Involvement of Carrier-Mediated Transport at the Blood–Cerebrospinal Fluid Barrier in Spermine Clearance from Rat Brain

Abstract

Spermine is the end-product in the polyamine biosynthetic pathway, and its excess accumulation induces neuroexcitatory responses and neurotoxicity. The purpose of this study was to elucidate the involvement of transport systems at the brain barriers in the clearance of spermine. In vivo rat spermine elimination from brain parenchyma across the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barrier (BCSFB) was assessed by intracerebral and intracerebroventricular administration techniques, respectively. To characterize spermine transport at the BCSFB, a transport study using rat choroid plexus was performed. After the intracerebral microinjection of [³H]spermine, no time-dependent decrease in [³H]spermine in the ipsilateral cerebrum was observed, suggesting the low contribution of the BBB to spermine clearance from the brain. In contrast, the [³H]spermine concentration in the CSF after intracerebroventricular administration was time-dependently decreased with an elimination rate constant of 0.352 min⁻¹, and the elimination clearance of [³H]spermine was 6.6-fold greater than that of [¹⁴C]D-mannitol, reflecting bulk flow of the CSF. This [³H]spermine elimination was attenuated by co-administration of unlabeled excess spermine, indicating carrier-mediated elimination of spermine from the CSF. [³H]Spermine transport into the choroid plexus was strongly inhibited by unlabeled spermine, other polyamines (spermidine and putrescine), and organic cation transporter substrates such as corticosterone and 1-methyl-4-phenylpyridinium. However, other substrates/inhibitors for organic cation transporters (decynium-22 and tetraethylammonium) had little effect. Consequently, our study indicates that transporting molecules at the BCSFB, distinct from typical organic cation transporters, are involved in spermine clearance from the CSF.

Spermine is a natural polyamine, and the end-product in the polyamine biosynthetic pathway.¹⁾ In the brain, spermine binds to N-methyl-D-aspartate receptors, and is involved in the modulation of learning and memory.^2,3) In addition, excess accumulation of spermine induces neuroexcitatory responses and, thus, neurotoxicity.⁴⁾ Therefore, it is considered that some mechanisms for control of the cerebral spermine concentration are present in the brain to maintain homeostasis of cerebral function via spermine-related neuro-responses. The precursor of spermine is spermidine,⁵⁾ which is synthesized from putrescine and it has been reported that the biosynthesis of spermine from spermidine is superior to that of spermidine from spermine.^6–8) Hence, neural spermine transport system(s) could be important for the removal of spermine from the brain, and the modulation of cerebral spermine concentration.

Brain parenchyma and cerebrospinal fluid (CSF) are separated from the circulating blood by the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB), respectively. The BBB and BCSFB are formed by brain capillary endothelial and choroid plexus epithelial cells, respectively, and express some transporting molecules which are involved in active elimination of compound from the brain or CSF.⁹⁾ Regarding the cationic compound elimination system(s) at these barriers, mRNAs of plasma membrane monoamine transporter (PMAT/solute carrier (SLC)29A4), organic cation/carnitine transporter 1–2 (OCTN1–2/SLC22A4–5), organic cation transporter 1–3 (OCT1–3/SLC22A1–3), and multidrug and toxin extrusion 1–2 (MATE1–2/SLC47A1–2) are expressed in rat BBB and BCSFB cell lines.¹⁰⁾ Among these transporters, PMAT is, at least in part, involved in brain/CSF-to-blood transport of 1-methyl-4-phenylpyridinium (MPP⁺)¹⁰⁾ and CSF-to-blood transport of histamine.¹¹⁾ In addition, OCT3 takes part in the efflux transport of creatinine, a cationic guanidino compound.¹²⁾ Therefore, it is possible that cerebral spermine is eliminated across these brain barriers.

Some previous studies have reported that polyamines including spermine are transported in a carrier-mediated manner in some organs and it has been reported that carrier-mediated transport of polyamines is present in mammalian intestine, liver, and kidney.^13–15) In addition, we have demonstrated that the rat inner blood–retinal barrier, which directly separates the retinal interstitial fluid from the circulating blood, has carrier-mediated retina-to-blood transport system(s) for spermine.¹⁶⁾ Thus, information about membrane transporters on the plasma membrane of tissues will help us to understand the physiological importance of spermine transport in tissue. Among the transporters which are expressed at the BBB and BCSFB, OCT1–3 accepts spermine as a substrate/inhibitor with an affinity of more than 1 mM.^17–19) However, spermine transport at the BBB/BCSFB and the responsible molecule(s) for this process are unknown.

The purpose of this study was to clarify the role of brain barriers in spermine clearance from the brain. In vivo BBB-mediated spermine transport from rat brain was examined by microinjection into the cerebrum, called the brain efflux index (BEI) method. BCSFB-mediated spermine efflux transport was evaluated by in vivo intracerebroventricular administration and an ex vivo uptake by choroid plexus isolated from rats.

MATERIALS AND METHODS

Reagents

Butanol, n-[1-¹⁴C] ([¹⁴C]n-butanol, 5 mCi/mmol), mannitol, D-[1-¹⁴C] ([¹⁴C]D-mannitol, 55 mCi/mmol), and spermine tetrahydrochloride, [terminal methylene-³H] ([³H]spermine, 50 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.). All other reagents were commercially available.

Animals

Wistar rats (male, ca. 160 g) were obtained from Japan SLC (Hamamatsu, Japan). These rats were maintained in a controlled environment and the experiments in the study were approved by the Animal Care Committee of the University of Toyama.

In Vivo Microinjection into Rat Cerebrum

The brain efflux index method²⁰⁾ was performed for evaluating spermine elimination across the BBB, and the details of this procedure are included in Supplementary Materials. The percentage [³H]spermine remaining in the ipsilateral cerebrum (100-BEI, %) was obtained from Eq. 1.   

(1)

Tracer Administration to Rat Lateral Ventricles

The experimental conditions for the administration have been described previously in detail,²¹⁾ and are contained in Supplementary Materials. Since compounds administered into lateral ventricles are known to be eliminated with one-compartmental kinetics (Eq. 2), the following kinetic parameters, detailed information of which is also included in Supplementary Materials, for [³H]spermine and [¹⁴C]D-mannitol were determined from the data using Eq. 3:   

(2)

  

(3)

Transport Study Using Choroid Plexus

[³H]Spermine uptake by rat choroid plexus was performed based on a previous report,²²⁾ and the procedure in this study is described in Supplementary Materials.

The distribution volume of the compound, which is expressed as the tissue/medium ratio, was calculated from Eq. 4.   

(4)

Data Analysis

The kinetic parameters in this study were calculated using least-squares regression analysis, and are represented as the mean±standard deviation (S.D.) while others are given as the mean±standard error of the mean (S.E.M.). Statistical significance of differences between the means for 2 groups was evaluated using the unpaired two-tailed Student’s t-test. In the case of more than 2 groups, one-way ANOVA followed by Dunnett’s test was used.

RESULTS AND DISCUSSION

Brain-to-blood spermine elimination across rat BBB was examined by microinjection into the cerebrum (Fig. 1A). The values of [³H]spermine remaining in the ipsilateral cerebrum at 10, 20, 40, and 60 min were not significantly altered, indicating that [³H]spermine injected into the cerebrum is retained in the brain parenchyma. Thus, it is suggested that the contribution of the BBB to spermine clearance from the brain is minor. To examine spermine elimination from the CSF and the involvement of the BCSFB in its elimination, the residual [³H]spermine concentration in rat CSF after intracerebroventricular administration was monitored (Fig. 1B). The [³H]spermine concentration in rat CSF was time-dependently decreased until 5 min with a k_el,CSF of 3.52×10⁻¹±0.89×10⁻¹ min⁻¹, which is 4.9-fold greater than that for [³H]D-mannitol, which is a model compound for CSF bulk flow (7.13×10⁻²±6.73×10⁻² min⁻¹). These results suggest that elimination of spermine from the CSF, rather than that from the brain parenchyma via the BBB, is involved in spermine clearance from the brain. Laube et al. have reported that spermine and/or spermidine in the brain are distributed to neurons and neuropils observed by anti-spermine/spermidine antibodies.²³⁾ The spermine in the brain parenchyma could be transferred to the cerebroventricles, and then eliminated. The glymphatic system has been proposed as continuous interchange between the brain interstitial fluid and CSF,^24,25) so it is possible that this exchange system is proposed as a possible route of spermine transfer to the CSF. Moreover, from the V_d,CSF value ([³H]spermine, 232±55 µL/rat; [¹⁴C]D-mannitol, 175±31 µL/rat), the CL_CSF for [³H]spermine and [¹⁴C]D-mannitol was found to be 81.6±28.2 µL/(min·rat) and 12.4±12.0 µL/(min·rat), respectively. These results indicate that other elimination process(es), distinct from the CSF bulk flow, take part in spermine clearance from the brain.

Fig. 1. Evaluation of [³H]Spermine Elimination from Rat Brain Parenchyma and CSF

(A) Time course of [³H]spermine remaining in the ipsilateral cerebrum after simultaneous injection into rat brain of 0.1 µCi [³H]spermine and 0.005 µCi [¹⁴C]D-mannitol. (B) Residual concentration in rat CSF versus time of [³H]spermine (closed circle) and [¹⁴C]D-mannitol (open square) after administration into rat lateral ventricle. Ten microliter of the buffer containing 0.4 µCi [³H]spermine and 0.005 µCi [¹⁴C]D-mannitol was injected into the rat lateral ventricles. The initial elimination of each compound from the CSF was shown as the solid line. (C) Effect of unlabeled 1 mM spermine on the residual CSF concentration of [³H]spermine and [¹⁴C]D-mannitol at 2 min. Each point or column represents the mean±S.E.M. (n=3). * p<0.01, significant difference between groups.

The value of the residual CSF concentration of [³H]spermine in the presence of unlabeled spermine at 1 mM (444±7% dose/mL CSF) was not significantly altered compared with that of [¹⁴C]D-mannitol in the absence (420±82% dose/mL CSF) or presence of unlabeled spermine (484±26% dose/mL CSF) (Fig. 1C). This result suggests that spermine elimination from the CSF involves carrier-mediated processes, and is saturated under these conditions. Since the CSF volume of rats in this study was determined to be 175 µL, which is equal to the V_d,CSF value of [¹⁴C]D-mannitol, the concentration of unlabeled spermine in rat CSF after 10 µL intracerebroventricular administration of 1 mM spermine was estimated to be 57.1 µM. Sala-Rabanal et al. have been reported that MPP⁺ transport via mouse OCT1-2 and rat OCT3 was not significantly inhibited by 1 mM spermine,²⁶⁾ although rat OCT1,¹⁷⁾ human OCT2,¹⁸⁾ and human/rat OCT3¹⁹⁾ accept spermine as a substrate or inhibitor. Among these OCTs, it has been reported that OCT3 is localized on the CSF side of choroid plexus epithelial cells in rats, and is involved in creatinine transport from the CSF to the cells.¹²⁾ These lines of evidence support the minor contribution of OCT3 on choroid plexus epithelial cells to spermine elimination from the CSF. To examine whether BCSFB transporters participate in spermine elimination from the CSF, [³H]spermine uptake by choroid plexus isolated from rats was investigated. [³H]Spermine was taken up into the choroid plexus linearly until 2 min (Fig. 2A). Figure 2B shows the inhibition of the [³H]spermine uptake by several compounds. At first, to examine whether the carrier-mediated spermine transport is existed on the BCSFB, the effect of unlabeled spermine at excess concentration on [³H]spermine transport into isolated rat choroid plexus was evaluated. Unlabeled 1 mM spermine significantly inhibited the [³H]spermine uptake by 82%, indicating the carrier-mediated transport of spermine at the apical membrane of the BCSFB. This [³H]spermine uptake was also significantly decreased by more than 70% in the presence of 1 mM spermidine and putrescine. This result shows that spermine transport system(s) on the choroid plexus also recognize other polyamines. Apart from OCT3, it has been reported that OCTN1–2, MATE1–2, and PMAT mRNAs are expressed in a cell line of rat choroid plexus epithelial cells.¹⁰⁾ This [³H]spermine uptake was significantly decreased by 45–55% in the presence of 1 mM MPP⁺, a typical substrate of PMAT and OCT1–3 or 0.1 mM corticosterone, a substrate of OCT1–3.^11,27,28) However, [³H]spermine uptake was not significantly changed by 0.1 mM decynium-22, which is an inhibitor of PMAT and OCT1–3, and 1 mM tetraethylammonium (TEA), a substrate of OCTN1–2, OCT1–3, and MATE1–2.^29–32) From this inhibition study using the rat choroid plexus, it appears that spermine transport at the apical membrane of BCSFB involves polyamine- and several cationic compound-recognizing transporters, but not OCT1–3, OCTN1–2, MATE1–2, and PMAT.

Fig. 2. [³H]Spermine Transport in the Isolated Rat Choroid Plexus

(A) Time-dependent [³H]spermine uptake by the isolated rat choroid plexus. Rat choroid plexus was immersed in buffer containing 1 µCi [³H]spermine and 0.05 µCi [¹⁴C]n-butanol at 37°C. By using non-linear least-squares regression analysis, the initial uptake process (line) was evaluated. (B) Effect of several inhibitors on [³H]spermine uptake by the isolated rat choroid plexus. The [³H]spermine uptake (1 µCi/sample) was performed for 2 min at 37°C in the presence or absence (control) of inhibitors at a concentration of 1 mM, except for corticosterone and decynium-22 (0.1 mM). Each point or column represents the mean±S.E.M. (n=3). * p<0.01, significantly different from the control. MPP⁺, 1-methyl-4-phenylpyridinium; TEA, tetraethylammonium.

The initial uptake clearance of [³H]spermine uptake by rat choroid plexus was found to be 1.74±0.33 µL/µL ChP. By multiplying this [³H]spermine uptake clearance by the total choroid plexus volume in rats, which is reported to be 6 µL/rat,²¹⁾ the rat BCSFB-mediated spermine elimination clearance from the CSF was calculated as 10.4±2.0 µL/(min·rat). This estimated clearance value is 12.8% of the in vivo total elimination clearance from the CSF of spermine (81.6 µL/(min·rat)) although the CSF bulk flow rate, 12.4 µL/(min·rat) observed from the elimination clearance of [¹⁴C]D-mannitol, was included. As other remaining carrier-mediated elimination process(es), the uptake of spermine by several neural cells facing the CSF, could be considered. Recent reports have indicated that ependymal cells, which separate the CSF from brain parenchyma, express several transporters, such as L-glutamate transporters³³⁾ and urate transporters.³⁴⁾ In addition to these reports, Yasuda et al. have found that arachnoid barrier epithelial cells, which are present in meninges, express several kinds of transporters, such as P-glycoprotein and ATP-binding cassette transporter G2.³⁵⁾ Thus, there is a possibility that these epithelial cells also involves in vivo spermine elimination from the CSF. Further studies involving identification of spermine-recognizing transporters which are distinct from well-known transporters and the expression of these molecules on choroid plexus epithelial cells, ependymal cells, and arachnoid barrier epithelial cells, may help us to better understand the details of this spermine elimination from the CSF. Nevertheless, this study has indicated that spermine-recognition by unknown transporter(s) is present on the plasma membrane of choroid plexus epithelial cells, and takes part in spermine elimination from the CSF.

In conclusion, the results of the present study indicate that the elimination of spermine from the CSF involves spermine clearance from the brain. Moreover, it is suggested that transporters at the BCSFB play a role in spermine elimination from the CSF. From the ex vivo transport study, the well-characterized organic cation transporters, including OCT3, make only a minor contribution to the efflux transport process of spermine across the BCSFB. In humans, the concentration in CSF of spermine is greater than that of other polyamines, such as putrescine and spermidine,³⁶⁾ although the whole blood concentration of spermine is similar to that of spermidine.³⁷⁾ Since spermine is the end-product of polyamine bio-synthesis,^6–8) it is considered that this carrier-mediated spermine efflux transport at the BCSFB takes part in the modulation of polyamine concentrations in the brain. Therefore, this finding will help in our understanding of neural concentration homeostasis of polyamines, including spermine, and thus modulation of neuro-exciting signal transduction via spermine-related N-methyl-D-aspartate pathways.

Acknowledgments

This research was partially supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP16K15157 and JP16K08365 and Smoking Research Foundation.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Igarashi K, Kashiwagi K. Polyamines: mysterious modulators of cellular functions. Biochem. Biophys. Res. Commun., 271, 559–564 (2000).
• 2) Williams K, Zappia AM, Pritchett DB, Shen YM, Molinoff PB. Sensitivity of the N-methyl-D-aspartate receptor to polyamines is controlled by NR2 subunits. Mol. Pharmacol., 45, 803–809 (1994).
• 3) Masuko T, Kashiwagi K, Kuno T, Nguyen ND, Pahk AJ, Fukuchi J, Igarashi K, Williams K. A regulatory domain (R1–R2) in the amino terminus of the N-methyl-D-aspartate receptor: effects of spermine, protons, and ifenprodil, and structural similarity to bacterial leucine/isoleucine/valine binding protein. Mol. Pharmacol., 55, 957–969 (1999).
• 4) de Vera N, Martinez E, Sanfeliu C. Spermine induces cell death in cultured human embryonic cerebral cortical neurons through N-methyl-D-aspartate receptor activation. J. Neurosci. Res., 86, 861–872 (2008).
• 5) Tabor CW, Tabor H. Polyamines. Annu. Rev. Biochem., 53, 749–790 (1984).
• 6) Jänne J, Alhonen L, Keinanen TA, Pietila M, Uimari A, Pirinen E, Hyvonen MT, Jarvinen A. Animal disease models generated by genetic engineering of polyamine metabolism. J. Cell. Mol. Med., 9, 865–882 (2005).
• 7) Hannonen P, Janne J, Raina A. Partial purification and characterization of spermine synthase from rat brain. Biochim. Biophys. Acta, 289, 225–231 (1972).
• 8) Seiler N. Polyamine oxidase, properties and functions. Prog. Brain Res., 106, 333–344 (1995).
• 9) Ohtsuki S, Terasaki T. Contribution of carrier-mediated transport systems to the blood–brain barrier as a supporting and protecting interface for the brain; importance for CNS drug discovery and development. Pharm. Res., 24, 1745–1758 (2007).
• 10) Okura T, Kato S, Takano Y, Sato T, Yamashita A, Morimoto R, Ohtsuki S, Terasaki T, Deguchi Y. Functional characterization of rat plasma membrane monoamine transporter in the blood–brain and blood–cerebrospinal fluid barriers. J. Pharm. Sci., 100, 3924–3938 (2011).
• 11) Usui T, Nakazawa A, Okura T, Deguchi Y, Akanuma SI, Kubo Y, Hosoya KI. Histamine elimination from the cerebrospinal fluid across the blood–cerebrospinal fluid barrier: involvement of plasma membrane monoamine transporter (PMAT/SLC29A4). J. Neurochem., 139, 408–418 (2016).
• 12) Tachikawa M, Kasai Y, Takahashi M, Fujinawa J, Kitaichi K, Terasaki T, Hosoya K. The blood–cerebrospinal fluid barrier is a major pathway of cerebral creatinine clearance: involvement of transporter-mediated process. J. Neurochem., 107, 432–442 (2008).
• 13) Kobayashi M, Fujisaki H, Sugawara M, Iseki K, Miyazaki K. The presence of an Na⁺/spermine antiporter in the rat renal brush-border membrane. J. Pharm. Pharmacol., 51, 279–284 (1999).
• 14) Hoshino K, Momiyama E, Yoshida K, Nishimura K, Sakai S, Toida T, Kashiwagi K, Igarashi K. Polyamine transport by mammalian cells and mitochondria: role of antizyme and glycosaminoglycans. J. Biol. Chem., 280, 42801–42808 (2005).
• 15) Iseki K, Kobayashi M, Miyazaki K. Spermine uptake by rat intestinal brush-border membrane vesicles. Biochim. Biophys. Acta, 1068, 105–110 (1991).
• 16) Kubo Y, Tomise A, Tsuchiyama A, Akanuma S, Hosoya K. Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood–retinal barrier. Exp. Eye Res., 124, 17–23 (2014).
• 17) Busch AE, Quester S, Ulzheimer JC, Waldegger S, Gorboulev V, Arndt P, Lang F, Koepsell H. Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1. J. Biol. Chem., 271, 32599–32604 (1996).
• 18) Higashi K, Imamura M, Fudo S, Uemura T, Saiki R, Hoshino T, Toida T, Kashiwagi K, Igarashi K. Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2. PLOS ONE, 9, e102234 (2014).
• 19) Li DC, Nichols CG, Sala-Rabanal M. Role of a hydrophobic pocket in polyamine interactions with the polyspecific organic cation transporter OCT3. J. Biol. Chem., 290, 27633–27643 (2015).
• 20) Kakee A, Terasaki T, Sugiyama Y. Brain efflux index as a novel method of analyzing efflux transport at the blood–brain barrier. J. Pharmacol. Exp. Ther., 277, 1550–1559 (1996).
• 21) Ogawa M, Suzuki H, Sawada Y, Hanano M, Sugiyama Y. Kinetics of active efflux via choroid plexus of beta-lactam antibiotics from the CSF into the circulation. Am. J. Physiol., 266, R392–R399 (1994).
• 22) Suzuki H, Sawada Y, Sugiyama Y, Iga T, Hanano M. Transport of benzylpenicillin by the rat choroid plexus in vitro. J. Pharmacol. Exp. Ther., 242, 660–665 (1987).
• 23) Laube G, Bernstein HG, Wolf G, Veh RW. Differential distribution of spermidine/spermine-like immunoreactivity in neurons of the adult rat brain. J. Comp. Neurol., 444, 369–386 (2002).
• 24) Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, Nagelhus EA, Nedergaard M. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci. Transl. Med., 4, 147ra111 (2012).
• 25) Jessen NA, Munk AS, Lundgaard I, Nedergaard M. The glymphatic system: a Beginner’s guide. Neurochem. Res., 40, 2583–2599 (2015).
• 26) Sala-Rabanal M, Li DC, Dake GR, Kurata HT, Inyushin M, Skatchkov SN, Nichols CG. Polyamine transport by the polyspecific organic cation transporters OCT1, OCT2, and OCT3. Mol. Pharm., 10, 1450–1458 (2013).
• 27) Kekuda R, Prasad PD, Wu X, Wang H, Fei YJ, Leibach FH, Ganapathy V. Cloning and functional characterization of a potential-sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta. J. Biol. Chem., 273, 15971–15979 (1998).
• 28) Urakami Y, Okuda M, Masuda S, Saito H, Inui KI. Functional characteristics and membrane localization of rat multispecific organic cation transporters, OCT1 and OCT2, mediating tubular secretion of cationic drugs. J. Pharmacol. Exp. Ther., 287, 800–805 (1998).
• 29) Martel F, Vetter T, Russ H, Gründemann D, Azevedo I, Koepsell H, Schömig E. Transport of small organic cations in the rat liver. The role of the organic cation transporter OCT1. Naunyn-Schmiedeberg’s Arch. Pharmacol., 354, 320–326 (1996).
• 30) Gründemann D, Koster S, Kiefer N, Breidert T, Engelhardt M, Spitzenberger F, Obermuller N, Schomig E. Transport of monoamine transmitters by the organic cation transporter type 2, OCT2. J. Biol. Chem., 273, 30915–30920 (1998).
• 31) Wu X, Kekuda R, Huang W, Fei YJ, Leibach FH, Chen J, Conway SJ, Ganapathy V. Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain. J. Biol. Chem., 273, 32776–32786 (1998).
• 32) Koepsell H. The SLC22 family with transporters of organic cations, anions and zwitterions. Mol. Aspects Med., 34, 413–435 (2013).
• 33) Akanuma S, Sakurai T, Tachikawa M, Kubo Y, Hosoya K. Transporter-mediated L-glutamate elimination from cerebrospinal fluid: possible involvement of excitatory amino acid transporters expressed in ependymal cells and choroid plexus epithelial cells. Fluids Barriers CNS, 12, 11 (2015).
• 34) Tomioka NH, Tamura Y, Takada T, Shibata S, Suzuki H, Uchida S, Hosoyamada M. Immunohistochemical and in situ hybridization study of urate transporters GLUT9/URATv1, ABCG2, and URAT1 in the murine brain. Fluids Barriers CNS, 13, 22 (2016).
• 35) Yasuda K, Cline C, Vogel P, Onciu M, Fatima S, Sorrentino BP, Thirumaran RK, Ekins S, Urade Y, Fujimori K, Schuetz EG. Drug transporters on arachnoid barrier cells contribute to the blood–cerebrospinal fluid barrier. Drug Metab. Dispos., 41, 923–931 (2013).
• 36) Moulinoux JP, Quemener V, Le Calve M, Chatel M, Darcel F. Polyamines in human brain tumors. A correlative study between tumor, cerebrospinal fluid and red blood cell free polyamine levels. J. Neurooncol., 2, 153–158 (1984).
• 37) Cohen LF, Lundgren DW, Farrell PM. Distribution of spermidine and spermine in blood from cystic fibrosis patients and control subjects. Blood, 48, 469–475 (1976).