura, Y., Akanuma, S., Tachikawa, M., Hosoya, K., 2010. Blood-to-retina transport of biotin via Naþ-dependent multivitamin transporter (SMVT) at the inner blood-retinal barrier. Exp. Eye Res. 91, 387e392. Ohta, K., Inoue, K., Hayashi, Y., Yuasa, H., 2006. Molecular identification and functional characterization of rat multidrug and toxin extrusion type transporter 1 as an organic cation/Hþ antiporter in the kidney. Drug Metab. Dispos. 34, 1868e1874. Otsuka, M., Matsumoto, T., Morimoto, R., Arioka, S., Omote, H., Moriyama, Y., 2005. A human transporter protein that mediates the final excretion step for toxic organic cations. Proc. Natl. Acad. Sci. U. S. A. 102, 17923e17928. Pardridge, W.M., Fierer, G., 1985. Blood-brain barrier transport of butanol and water relative to N-isopropyl-p-iodoamphetamine as the internal reference. J. Cereb. Blood Flow Metab. 5, 275e281. Pegg, A.E., McCann, P.P., 1982. Polyamine metabolism and function. Am. J. Physiol. 243, C212eC221. Pineda, M., Fernández, E., Torrents, D., Estévez, R., López, C., Camps, M., Lloberas, J., Zorzano, A., Palacín, M., 1999. Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids. J. Biol. Chem. 274, 19738e19744. Quemener, V., Moulinoux, J.P., Havouis, R., Seiler, N., 1992. Polyamine deprivation enhances antitumoral efficacy of chemotherapy. Anticancer Res. 12, 1447e1453. Russell, D.H., Stern, L.Z., 1981. Altered polyamine excretion in Duchenne muscular dystrophy. Neurology 31, 80e83. Saito, A., Takagi, T., Chung, T.G., Ohta, K., 1983. Serum levels of polyamines in patients with chronic renal failure. Kidney Int. Suppl. 16, S234eS237. Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., Kanai, Y., 1999. Identification and functional characterization of a Naþ-independent neutral amino acid transporter with broad substrate selectivity. J. Biol. Chem. 274, 19745e19751. Seiler, N., 2004. Catabolism of polyamines. Amino Acids 26, 217e233. Stewart, P.A., Tuor, U.I., 1994. Blood-eye barriers in the rat: correlation of ultrastructure with function. J. Comp. Neurol. 340, 566e576. Sturman, J.A., Ingoglia, N.A., Lindquist, T.D., 1976. Interconversion of putrescine, spermidine and spermine in goldfish and rat retina. Life Sci. 19, 719e724. Swendseid, M.E., Panaqua, M., Kopple, J.D., 1980. Polyamine concentrations in red cells and urine of patients with chronic renal failure. Life Sci. 26, 533e539. Tabor, C.W., Tabor, H., 1976. 1,4-Diaminobutane (putrescine), spermidine, and spermine. Annu Rev. Biochem. 45, 285e306. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., Hirano, H., 1992. Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT1) in cells of the blood-retinal barrier in the rat. Investig. Ophthalmol. Vis. Sci. 33, 377e383. Tomi, M., Hosoya, K., 2010. The role of blood-ocular barrier transporters in retinal drug disposition: an overview. Expert. Opin. Drug Metab. Toxicol. 6, 1111e1124. Tomi, M., Mori, M., Tachikawa, M., Katayama, K., Terasaki, T., Hosoya, K., 2005. Ltype amino acid transporter 1-mediated L-leucine transport at the inner bloodretinal barrier. Investig. Ophthalmol. Vis. Sci. 46, 2522e2530. Tomi, M., Arai, K., Tachikawa, M., Hosoya, K., 2007a. Naþ-independent choline transport in rat retinal capillary endothelial cells. Neurochem. Res. 32, 1833e1842. Q9 Tomi, M., Terayama, T., Isobe, T., Egami, F., Morito, A., Kurachi, M., Ohtsuki, S., Kang, Y.S., Terasaki, T., Hosoya, K., 2007b. Function and regulation of taurine transport at the inner blood-retinal barrier. Microvasc. Res. 73, 100e106. Q10 Tomi, M., Kitade, N., Hirose, S., Yokota, N., Akanuma, S., Tachikawa, M., Hosoya, K., 2009. Cationic amino acid transporter 1-mediated L-arginine transport at the inner blood-retinal barrier. J. Neurochem. 111, 716e725. Usui, T., Kubo, Y., Akanuma, S., Hosoya, K., 2013. b-Alanine and l-histidine transport across the inner blood-retinal barrier: potential involvement in L-carnosine supply. Exp. Eye Res. 113, 135e142. Wallace, H.M., Caslake, R., 2001. Polyamines and colon cancer. Eur. J. Gastroenterol. Hepatol. 13, 1033e1039. Withrow, C., Ashraf, S., O’Leary, T., Johnson, L.R., Fitzgerald, M.E., Johnson, D.A., 2002. Effect of polyamine depletion on cone photoreceptors of the developing rabbit retina. Investig. Ophthalmol. Vis. Sci. 43, 3081e3090. Yamaoka, K., Tanigawara, Y., Nakagawa, T., Uno, T., 1981. A pharmacokinetic analysis program (multi) for microcomputer. J. Pharmacobiodyn. 4, 879e885. Yanagihara, N., Moriwaki, M., Shiraki, K., Miki, T., Otani, S., 1996. The involvement of polyamines in the proliferation of cultured retinal pigment epithelial cells. Investig. Ophthalmol. Vis. Sci. 37, 1975e1983. Yoneyama, D., Shinozaki, Y., Lu, W.L., Tomi, M., Tachikawa, M., Hosoya, K., 2010. Involvement of system A in the retina-to-blood transport of l-proline across the inner blood-retinal barrier. Exp. Eye Res. 90, 507e513.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
YEXER6426_proof ■ 16 May 2014 ■ 1/7
Experimental Eye Research xxx (2014) 1e7
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier Q12 Q1
Yoshiyuki Kubo 1, Ayaka Tomise 1, Ai Tsuchiyama, Shin-ichi Akanuma, Ken-ichi Hosoya* Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2013 Accepted in revised form 4 May 2014 Available online xxx
The elimination of spermine, the end product of cellular polyamine, from the retina to the blood across the blood-retinal barrier (BRB) was investigated. The in vivo microdialysis study revealed that the elimination of [3H]spermine from vitreous humor after vitreous bolus injection was in a biexponential manner. The rate constant for the elimination of [3H]spermine during the terminal phase was estimated to be 1.67-fold greater than that of [14C]D-mannitol, a bulk flow marker, and the difference in the terminal elimination rate constant between [3H]spermine and [14C]D-mannitol was reduced in the presence of 50 mM spermine, suggesting a retina-to-blood transport system for [3H]spermine across the BRB. The retina-to-blood transport of [3H]spermine was also supported by a study of the retinal uptake index (RUI). The in vitro transport study with TR-iBRB2 cells, a model cell line of the inner BRB, revealed time-, concentration- and temperature-dependent transport of [3H]spermine, suggesting the involvement of carrier-mediated processes in spermine transport across the inner BRB. The in vitro study also suggested that the transport of spermine at the inner BRB is pH-, membrane potential- and Cl-sensitive and Naþinsensitive, and these functional properties of spermine transport suggest only a minor contribution of spermine transporters, such as CCC9 (SLC12A8), the expression of which was suggested at the inner BRB. In the inhibition study, [3H]spermine transport was markedly inhibited by putrescine, spermidine, spermine and agmatine while substrates of a well-characterized organic cation transporter (OCTs/ SLC22A) and a cationic amino acid transporter (CATs/SLC7A) had no effect, suggesting the involvement of unknown transporters in spermine elimination from the retina. Ó 2014 Published by Elsevier Ltd.
Keywords: polyamine spermine transport blood-retinal barrier polyamine transporter
1. Introduction Polyamines, such as putrescine, spermidine and spermine, are known as bioactive amines that are ubiquitously distributed in the animal tissues (de Vera et al., 1995; Moinard et al., 2005). Polyamines have been suggested to play an important role in cellular proliferation and differentiation (Tabor and Tabor, 1976; Pegg and McCann, 1982), and alterations of polyamine concentration, in cells, blood and urine, have been reported in patients with diseases including cancer, psoriasis, multiple sclerosis, Duchenne muscular dystrophy and chronic renal failure (Wallace and Caslake, 2001; Noga et al., 2012; Russell and Stern, 1981; Swendseid et al., 1980; Saito et al., 1983). In cancer cells, the cellular concentration of polyamine was reported to be elevated by the activation of
* Corresponding author. Tel.: þ81 76 434 7505; fax: þ81 76 434 5172. E-mail address: [email protected] (K.-i. Hosoya). 1 Kubo Y and Tomise A contributed equally to this work.
ornithine decarboxylase that catalyzes the biosynthesis of putrescine (LaMuraglia et al., 1986; Cañizares et al., 1999), and the proliferation of cancer cells was reported to be suppressed in the presence of an ornithine decarboxylase inhibitor, such as adifluoromethylornithine (Quemener et al., 1992; Meyskens et al., 2008). In the retina associated with vision sense, the physiological significance of polyamines has been reported, and the loss of cone cells, photoreceptor cells, is known to be caused by polyamine deficiency in the developing rabbit retina (Withrow et al., 2002). In a study with multiple sclerosis model mice, the cell loss in the retinal ganglion cell layer has been shown to be inhibited by the oral administration of spermidine, suggesting the antioxidative effect of polyamines (Guo et al., 2011). In addition, the involvement of spermine was suggested in the retinal degeneration observed in gyrate atrophy of the choroid and retina since the excess amount of spermine induced cell apoptosis in a study involving the bovine retina (Kaneko et al., 2007). These reports suggest that the regulation of polyamine concentration is important in the retina. In the
http://dx.doi.org/10.1016/j.exer.2014.05.002 0014-4835/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Q2 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 2/7
2
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
cellular biosynthesis of polyamines, it is known that putrescine is produced from ornithine by ornithine decarboxylase (ODC), and putrescine is converted into spermidine by spermidine synthase (Sturman et al., 1976; Pegg and McCann, 1982; Seiler, 2004). ODC is one of the rate-limiting enzymes in biosynthesis of polyamine, and its cellular expression and activity were detectable in the retina (Yanagihara et al., 1996; Biedermann et al., 1998). In addition, the localization of ODC and spermine/spermidine in Müller cells was reported to suggest the role of spermine/spermidine in regulation of Kþ channels (Biedermann et al., 1998). Spermine synthase catalyzes the conversion of spermidine into spermine that is known to be the end product, suggesting the involvement of a spermine elimination system from the retina in the regulation of polyamine concentration (Sturman et al., 1976; Pegg and McCann, 1982; Seiler, 2004). In the retina, the blood-retinal barrier (BRB) separates the retinal tissue and the circulating blood, and the BRB includes two barrier structures, the inner and outer BRB, that are formed by the retinal capillary epithelial cells and retinal pigment epithelial (RPE) cells, respectively. In order to maintain the retinal function, membrane transporters expressed by the BRB contribute to the nutrient supply and endobiotic removal across the BRB since the retinal capillary epithelial cells and RPE cells form the tight junction to restrict the paracellular solute transport at the inner and outer BRB, respectively (Stewart and Tuor, 1994; Cunha-Vaz, 2004; Hosoya et al., 2011, 2012). In particular, it is known that the inner BRB is involved in nourishing two-thirds of the retina (Tomi and Hosoya, 2010), and previous reports about retinal capillary endothelial cells revealed the expression of various membrane transporters, such as cationic amino acid transporter 1 (CAT1/SLC7A1), neutral and basic amino acid transporter (yþLAT2/ SLC7A6), monocarboxylate transporter 1 (MCT1/Slc16a1), equilibrative nucleoside transporter 2 (ENT2/SLC29A2), L (leucinereferring)-type amino acid transporter (LAT1/SLC7A5), taurine transporter (TAUT/SLC6A6) and glucose transporter (GLUT1/ SLC2A1) (Tomi et al., 2009; Hosoya et al., 2001a; Nagase et al., 2006; Tomi et al., 2005, 2007a, 2007b; Usui et al., 2013; Takata et al., 1992; Kumagai et al., 1996). Regarding polyamine transport, polyamine transporters have been identified in various organisms, including prokaryotes and eukaryotes (Igarashi and Kashiwagi, 2010). In mammals, several transport systems for polyamines have been reported in tissues, such as lymphocytes, intestine, kidney and brain (Kakinuma et al., 1988; Iseki et al., 1991; Kobayashi et al., 1999; Masuko et al., 2003), and organic cation transporter 1 (OCT1/SLC22A1), L-carnitine transporter 2 (CT2/SLC22A16) and the splice variant of cation-chloride cotransporter 9 (CCC9/SLC12A8) have been reported to recognize spermine as a substrate (Busch et al., 1996; Aouida et al., 2010; Daigle et al., 2009). However, little is known about the mechanism for the elimination of spermine from the retina to the circulating blood, and any clarification of spermine elimination will be beneficial in maintenance of the retina and the clinical treatment of retinal diseases, such as diabetic retinopathy and gyrate atrophy of choroid and retina (Matsushita et al., 2010; Nicoletti et al., 2003; Kaneko et al., 2007). In this study, in vivo and in vitro studies were performed to investigate the elimination of spermine from the retina to the circulating blood. In the in vivo study, the blood-to-retina and retina-to-blood transports of spermine were analyzed by means of the retinal uptake index (RUI) and microdialysis, respectively. In the in vitro study, spermine transport was analyzed in a conditionally immortalized cell line of retinal capillary endothelial cells (TRiBRB2 cells), an in vitro model cell line of the inner BRB (Hosoya et al., 2001a, 2001b).
2. Materials and methods 2.1. Animals Male Wistar rats (160e300 g) were purchased from Japan SLC (Hamamatsu, Japan). The use of experimental animals followed the guidelines instituted by the Animal Care Committee in the University of Toyama and by the Association for Research in vision and Ophthalmology (ARVO) Statement. 2.2. Reagents Chemicals were of reagent grade and available commercially, and [1-14C]D-mannitol ([14C]D-mannitol, 55 mCi/mmol) and [1-14C] n-butanol ([14C]n-butanol, 2 mCi/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). [Terminal methylene-3H]spermine ([3H]spermine, 50 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). 2.3. Microdialysis study A microdialysis study was performed as described elsewhere (Katayama et al., 2006; Hosoya et al., 2009; Yoneyama et al., 2010; Akanuma et al., 2013). In brief, after anesthetizing rats (250e300 g) with sodium pentobarbital (60 mg/kg), their heads were placed on a stereotaxic frame (Narishige, Tokyo, Japan). To prevent eye blinking, 2% xylocaine was instilled to locally anesthetize their eyelids, and a 25G needle was inserted through the pars plana at a depth of 3.0 mm. After removal of the needle, 1 mL Ringer-HEPES solution (141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 10 mM HEPES, pH 7.4) containing [3H]spermine (2.0 mCi) and [14C]D-mannitol (0.2 mCi) were administered by means of a microsyringe (Hamilton, Reno, NE) at a depth of 3.0 mm from the eye surface. Immediately after, a microdialysis probe (TEP-50; Eicom, Kyoto, Japan) was implanted into the vitreous chamber, and the probe was fixed on the eye surface by means of surgical glue (Daiichi-Sankyo, Tokyo, Japan). To continuously supply Ringer-HEPES solution to the probe (2 mL/min, 37 C), an infusion pump (Harvard, Holliston, MA) and polyethylene tubing (Natsume, Tokyo, Japan) were used. The radioactivity in the dialyzate collected at designated times was determined with a liquid scintillation counter (LSC-5200, Aloka, Tokyo, Japan). Data analysis was performed as described previously (Yoneyama et al., 2010; Akanuma et al., 2013), and the data obtained were used in the calculation of CP, the vitreous concentrations normalized by the injected dose (% of dose/mL), by means of Eq. (1), where Dosetracer and CT are the total radioactivity in the solution after injection (dpm) and the concentration in the dialyzate (dpm/mL), respectively.
CP ¼ CT =Dosetracer 100
(1)
CP(t) defined as the CP at time t, was subjected to the nonlinear least-square regression analysis program, MULTI (Yamaoka et al., 1981), and was fitted to Eq. (2), where a and b are the apparent first-order rate constants for the initial and terminal phase, respectively, and A and B are intercepts on the y-axis for each exponential segment.
CP ðtÞ ¼ A eat þ B ebt
(2)
The probe recovery was estimated from Eq. (3), and CV (dpm/ mL) is the concentration in the test solution. In the present study, the recovery values were constant over 180 min, and the value for [3H]spermine and [14C]D-mannitol were 5.22% and 8.69%, respectively.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 3/7
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
Recovery ð%Þ ¼ CT =CV 100
(3)
In the inhibition study, Ringer-HEPES solution containing unlabeled compounds was supplied to the probe. The inhibitory effect on [3H]spermine elimination from the vitreous humor was evaluated from the difference in b values between [3H]spermine and [14C]D-mannitol (Db), and the effect was expressed as a percentage of the control estimated from Eq. (4).
ð% of controlÞ ¼ ðDb in the presence of inhibitorÞ= ðDb in the absence of inhibitorÞ 100
(4)
2.4. In vivo study of blood-to-retina transport of [3H]spermine The carotid artery injection method in rats was adopted to study the permeability of spermine to the retina as described previously (Hosoya et al., 2010; Alm and Törnquist, 1981; Pardridge and Fierer, 1985). In brief, a test solution was prepared by adding [3H]spermine (5.0 mCi, 0.5 mM) and a diffusible internal reference [14C]n-butanol (0.5 mCi) to 200 mL Ringer-HEPES buffer (pH 7.4). The test solution was injected into the common carotid artery of anesthetized rats (160e180 g), and decapitation was performed 15 s after injection. The retinas were collected to be lysed and neutralized with 2 N sodium hydroxide and hydrochloric acid, and the radioactivity in samples was measured by liquid scintillation counting. As described previously (Hosoya et al., 2010; Kubo et al., 2013), to express the distribution characteristics of compounds, the retinal uptake index (RUI) was used and was estimated from Eq. (5).
RUI ð%Þ ¼ ð½3 H=½14 Cðdpm in the retinaÞÞ=ð½3 H=½14 C ðdpm in the injectate solutionÞÞ 100
(5)
2.5. Study of [3H]spermine transport in TR-iBRB2 cells The [3H]spermine uptake by TR-iBRB2 cells, a conditionally immortalized rat capillary endothelial cell line, was investigated as described previously (Tomi et al., 2009; Usui et al., 2013; Kubo et al., 2013). In brief, TR-iBRB2 cells (passage number 23e35) were maintained in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceuticals, Tokyo, Japan) with 10% fetal bovine serum, 20 mM NaHCO3, and antibiotics at 33 C (Hosoya et al., 2001a, 2001b). An uptake study was performed by means of extracellular fluid (ECF)buffer (122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, 10 mM D-glucose and 10 mM HEPES (pH 7.4)). The cells to be cultured were rinsed with ECFbuffer, and incubated with 10 nM [3H]spermine (0.1 mCi) in 200 mL ECF-buffer at 37 C. The ion-dependence of [3H]spermine uptake by TR-iBRB2 cells was examined in Naþ-free ECF buffer as described previously (Ohkura et al., 2010; Kubo et al., 2013). Uptake was terminated at the designated time, and the cells were lysed and neutralized in 1 N sodium hydroxide and hydrochloric acid. The cellular radioactivity and protein content were determined by means of liquid scintillation counting and a DC protein assay kit (Bio-Rad, Hercules, CA), respectively. As described elsewhere (Hosoya et al., 2001b; Usui et al., 2013), the cellular uptake of [3H] spermine was expressed as the cell-to-medium (cell/medium) ratio calculated from Eq. (6).
Cell=medium ratio ¼ ð½3 H dpm per cell protein ðmgÞÞ= ð½3 H dpm per mL mediumÞ
(6)
In the estimation of the kinetic parameters for cellular spermine uptake by means of MULTI (Yamaoka et al., 1981), the data obtained were fitted to a two saturable processes model (Eq. (7)), where V,
3
[S], Vmax1, Vmax2, Km1 and Km2 are the uptake rate of spermine, the concentration of spermine in ECF-buffer, the maximum uptake rate for the high-affinity process, the maximum uptake rate for the lowaffinity process, the MichaeliseMenten constant for the highaffinity process and the MichaeliseMenten constant for the lowaffinity process, respectively.
V ¼ Vmax1 ½S=ðKm1 þ ½SÞ þ Vmax2 ½S=ðKm2 þ ½SÞ
(7)
2.6. Expression analysis by reverse transcription polymerase chain reaction (RT-PCR) The investigation of the transcript expression of CT2 and CCC9 was performed as described previously (Tomi et al., 2007a, 2007b; Yoneyama et al., 2010). In brief, TRIzol reagent (Invitrogen, Carlsbad, CA) and an RNeasy Mini kit (Qiagen, Hilden, Germany) were used in the preparation of total RNA from TR-iBRB2 cells and primary-cultured RPE cells (Babu et al., 2011; Usui et al., 2013), and 1 mg total RNA was subjected to single-strand cDNA synthesis by means of reverse transcription (RT) and oligo dT primer. GeneAmp PCR system 9700 (Applied Biosystems, Foster, CA) was used in the polymerase chain reaction for rat CT2 through 35 cycles at 94 C for 30 s, 47 C for 30 s, and 72 C for 30 s, and for rat CCC9 through 35 cycles at 94 C for 30 s, 61 C for 30 s, and 72 C for 30 s. Primers for rat CT2 were designed by referring to mouse CT2 (GenBank ID: NM_027572), and the sense and antisense sequences were 50 AGTCCTTGCTTCGATGGCTA-30 and 50 -AGCGTGTGATCATGAAGCTG30 , respectively. Primers for rat CCC9 were designed by referring to rat CCC9 (SLC12A8, GenBank ID: NM_153625), and the sense and antisense sequences were 50 -CGAGAGGCCCTCCGCTCTGA-30 and 50 TCCGGGCCTTGTCTGGGCAT-30 , respectively. Agarose gel electrophoresis with ethidium bromide was performed to separate and visualize the PCR products under ultraviolet light, and the resultant products were confirmed by sequence analysis using a DNA sequencer (ABI PRISM 3130; Applied Biosystems). 2.7. Statistical analysis The kinetic parameters are represented as the mean S.D., and others are shown as the mean S.E.M. An unpaired Student’s t-test was performed in the statistical analysis of two groups, and oneway analysis of variance followed by Dunnett’s test was performed in the analysis of several groups. 3. Results 3.1. Retina-to-blood transport of [3H]spermine from the vitreous humor The time-profile of the [3H]spermine remaining in the vitreous humor after bolus injection was examined (Fig. 1A), and [3H]spermine exhibited a biexponential elimination from the vitreous humor with an apparent elimination constant (b) during terminal phase of 15.3 103 1.03 103 min1 (Fig. 1B). [14C]D-Mannitol, co-injected as a bulk flow marker, also exhibited a biexponential elimination from the vitreous humor with an apparent elimination constant (b) during terminal phase of 9.14 103 0.70 103 min1, and the b value of [3H]spermine was 1.67-fold greater than that of [14C]D-mannitol (Fig. 1B), showing that the decline in [3H] spermine was significantly greater than that of [14C]D-mannitol. In the inhibition study, unlabeled spermine showed a significant reduction in the b value difference between [3H]spermine and [14C] D-mannitol while no significant effect was observed in the presence of tetraethylammonium (TEA) (Table 1). These results suggest the
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 4/7
4
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
Fig. 1. Time-profile of [3H]spermine and [14C]D-mannitol in the vitreous humor after vitreous bolus injection (A) and elimination rate constants (b) of [3H]spermine and [14C]Dmannitol in the terminal phase (B). Open circles and triangles represent the concentration in the dialyzate of [14C]D-mannitol and [3H]spermine, respectively. Each point and column represents the mean S.E.M. (n ¼ 3). *p < 0.01, significantly different from [14C]D-mannitol.
involvement of the retina-to-blood carrier-mediated transport process at the BRB in spermine elimination from the retina. 3.2. Blood-to-retina transport of [3H]spermine To examine the in vivo blood-to-retina transport of spermine at the BRB, the retinal uptake index (RUI) was estimated using a carotid artery single injection in rats. In Table 2, the RUI value of [3H] spermine was shown to be 42.5%, and no significant effect on the value (43.2%) was observed in the presence of 50 mM spermine, suggesting that the blood-to-retina carrier-mediated transport of spermine is minor at the BRB. 3
3.3. Uptake of [ H]spermine by TR-iBRB2 cells In order to investigate the transport of spermine at the inner BRB, the uptake study with TR-iBRB2 cells, an in vitro model cell line, was performed. As seen from the results, [3H]spermine uptake by TR-iBRB2 cells exhibited a time-dependent increase for 10 min at least, with an initial uptake rate of 24.9 4.2 mL/(min mg protein) (Fig. 2A). TR-iBRB2 cells showed a temperature-dependent uptake of [3H]spermine, exhibiting a [3H]spermine uptake significantly reduced by 90.0% at 4 C, (Fig. 2A). In the study of pH-dependence, [3H]spermine transport in TR-iBRB2 cells was significantly increased and decreased at pH 6.4 and 8.4, respectively, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a proton uncoupler, canceled the increased uptake at pH 6.4 (Fig. 2B). In the study of ion-dependence, the transport of [3H]spermine was significantly reduced in Cl-free buffer and Kþ-replacement buffer by 20.5% and 64.8%, respectively, and no alteration was observed in Naþ-free buffer (Fig. 2C). In the study of spermine transport over Table 1 Inhibitory effect on the elimination rate constant (b) difference between [3H]spermine and [14C]D-mannitol during the terminal phase. % of control Control Spermine TEA
100 18 26.6 6.4* 91.7 23.2
Each inhibitor was perfused in the microdialysis probe. Inhibitory effect was examined in the presence of spermine (50 mM) and TEA (50 mM). Each value represents the mean S.E.M. (n ¼ 4e5). *p < 0.01, significantly different from the control. TEA, tetraethylammonium.
the concentration range of 1e200 mM (Fig. 3), the transport of [3H] spermine was shown to be concentration-dependent, exhibiting a Km1 of 0.968 0.369 mM, a Vmax1 of 42.7 7.3 pmol/ (min mg protein), a Km2 of 158 67 mM and a Vmax2 of 382 88 pmol/(min mg protein). These results suggest the involvement of pH-, Cl- and a membrane potential-dependent carrier-mediated transport systems in the [3H]spermine transport in TR-iBRB2 cells. 3.4. Expression of spermine transport molecules To investigate the expression of rat CT2 (SLC22A16) and rat CCC9 (SLC12A8) at the BRB, RT-PCR analysis was performed. The results obtained showed that the PCR product for CT2 (271 bp) was not detected in TR-iBRB2 cells and RPE cells, suggesting that the expression of CT2 is minor at the inner and outer BRB (Fig. 4A). On the other hand, the PCR product for CCC9 (401 bp) was detected in TR-iBRB2 cells and RPE cells (Fig. 4B), suggesting the expression of CCC9 at the inner and outer BRB. 3.5. Inhibitory effect on the transport of [3H]spermine in TR-iBRB2 cells The inhibition study of [3H]spermine transport in TR-iBRB2 cells was performed to examine the transport characteristics of spermine at the inner BRB. As the results, in the presence of spermine, spermidine, putrescine and agmatine, [3H]spermine transport was strongly reduced by more than 87% (Table 3). Tetraethylammonium (TEA) exhibited a significant but weak inhibitory effect on [3H] spermine transport by 18% while no significant inhibition was observed in the presence of 1-methyl-4-phenylpyridinium (MPPþ), serotonin, L-carnitine, L-histidine, choline, L-arginine, cimetidine and L-ornithine (Table 3).
Table 2 In vivo [3H]spermine uptake by rat retina. RUI (%) Control Spermine
42.5 1.7 43.2 2.6
[3H]Spermine (5.0 mCi/rat) and [14C]n-butanol (0.5 mCi/rat) were injected into the common carotid artery in the absence (control) or presence of spermine (50 mM). Each value represents the mean S.E.M. (n ¼ 3e4).
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 5/7
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
5
Fig. 3. Concentration-dependent uptake of spermine. The uptake of [3H]spermine (0.1 mCi, 10 nM) was examined at 37 C for 5 min, over the concentration range 1e 200 mM. The data obtained were analyzed by means of MichaeliseMenten (main graph) and EadieeScatchard (inset graph) analyses. Each point represents the mean S.E.M (n ¼ 3).
the elimination system for spermine across the BRB is thought to have a significant influence in the polyamine concentration in the retina. Therefore, in the present study, in vivo and in vitro analyses were performed to investigate the mechanism for the retina-toblood transport of spermine at the inner BRB that nourishes twothirds of the retina (Tomi and Hosoya, 2010). In the in vivo analysis, microdialysis was performed to study the retina-to-blood transport of [3H]spermine at the BRB using [14C]Dmannitol as a bulk flow marker for passage from the vitreous humor to Schlemm’s canal and/or the uveoscleral outflow route. The concentrationetime profile for [3H]spermine and [14C]D-mannitol in the dialyzate showed that their concentration in the vitreous humor was reduced in a biexponetial manner, and [3H]spermine and [14C]D-mannitol exhibited a steeper initial slope (a) than the later slope (b), supporting the hypothesis that the initial and later declines reflect diffusion into the vitreous humor, including the microdialysis tube, after vitreous bolus injection and elimination from the vitreous humor of both [3H]spermine and [14C]D-mannitol (Fig. 1A). In the comparison of the b values, [3H]spermine exhibited a 1.67-fold greater b value than that of [14C]D-mannitol (Fig. 1B), suggesting that [3H]spermine was subjected to retina-to-blood transport at the BRB in addition to its elimination from the vitreous humor via bulk flow and passive diffusion. In the inhibition study, the results suggest the involvement of a spermine-sensitive and carrier-mediated transport system in the retina-to-blood transport of spermine at the BRB since unlabeled spermine significantly reduced the difference in the b value between [3H]spermine
Fig. 2. Uptake of [3H]spermine by TR-iBRB2 cells. (A) Time-dependent uptake of [3H] spermine (0.1 mCi, 10 nM) was investigated at 37 C (closed circles), and temperaturedependence was examined at 4 C (open circle). Extracellular pH-dependence (B) and Naþ-, Cl- and membrane potential-dependence (C) of [3H]spermine uptake was performed at 37 C for 5 min. Extracellular pH-dependence was examined with or without 50 mM FCCP. Each point and column represents the mean S.E.M (n ¼ 3). *p < 0.01, significantly different from the control.
4. Discussion Polyamines are cationic compounds that are ubiquitously distributed in tissues, and their involvement has been reported in the maintenance of retinal function and the pathogenesis of severe retinal diseases, such as gyrate atrophy of choroid and retina (Withrow et al., 2002; Kaneko et al., 2007; Guo et al., 2011), showing that the regulation of polyamine concentration is important in the maintenance of a healthy retina. In particular, spermine is the end product in the cellular biosynthesis of polyamines, and
Fig. 4. Expression of rat CT2 (SLC22A16) and rat CCC9 (SLC12A8). Expression of CT2 (A) and CCC9 (B) was analyzed by RT-PCR. (þ) and () represent PCR, with and without reverse transcription, respectively.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 6/7
6
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7 Table 3 Inhibitory effect on [3H]spermine uptake by TR-iBRB2 cells. % of control Control Putrescine Spermidine Spermine Agmatine TEA MPPþ Serotonin L-Carnitine Choline (5 mM) Cimetidine L-Arginine L-Ornithine L-Histidine
100 11.8 3.61 3.27 12.3 81.2 91.0 93.7 101 109 119 115 119 103
2 0.5* 0.19* 0.18* 0.36* 3.7* 1.2 4.1 3 3 0.6 5 6 4
The uptake of [3H]spermine (10 nM, 0.1 mCi) by TR-iBRB2 cells was examined in the absence (control) or presence of 1 mM compounds at 37 C for 5 min. Choline was used at a concentration of 5 mM. Each value represents the means S.E.M. (n ¼ 3e15). *p < 0.01, significantly different from the control. TEA, tetraethylammonium; MPPþ, 1methyl-1-phynylpyridinium.
and [14C]D-mannitol in spite of no significant effect being exhibited by TEA (Table 1). The study with a carotid single injection suggested the minor contribution of the blood-to-retina carrier-mediated transport of spermine at the BRB because unlabeled spermine had no significant effect on the RUI value of [3H]spermine (Table 2). The in vivo studies suggest the involvement of the retina-toblood carrier-mediated transport process in the elimination of spermine from the retina, and the in vitro transport study with TRiBRB2 cells, an in vitro model of the inner BRB (Hosoya et al., 2001a, 2001b), was used in order to investigate the functional properties of spermine transport. From the results, the involvement of carriermediate transport processes were suggested in spermine transport at the inner BRB since the transport of [3H]spermine occurred in a time-, concentration-, and temperature-dependent manner, with high-affinity (Km1 ¼ 0.968 mM) and low-affinity (Km2 ¼ 158 mM) processes (Figs. 2A and 3). In the study of ion coupling, the pH-sensitivity of spermine transport was suggested at the inner BRB since the transport of [3H]spermine in TR-iBRB2 cells was significantly altered under acidic and alkaline extracellular pH conditions and a proton uncoupler showed the cancelation of the enhanced transport observed at acidic extracellular pH (Fig. 2B). The significant decrease in [3H]spermine transport, in the Kþ- and Cl-free buffers, suggested the membrane potential- and Cl-sensitivities of spermine transport at the inner BRB (Fig. 2C). The study also suggests that spermine transport is Naþ-insensitive since the transport of [3H]spermine in TR-iBRB2 cells exhibited no significant change in the Naþ-free buffer (Fig. 2C). These results suggest the involvement of pH-, membrane potential- and Cl-sensitive carrier-mediated processes in the retina-to-blood transport of spermine at the inner BRB. Recent research on transporters suggests that OCT1 (SLC22A1), CT2 (SLC22A16) and the splice variant of CCC9 (SLC12A8) recognize spermine (Busch et al., 1996; Aouida et al., 2010; Daigle et al., 2009), and the undetectable expression of OCT1 at the inner BRB has been reported previously (Tomi et al., 2007a, 2007b). The detectable and undetectable expressions of CCC9 and CT2 in TR-iBRB2 cells suggest the possible contribution of CCC9 at the inner BRB (Fig. 4). However, TR-iBRB2 cells exhibited the Cl-sensitity of [3H]spermine transport to suggest that CCC9 is not responsible for spermine transport at the inner BRB because the Cl-insensitivity of CCC9 was previously reported although CCC9 is a member of the cation-coupled chloride cotransporters family (Daigle et al., 2009).
In the inhibition study, the results obtained suggest the involvement of a polyamine transporter in the retina-to-blood transport of spermine since the transport of [3H]spermine in TRiBRB2 cells was markedly inhibited by polyamines, such as putrescine, spermidine, spermine and agmatine (Table 3). Although TEA exhibited a slight effect on the transport of [3H]spermine in TRiBRB2 cells (Table 3), the contribution of organic cation transporters is thought to be minor at the inner BRB because no significant decrease in the [3H]spermine transport was shown by MPPþ, serotonin, L-carnitine, choline and cimetidine (Table 3), which are typical substrates of identified organic cation transporters (Otsuka et al., 2005; Engel and Wang, 2005; Ohta et al., 2006; Koepsell, 2013). In addition, no significant effect on the transport of [3H] spermine was exhibited by L-arginine, L-ornithine and L-histidine (Table 3), which are known as substrates of SLC7A family members, such as LAT1 (SLC7A5), CATs (SLC7A1-4) and yþLAT2 (SLC7A6) (Kanai et al., 1998; Segawa et al., 1999; Pineda et al., 1999; Tomi et al., 2009), and this suggests that the well-characterized members of SLC7A family are not responsible for the retina-to-blood transport of spermine, implying the involvement of polyaminespecific but unknown transporters at the inner BRB. In conclusion, the present analyses suggest the involvement of carrier-mediated transport processes in the elimination of spermine, the end product of polyamine biosynthesis, across the inner BRB. The inhibition study suggests the minor contribution of wellidentified transporters to the retina-to-blood transport of spermine, and show that unknown transporters contribute to spermine elimination from the retina. The present findings will be helpful to improve our understanding of the regulation of spermine concentration in the retina, and for the maintenance of a healthy retina. Conflict of interest The authors declare that they have no conflict of interest. Uncited references Hayasaka et al., 2011; Hosoya and Tomi, 2005.
Q11
Acknowledgments This study was supported, in part, by a Grant-in Aid for Scientific Q3,4 Research from the Ministry of Education, Science, Sports, and Cul- Q5,6 ture, the Japan Society for the Promotion of Science, and The Nakatomi Foundation, Japan. Q7 References Akanuma, S., Soutome, T., Hisada, E., Tachikawa, M., Kubo, Y., Hosoya, K., 2013. Naþindependent nucleoside transporters regulate adenosine and hypoxanthine levels in Müller cells and the inner blood-retinal barrier. Investig. Ophthalmol. Vis. Sci. 54, 1469e1477. Alm, A., Törnquist, P., 1981. The uptake index method applied to studies on the blood-retinal barrier. I. A methodological study. Acta Physiol. Scand. 113, 73e79. Aouida, M., Poulin, R., Ramotar, D., 2010. The human carnitine transporter SLC22A16 mediates high affinity uptake of the anticancer polyamine analogue bleomycinA5. J. Biol. Chem. 285, 6275e6284. Babu, E., Ananth, S., Veeranan-Karmegam, R., Coothankandaswamy, V., Smith, S.B., Boettger, T., Ganapathy, V., Martin, P.M., 2011. Transport via SLC5A8 (SMCT1) is obligatory for 2-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells. Investig. Ophthalmol. Vis. Sci. 52, 5749e5757. Biedermann, B., Skatchkov, S.N., Brunk, I., Bringmann, A., Pannicke, T., Bernstein, H.G., Faude, F., Germer, A., Veh, R., Reichenbach, A., 1998. Spermine/ spermidine is expressed by retinal glial (Müller) cells and controls distinct Kþ channels of their membrane. Glia 23, 209e220. Busch, A.E., Quester, S., Ulzheimer, J.C., Waldegger, S., Gorboulev, V., Arndt, P., Lang, F., Koepsell, H., 1996. Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1. J. Biol. Chem. 271, 32599e32604.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
YEXER6426_proof ■ 16 May 2014 ■ 7/7
Y. Kubo et al. / Experimental Eye Research xxx (2014) 1e7
Q8
Cañizares, F., Salinas, J., de las Heras, M., Diaz, J., Tovar, I., Martinez, P., Peñafiel, R., 1999. Prognostic value of ornithine decarboxylase and polyamines in human breast cancer: correlation with clinicopathologic parameters. Clin. Cancer Res. 5, 2035e2041. Cunha-Vaz, J.G., 2004. The blood-retinal barriers system. Basic concepts and clinical evaluation. Exp. Eye Res. 78, 715e721. Daigle, N.D., Carpentier, G.A., Frenette-Cotton, R., Simard, M.G., Lefoll, M.H., Noël, M., Caron, L., Noël, J., Isenring, P., 2009. Molecular characterization of a human cation-Cl- cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport. J. Cell Physiol. 220, 680e689. de Vera, N., Camón, L., Martínez, E., 1995. Blood polyamines in the rat. J. Pharm. Pharmacol. 47, 204e205. Engel, K., Wang, J., 2005. Interaction of organic cations with a newly identified plasma membrane monoamine transporter. Mol. Pharmacol. 68, 1397e1407. Guo, X., Harada, C., Namekata, K., Kimura, A., Mitamura, Y., Yoshida, H., Matsumoto, Y., Harada, T., 2011. Spermidine alleviates severity of murine experimental autoimmune encephalomyelitis. Investig. Ophthalmol. Vis. Sci. 52, 2696e2703. Hayasaka, S., Kodama, T., Ohira, A., 2011. Retinal risks of high-dose ornithine supplements: a review. Br. J. Nutr. 106, 801e811. Hosoya, K., Tachikawa, M., 2012. The inner blood-retinal barrier: molecular structure and transport biology. Adv. Exp. Med. Biol. 763, 85e104. Hosoya, K., Tomi, M., 2005. Advances in the cell biology of transport via the inner blood-retinal barrier: establishment of cell lines and transport functions. Biol. Pharm. Bull. 28, 1e8. Hosoya, K., Kondo, T., Tomi, M., Takanaga, H., Ohtsuki, S., Terasaki, T., 2001a. MCT1mediated transport of L-lactic acid at the inner blood-retinal barrier: a possible route for delivery of monocarboxylic acid drugs to the retina. Pharm. Res. 18, 1669e1676. Hosoya, K., Tomi, M., Ohtsuki, S., Takanaga, H., Ueda, M., Yanai, N., Obinata, M., Terasaki, T., 2001b. Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat. Exp. Eye Res. 72, 163e172. Hosoya, K., Makihara, A., Tsujikawa, Y., Yoneyama, D., Mori, S., Terasaki, T., Akanuma, S., Tomi, M., Tachikawa, M., 2009. Roles of inner blood-retinal barrier organic anion transporter 3 in the vitreous/retina-to-blood efflux transport of p-aminohippuric acid, benzylpenicillin, and 6-mercaptopurine. J. Pharmacol. Exp. Ther. 329, 87e93. Hosoya, K., Yamamoto, A., Akanuma, S., Tachikawa, M., 2010. Lipophilicity and transporter influence on blood-retinal barrier permeability: a comparison with blood-brain barrier permeability. Pharm. Res. 27, 2715e2724. Hosoya, K., Tomi, M., Tachikawa, M., 2011. Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier. Expert Opin. Drug Deliv. 8, 1571e1587. Igarashi, K., Kashiwagi, K., 2010. Characteristics of cellular polyamine transport in prokaryotes and eukaryotes. Plant Physiol. Biochem. 48, 506e512. Iseki, K., Kobayashi, M., Miyazaki, K., 1991. Spermine uptake by rat intestinal brushborder membrane vesicles. Biochim. Biophys. Acta 1068, 105e110. Kakinuma, Y., Hoshino, K., Igarashi, K., 1988. Characterization of the inducible polyamine transporter in bovine lymphocytes. Eur. J. Biochem. 176, 409e414. Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., Endou, H., 1998. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem. 273, 23629e23632. Kaneko, S., Ueda-Yamada, M., Ando, A., Matsumura, S., Okuda-Ashitaka, E., Matsumura, M., Uyama, M., Ito, S., 2007. Cytotoxic effect of spermine on retinal pigment epithelial cells. Investig. Ophthalmol. Vis. Sci. 48, 455e463. Katayama, K., Ohshima, Y., Tomi, M., Hosoya, K., 2006. Application of microdialysis to evaluate the efflux transport of estradiol 17-beta glucuronide across the rat blood-retinal barrier. J. Neurosci. Methods 156, 249e256. Kobayashi, M., Fujisaki, H., Sugawara, M., Iseki, K., Miyazaki, K., 1999. The presence of an Naþ/spermine antiporter in the rat renal brush-border membrane. J. Pharm. Pharmacol. 51, 279e284. Koepsell, H., 2013. The SLC22 family with transporters of organic cations, anions and zwitterions. Mol. Aspects Med. 34, 413e435. Kubo, Y., Kusagawa, Y., Tachikawa, M., Akanuma, S., Hosoya, K., 2013. Involvement of a novel organic cation transporter in verapamil transport across the inner blood-retinal barrier. Pharm. Res. 30, 847e856. Kumagai, A.K., Vinores, S.A., Pardridge, W.M., 1996. Pathological upregulation of inner blood-retinal barrier Glut1 glucose transporter expression in diabetes mellitus. Brain Res. 706, 313e317. LaMuraglia, G.M., Lacaine, F., Malt, R.A., 1986. High ornithine decarboxylase activity and polyamine levels in human colorectal neoplasia. Ann. Surg. 204, 89e93. Masuko, T., Kusama-Eguchi, K., Sakata, K., Kusama, T., Chaki, S., Okuyama, S., Williams, K., Kashiwagi, K., Igarashi, K., 2003. Polyamine transport, accumulation, and release in brain. J. Neurochem. 84, 610e617. Matsushita, K., Fukumoto, M., Kobayashi, T., Kobayashi, M., Ishizaki, E., Minami, M., Katsumura, K., Liao, S.D., Wu, D.M., Zhang, T., Puro, D.G., 2010. Diabetesinduced inhibition of voltage-dependent calcium channels in the retinal microvasculature: role of spermine. Investig. Ophthalmol. Vis. Sci. 51, 5979e 5990. Meyskens Jr., F.L., McLaren, C.E., Pelot, D., Fujikawa-Brooks, S., Carpenter, P.M., Hawk, E., Kelloff, G., Lawson, M.J., Kidao, J., McCracken, J., Albers, C.G., Ahnen, D.J., Turgeon, D.K., Goldschmid, S., Lance, P., Hagedorn, C.H., Gillen, D.L., Gerner, E.W., 2008. Difluoromethylornithine plus sulindac for the prevention of
7
sporadic colorectal adenomas: a randomized placebo-controlled, double-blind trial. Cancer Prev. Res. (Phila) 1, 32e38. Moinard, C., Cynober, L., de Bandt, J.P., 2005. Polyamines: metabolism and implications in human diseases. Clin. Nutr. 24, 184e197. Nagase, K., Tomi, M., Tachikawa, M., Hosoya, K., 2006. Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier. Biochim. Biophys. Acta 1758, 13e19. Nicoletti, R., Venza, I., Ceci, G., Visalli, M., Teti, D., Reibaldi, A., 2003. Vitreous polyamines spermidine, putrescine, and spermine in human proliferative disorders of the retina. Br. J. Ophthalmol. 87, 1038e1042. Noga, M.J., Dane, A., Shi, S., Attali, A., van Aken, H., Suidgeest, E., Tuinstra, T., Muilwijk, B., Coulier, L., Luider, T., Reijmers, T.H., Vreeken, R.J., Hankemeier, T., 2012. Metabolomics of cerebrospinal fluid reveals changes in the central nervous system metabolism in a rat model of multiple sclerosis. Metabolomics 8, 253e263. Ohkura, Y., Akanuma, S., Tachikawa, M., Hosoya, K., 2010. Blood-to-retina transport of biotin via Naþ-dependent multivitamin transporter (SMVT) at the inner blood-retinal barrier. Exp. Eye Res. 91, 387e392. Ohta, K., Inoue, K., Hayashi, Y., Yuasa, H., 2006. Molecular identification and functional characterization of rat multidrug and toxin extrusion type transporter 1 as an organic cation/Hþ antiporter in the kidney. Drug Metab. Dispos. 34, 1868e1874. Otsuka, M., Matsumoto, T., Morimoto, R., Arioka, S., Omote, H., Moriyama, Y., 2005. A human transporter protein that mediates the final excretion step for toxic organic cations. Proc. Natl. Acad. Sci. U. S. A. 102, 17923e17928. Pardridge, W.M., Fierer, G., 1985. Blood-brain barrier transport of butanol and water relative to N-isopropyl-p-iodoamphetamine as the internal reference. J. Cereb. Blood Flow Metab. 5, 275e281. Pegg, A.E., McCann, P.P., 1982. Polyamine metabolism and function. Am. J. Physiol. 243, C212eC221. Pineda, M., Fernández, E., Torrents, D., Estévez, R., López, C., Camps, M., Lloberas, J., Zorzano, A., Palacín, M., 1999. Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids. J. Biol. Chem. 274, 19738e19744. Quemener, V., Moulinoux, J.P., Havouis, R., Seiler, N., 1992. Polyamine deprivation enhances antitumoral efficacy of chemotherapy. Anticancer Res. 12, 1447e1453. Russell, D.H., Stern, L.Z., 1981. Altered polyamine excretion in Duchenne muscular dystrophy. Neurology 31, 80e83. Saito, A., Takagi, T., Chung, T.G., Ohta, K., 1983. Serum levels of polyamines in patients with chronic renal failure. Kidney Int. Suppl. 16, S234eS237. Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., Kanai, Y., 1999. Identification and functional characterization of a Naþ-independent neutral amino acid transporter with broad substrate selectivity. J. Biol. Chem. 274, 19745e19751. Seiler, N., 2004. Catabolism of polyamines. Amino Acids 26, 217e233. Stewart, P.A., Tuor, U.I., 1994. Blood-eye barriers in the rat: correlation of ultrastructure with function. J. Comp. Neurol. 340, 566e576. Sturman, J.A., Ingoglia, N.A., Lindquist, T.D., 1976. Interconversion of putrescine, spermidine and spermine in goldfish and rat retina. Life Sci. 19, 719e724. Swendseid, M.E., Panaqua, M., Kopple, J.D., 1980. Polyamine concentrations in red cells and urine of patients with chronic renal failure. Life Sci. 26, 533e539. Tabor, C.W., Tabor, H., 1976. 1,4-Diaminobutane (putrescine), spermidine, and spermine. Annu Rev. Biochem. 45, 285e306. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., Hirano, H., 1992. Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT1) in cells of the blood-retinal barrier in the rat. Investig. Ophthalmol. Vis. Sci. 33, 377e383. Tomi, M., Hosoya, K., 2010. The role of blood-ocular barrier transporters in retinal drug disposition: an overview. Expert. Opin. Drug Metab. Toxicol. 6, 1111e1124. Tomi, M., Mori, M., Tachikawa, M., Katayama, K., Terasaki, T., Hosoya, K., 2005. Ltype amino acid transporter 1-mediated L-leucine transport at the inner bloodretinal barrier. Investig. Ophthalmol. Vis. Sci. 46, 2522e2530. Tomi, M., Arai, K., Tachikawa, M., Hosoya, K., 2007a. Naþ-independent choline transport in rat retinal capillary endothelial cells. Neurochem. Res. 32, 1833e1842. Q9 Tomi, M., Terayama, T., Isobe, T., Egami, F., Morito, A., Kurachi, M., Ohtsuki, S., Kang, Y.S., Terasaki, T., Hosoya, K., 2007b. Function and regulation of taurine transport at the inner blood-retinal barrier. Microvasc. Res. 73, 100e106. Q10 Tomi, M., Kitade, N., Hirose, S., Yokota, N., Akanuma, S., Tachikawa, M., Hosoya, K., 2009. Cationic amino acid transporter 1-mediated L-arginine transport at the inner blood-retinal barrier. J. Neurochem. 111, 716e725. Usui, T., Kubo, Y., Akanuma, S., Hosoya, K., 2013. b-Alanine and l-histidine transport across the inner blood-retinal barrier: potential involvement in L-carnosine supply. Exp. Eye Res. 113, 135e142. Wallace, H.M., Caslake, R., 2001. Polyamines and colon cancer. Eur. J. Gastroenterol. Hepatol. 13, 1033e1039. Withrow, C., Ashraf, S., O’Leary, T., Johnson, L.R., Fitzgerald, M.E., Johnson, D.A., 2002. Effect of polyamine depletion on cone photoreceptors of the developing rabbit retina. Investig. Ophthalmol. Vis. Sci. 43, 3081e3090. Yamaoka, K., Tanigawara, Y., Nakagawa, T., Uno, T., 1981. A pharmacokinetic analysis program (multi) for microcomputer. J. Pharmacobiodyn. 4, 879e885. Yanagihara, N., Moriwaki, M., Shiraki, K., Miki, T., Otani, S., 1996. The involvement of polyamines in the proliferation of cultured retinal pigment epithelial cells. Investig. Ophthalmol. Vis. Sci. 37, 1975e1983. Yoneyama, D., Shinozaki, Y., Lu, W.L., Tomi, M., Tachikawa, M., Hosoya, K., 2010. Involvement of system A in the retina-to-blood transport of l-proline across the inner blood-retinal barrier. Exp. Eye Res. 90, 507e513.
Please cite this article in press as: Kubo, Y., et al., Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier, Experimental Eye Research (2014), http://dx.doi.org/10.1016/j.exer.2014.05.002
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
