Sodium Transporters Are Involved in Lithium Influx ... - ACS Publications

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Sodium transporters are involved in lithium influx in brain endothelial cells Huilong Luo, Matthieu Gauthier, Tan Xi, Christophe Landry, Joel Poupon, Marie-Pierre Dehouck, Fabien Gosselet, Nicolas Perriere, Frank Bellivier, Salvatore Cisternino, and Xavier Decleves Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00018 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 11, 2018

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Molecular Pharmaceutics

Sodium transporters are involved in lithium influx in brain endothelial cells

Huilong Luo1,2, Matthieu Gauthier1,2, Xi Tan1,2, Christophe Landry4, Joël Poupon5, MariePierre Dehouck4, Fabien Gosselet4, Nicolas Perrière6, Frank Bellivier1,3, Salvatore Cisternino1,2,#, Xavier Declèves1,2,*,#

1

Inserm, U1144, Paris, F-75006, France

2

Université Paris Descartes, UMR-S 1144, Paris, F-75006, France

3

Université Paris Diderot, UMR-S 1144, Paris, F-75006, France

4

Université Artois, EA 2465, Laboratoire de la Barrière Hémato-Encéphalique (LBHE), F-

62300 Lens, France 5

Laboratoire de Toxicologie, Hôpital Lariboisière, Paris, France

6

BrainPlotting, Paris, France

#

These authors are co-last authors

Running title: Lithium transport at the blood-brain barrier

Corresponding Author * Xavier Declèves Inserm UMR-S1144, Faculté de Pharmacie, 4 avenue de l’observatoire, 75006 Paris, France. Telephone: +33-1-53-73-99-91. Fax: 33-1-53-73-97-19. E-mail: [email protected]

ACS Paragon Plus Environment

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Molecular Pharmaceutics 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

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Table of contents / abstract graphic:

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Abstract:

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Variability in drug response to lithium (Li+) is poorly understood and significant as only 40%

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of patients with bipolar disorder highly respond to Li+. Li+ can be transported by sodium

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(Na+) transporters in kidney tubules or red blood cells but its transport has not been

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investigated at the blood-brain barrier (BBB). Inhibition and/or transcriptomic strategies for

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Na+-transporters such as NHE (SLC9), NBC (SLC4) and NKCC (SLC12) were used to assess

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their role on Li+ transport in human brain endothelial cells. Na+-free buffer was also used to

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examine Na+/Li+ countertransport (NLCT) activity. The BBB permeability of Li+ evaluated in

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the rat was 2% that of diazepam, a high passive diffusion lipophilic compound. Gene

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expression of several Na+ transporters was determined in hCMEC/D3 cells, human

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hematopoietic stem cells-derived BBB models (HBLEC) and human primary brain

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microvascular endothelial cells (hPBMEC) and showed the following rank order with close

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expression profile: NHE1 > NKCC1 > NHE5 > NBCn1 while NHE2-4, NBCn2, and NBCe1-2

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were barely detected. Li+ influx in hCMEC/D3 cells was increased in Na+-free buffer by 3.3-

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fold while depletion of chloride or bicarbonate had no effect. DMA (NHE inhibitor), DIDS

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(anionic carriers inhibitor) and bumetanide (NKCC inhibitor) decreased significantly Li+

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uptake in hCMEC/D3 by 52%, 51% and 47%, respectively, while S0859 (NBC inhibitor)

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increased 2.3-fold Li+ influx. Zoniporide (NHE1 inhibitor) and siRNA against NHE1 had no

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effect on Li+ influx in hCMEC/D3 cells. Our study shows that NHE1 and/or NHE5, NBCn1,

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and NKCC1 may play a significant role in the transport of Li+ through plasma membrane of

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brain endothelial cells.

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Keywords: Lithium, blood-brain barrier, NHE, NBC, NKCC1


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Page 4 of 43

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Introduction

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Bipolar disorder (BD) is a highly prevalent disorder, its early age at onset (peak 15-19 years)

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and chronicity make it the 4th most burdensome condition worldwide in individuals aged less

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than 25 years and the 6th most burdensome disorder in working age adults.1, 2 Mood stabilizers

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are the mainstay of treatment of BD and Li+ is the most frequently recommended first-line

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treatment in clinical practice guidelines. Unfortunately, it is now well established that about

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40% of Li+-treated BD patients did not show any prophylactic response despite adequate

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treatment duration and plasma levels.3 Several studies attempted to identify clinical and/or

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biological factors associated with this Li+ variability of response. Some of these factors could

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be inadequate pharmacokinetic (PK) and/or pharmacodynamic profile of Li+ in non-responder

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patients. Despite adequate Li+ serum concentrations within the therapeutic window, some

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patients do not respond to Li+ therapy suggesting weak correlation between blood and

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cerebral concentrations in non-responders.4 A recent study in rat showed both a non-parallel

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PK profile of Li+ in blood as compared to brain after a single intraperitoneally dose of lithium

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chloride (LiCl) and a delayed time of maximal Li+ concentrations in the CSF and brain

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homogenate as compared to that achieved in the rat serum.5 Interestingly, nuclear magnetic

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resonance (NMR) imaging of Li+ in BD patients showed that cerebral concentrations of Li+

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were lower in non-responders to Li+ therapy as compared to responders, even though they had

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similar blood concentrations.6 This latter study also confirmed the lack of correlation between

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serum and cerebral concentrations of Li+ in non-responders.

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Several mechanisms could explain such differences: (i) influx and/or efflux mechanisms of

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Li+ exchange through the blood-brain barrier (BBB) and/or the blood cerebrospinal fluid

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barrier (BCSFB), (ii) binding of Li+ to its brain parenchyma targets. Previous works proposed

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that Li+ is actively exchanged through cell membranes by sodium-coupled transporters in rat

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kidney.7 The body’s water and sodium (Na+) balance were mainly regulated through


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reabsorption and excretion processes in kidney, which were mediated by transporters and

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channels expressed in the proximal renal tubules, such as the amiloride-sensitive Na+-coupled

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transporter, Na+/H+ exchangers (NHE) and Na+/K+/Cl- co-transporters (NKCC).8,

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BBB, apart from the Na+/K+ ATPase pump mainly localized at the abluminal side of the brain

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capillary endothelial cells, several Na+-coupled transporters have been evidenced and

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proposed to be expressed either at their luminal and/or abluminal membrane such as Na+/H+

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antiporters (NHE,SLC9A), sodium/bicarbonate symporters (NBC, SLC4A), NKCC

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transporters (SLC12A), and the Na+/Ca2+ antiporters (SLC8A).10 Some transporters members

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of these families are involved in the transport of Na+ through the BBB to partly support Na+

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exchange between the blood and the brain parenchyma. At our knowledge, mechanisms by

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which Li+ is taken up from the blood into the brain has not yet been investigated. In this study,

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we studied the role of Na+ transporters in the membrane transport of Li+ using both in situ

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brain perfusion in the rat and in vitro human brain endothelial cell models, including a stable

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and widely-used brain microvascular endothelial cell line hCMEC/D311 and a newly-

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established human BBB model using cord blood-derived hematopoietic stem cells (Human

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Brain-Like Endothelial Cells model, HBLECs).12

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At the

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Materials and methods

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Animals

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Male Sprague-Dawley rats (7 to 8 weeks old; 310 ± 10 g) were obtained from Janvier (Genest,

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France). The rats were housed in a controlled environment (19±21°C, 55±10% relative

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humidity) with a 12-hour light/ dark cycle, and access to food and tap water ad libitum. All

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experimental procedures complied with the ethical rules of the French agency for

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experimentation with laboratory animals, and complied with the ethical rules of the European

8

directive (210/63/EU) for experimentation with laboratory animals; they were approved by

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the ethics review committee of Paris Descartes University (approval no. 12-185/12-2012) and

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ARRIVE guidelines.

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Chemicals and reagents

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[14C]-sucrose was obtained from Perkin Elmer (Courtaboeuf, France). N-Methyl-D-glucamine

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Hydrochloride (NMDG-Cl) was purchased from TCI Europe (Eschborn, Germany). The

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sodium transporter inhibitors 5-(N,N-Dimethyl) amiloride hydrochloride (DMA), 4,4’-

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diisothiocyanatostilbene-2,2’-disulfonic acid disodium salt hydrate (DIDS), ouabain,

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bumetanide, S0859 and zoniporide were purchased from Sigma-Aldrich (purity > 98%, Saint

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Quentin Fallavier, France). KCl, NaHCO3, KH2PO4, NaH2PO4 and CaCl2 were purchased from

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Merck (Darmstadt, Germany).

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RNA extraction kits and control-siRNA (Neg. NHE1 siRNA AF 488, reference 1027284)

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were purchased from Qiagen (Courtaboeuf, France). siRNA for NHE1 (Silencer® Pre-

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designed siRNA reference s4582) was purchased from Ambion (Applied Biosystems,

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Courtaboeuf, France). Lipofectamine 2000 transfection reagent, RT-PCR reagents and

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primers were purchased from Invitrogen Life Technologies (Cergy-Pontoise, France). The

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Power SYBR Green PCR Master Mix was purchased from Applied Biosystems (Applied

25

Biosystems, France). All other chemicals and reagents were purchased from Sigma-Aldrich.


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In situ rat brain perfusion

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Rats were anaesthetized with diazepam (5 mg/kg i.p.) and ketamine (100 mg/kg i.p.). In situ

3

brain perfusion was performed as previously described.13 Briefly, the right external carotid

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branch and occipital artery were ligated at the level of the bifurcation with the internal carotid

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and the cerebral hemisphere was perfused through the catheterized right common carotid

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artery. The syringe containing the perfusion fluid was placed in an infusion pump and

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connected to the catheter. The thorax was opened, the heart was cut, and perfusion started

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immediately at a flow rate of 10 mL/min. Perfusion was terminated by decapitating the rat at

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90s. The brain was quickly removed and dissected out on ice. The right cerebral hemisphere

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and aliquots of the perfusion ﬂuid was placed in tared vials and weighed and kept at -20°C

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until Li+ assay.

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These experiments were made by a trained person and appropriate procedures followed so

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that these experiments were performed without loss of the integrity of the BBB as measured

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on dedicated rats with the [14C]-sucrose marker integrity assay.

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sucrose brain distribution volume was measured after 90s of in situ brain perfusion. The right

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brain and aliquots of perfusion fluid were weighted, digested (Solvable®; Perkin Elmer) and

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mixed with Ultima-gold XR® (Perkin Elmer). Radiolabel counting was carried out in a Tri-

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Carb 2810TR (Perkin Elmer) to measure disintegrations per minute (dpm). The ratio of the

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[14C]-sucrose amount into the right brain (X*; dpm.g-1) to the perfusion fluid concentration

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(C*perf; dpm.µL-1) allows to estimate the vascular brain volume (Vv; µL.g-1) and integrity of

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the BBB: Vv =

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13,14

In this assay, the [14C]-

X* C *perf

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The control perfusion fluid was Krebs carbonate-buffered physiological saline (mmol/L: 128

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NaCl, 24 NaHCO3, 4.2 KCl, 2.4 NaH2PO4, 1.5 CaCl2, 0.9 MgSO4, 9 D-glucose), warmed to


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Page 8 of 43

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37°C and gassed with 95% O2/5% CO2 (pH 7.40 ± 0.05). In some experiments, sodium was

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iso-osmotically replaced mainly by NMDG-Cl.

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The perfusion fluid contained Li+ (1 mM). The Li+ intrinsic transport rate also called brain

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clearance (Kin; µL. s-1. g-1) was measured in perfusion fluid with (Control) or without Na+

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(NMDG-Cl buffer). Briefly, the apparent distribution volume of Li+ (Vbrain; µL.g-1) was

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calculated as ratio of the Li+ tissue on perfusion fluid concentrations and its brain transport

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rate was calculated as : Kin=Vbrain/90s.

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Cell culture conditions

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hCMEC/D3 cells. The hCMEC/D3 cell line was kindly provided by Dr Pierre-Olivier

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Couraud (Institut Cochin, Paris, France), and was used for experiments between passages 27

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and 35. The hCMEC/D3 cells were cultured according to previously reported methods.11

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Briefly, the growth medium for hCMEC/D3 was EBM-2 medium (Lonza, Basel, Switzerland)

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supplemented with 5 µg/mL ascorbic acid, 1.4 µM hydrocortisone, 1 ng/mL basic FGF

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(Sigma), 5% fetal bovine serum (Eurobio, Les Ulis, France) 10 mM HEPES (PAA, Pasching,

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Austria) and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) under 37℃ and 5%

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CO2. The flask and plate for the hCMEC/D3 culture was pre-coated with rat tail collagen type

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I (150 µg/mL). Cells were passaged every 3-4 days using trypsin/EDTA to detach the cells

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from the flasks. All transport experiments were conducted on confluent cultures, 3 days after

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incubation in 12-well plates at 5×104 cells/cm2.

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HEK-293 cells. Human embryonic kidney HEK293 cells were cultured with Dulbecco’s

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modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum

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(Thermo Electron, Melbourne, Australia) and 1% penicillin-streptomycin (Gibco) under 37 ℃

23

and 5% CO2.

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Human Brain-Like Endothelial Cells model (HBLECs). Here, we used human umbilical

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cord blood stem cells as an in vitro model of the human BBB.


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Briefly, CD34+ cells were

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isolated from human umbilical cord blood using the mini-MACS immunomagnetic separation

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system (Miltenyi Biotec, Bergisch Gladbach, Germany) and differentiated into endothelial

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cells. At this stage, the cells have a cobblestone-like morphology and express high levels of

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endothelial cell markers, including CD31, vascular endothelial cadherin and von Willebrand

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factor. Secondly, these CD34+-derived endothelial cells were seeded onto Transwell® inserts

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(pore size: 0.4µm; ref. 3401, Corning Inc., Corning, NY) coated with Matrigel™ (BD

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Biosciences, Franklin Lakes, NJ) diluted 1:48 in endothelial cell medium (ECM-5), composed

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of ECM basal medium (Sciencell, Carlsbad, CA) supplemented with 5% (v/v) heat-

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inactivated fetal calf serum, 1% (v/v) endothelial cell growth supplement (Sciencell) and 0.5%

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(v/v) gentamycin in a humidified 5% CO2 atmosphere and cultured in presence of brain

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pericytes at the bottom of the wells. Brain pericytes were seeded in the morning in 12-well

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plates (50,000 cells per well) and endothelial cells were seeded on matrigel™-cotaed inserts

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in the afternoon of the same day (80,000 cells per insert). Brain pericytes were isolated from

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freshly bovine brain capillaries as previously described.14 After 6 days of co-culture, the

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endothelial cells acquire the tight junctions and transporters typically observed in brain

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endothelium and display of most of the BBB’s in vivo properties for at least 20 days. 12 ECM-

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5 medium was changed every 2 days.

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Human Primary Brain Microvascular Endothelial Cells (hPBMECs). Endothelial cells

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were isolated from a surgical resection of a patient (female, 70 years old) suffering from

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glioma. Our experimentation was performed in compliance with the French legislation and

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the protocol was approved by the French Ministry of Higher Education and Research

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(CODECOH DC-2014-2229). Briefly, brain capillaries were isolated after soft digestion of

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brain tissues and then seeded. Brain endothelial cells were shortly amplified and seeded on

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transwell microporous membranes in monoculture or in coculture with fresh human astrocytes


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Page 10 of 43

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from the same patient. Cells were grown in EBM-2 medium (Lonza) supplemented with

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serum and growth factors (Sigma). Dry pellets were prepared for RT-qPCR experiments.

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RNA extraction and reverse transcription

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RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) was used to isolate total RNA. The purity

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and concentrations of the RNA samples were detected spectrophotometrically at 260 nm and

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280 nm by the Nanodrop ND-1000 instrument (NanoDrop Technologies, USA). Reverse

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transcription was realised on total RNA in reaction mixture system as described previously.15

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RT negative controls were prepared by replacing the reverse transcriptase in the mix to

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nuclease-free water. A thermal cycler (PTC-100 programmable thermal controller, MJ

10

research INC, USA) was applied with incubation condition as follows: 25°C for 10 min, then

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at 42°C for 30 min and at 99°C for 5 min. cDNAs were then stored at -80°C.

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Real-time quantitative RT-PCR (qPCR)

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Gene expression was assessed by SYBR Green fluorescence detection on an ABI Prism 7900

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HT Sequence Detection System (Applied Biosystems, Foster City, CA) as previously

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described.15 The final reaction mixture system included diluted cDNA, Power SYBR Green

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PCR Master mix kit (Thermo Fisher Scientific), and primers (Supplemental Table 1). Primers

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were designed using OLIGO 6.42 software (MedProbe, Norway). No-template control assays

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and RT negative controls showed negligible signals (Ct value > 40). Reaction specificity was

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ensured by melting curve analysis. Primers were validated with RNA from HEK293 cells.

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The Ct of the first standard in the calibration corresponding to cDNA diluted (1/20) was

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between 22 and 29 for all the genes of interest in the HEK293 cell line. Gene expression was

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evaluated using the Ct value. It was considered quantifiable for Ct less than 31 (cDNA 1/20).

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The ∆∆Ct method was used to compare gene expression in hCMEC/D3, HEK293, HBLEC

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and hPBMEC normalised with the housekeeping gene encoding TATA box-binding protein


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(TBP). The efficacy of each PCR was better than 95% and results are expressed as arbitrary

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units.

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Influx of Li+ in hCMEC/D3 cells

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Time course of Li+ influx. hCMEC/D3 cells cultured at confluence in 12-well plate were pre-

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incubated for 5 min with 500 µL/well of Krebs-HEPES buffer (KH; control) prepared with

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124 mM NaCl, 4.2 mM KCl, 24 mM NaHCO3, 2.4 mM NaH2PO4, 1.5 mM CaCl2, 0.9 mM

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MgSO4, 10 mM HEPES, 4.5 mM D-Glucose and adjusted to pH 7.4 with HCl/NaOH. After

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removal of the pre-incubation KH buffer cells were then incubated with KH buffer containing

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0.1 mmol/L Li+ for 3, 5, 10, 15 or 30 minutes. At the end of the incubation, exposure of Li+

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was stopped by rapidly removing the KH buffer and cells were placed on ice. Cells were

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washed on ice twice consecutively with ice-cold 500 µL Dulbecco’s PBS (D-PBS, prepared

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with 138 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4) and subsequently

13

lysed with 500 µL of 0.1% Triton in water at 60 °C for 1 hour. Twenty µL of cell lysates were

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collected for protein assay by UV spectroscopy (VictorTM X2 Multilabel Plate Reader,

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PerkinElmer) using the micro BCA protein assay and standard solutions of bovine serum

16

albumin (BSA) (Pierce®, ThermoFisher Scientific) according to the manufacturer instructions.

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Li+ was quantified by ICP-MS (see method) and results were expressed as µmol/mg of total

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proteins.

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Li+ influx in hCMEC/D3 with Na+-free, Cl--free and HCO3--free KH buffers.

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hCMEC/D3 cells in 12-wells plate were also incubated with KH buffers lacking one selected

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inorganic ion replaced by iso-osmotic substitution: (i) a KH buffer lacking sodium (Na+-free

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KH ) prepared with 130 mM NMDG-Cl, 24 mM KHCO3, 2.4 mM KH2PO4, 1.5 mM CaCl2,

23

0.9 mM MgSO4, 10 mM HEPES, 4.5 mM D-Glucose adjusted to pH 7.4 with HCl/KOH and

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(ii) a KH buffer without chloride (Cl--free KH) prepared with 132.2 mM NaNO3, 22.2 mM

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NaHCO3, 0.9 mM MgSO4, 1.8 mM CaCO3, 2.4 mM KH2PO4, 1.5 mM Calcium gluconate, 10


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Page 12 of 43

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mM HEPES, 4.5 mM D-Glucose adjusted to pH 7.4 with HNO3/NaOH, (iii) a KH buffer

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without carbonate (HCO3-free KH) prepared with 128 mM NaCl, 4.2 mM KCl, 2.4 mM

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NaH2PO4, 1.5 mM CaCl2, 0.9 mM MgSO4, 10 mM HEPES, 4.5 mM D-Glucose adjusted to

4

pH 7.4 with HCl/NaOH. The procedure of Li+ exposure to hCMEC/D3 cells with KH buffer

5

lacking sodium, chloride or carbonate was the same as that used with the KH buffer. Cells

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were incubated at 37°C for 3 min with Li+ 0.1 mmol/L. The cell lysates were then collected

7

and analysed as mentioned below.

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Incubation with Na+-coupled transporter inhibitors

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Cells in 12-wells plate were firstly pre-incubated for 5 min with 500 µL/well of the KH

10

buffer. NCT Inhibitors were added to the KH buffer with final concentrations as follows: 0.1

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mM DMA, 0.1 mM ouabain, 0.1 mM DIDS, 10 µM bumetanide, 30 µM S0859, or 50 µM

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zoniporide. DMA and DIDS were prepared directly to the final concentration by dissolving

13

them in KH buffer, while bumetanide, S0859 and zoniporide were firstly prepared as stock

14

solution in the KH buffer containing 0.5% DMSO. After 3 min of incubation with the KH

15

buffer containing the inhibitors at 37 °C, the cell lysates were collected and analysed as

16

mentioned above.

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Measurement of intracellular pH in hCMEC/D3 cells

18

Measurement of intracellular pH was evaluated as previously described.16 hCMEC/D3 cells

19

cultured in 24-well plates, were initially incubated with 2 µmol/L BCECF-AM (Invitrogen), a

20

precusor of the intracellular pH (pHi) indicator BCECF in HBSS (containing 10 mM HEPES,

21

pH 7.4) for 30 minutes at 37 °C, then placed in a closable, temperature-controlled chamber in

22

a microplate fluorometer (Tecan Safire, Zurich, Switzerland, maintained at 37 °C). Cells were

23

then washed and incubated with selected KH buffer lacking or not (KH = control) ionorganic

24

ion: Na+-free KH, Cl-free KH, HCO3-free KH (all adjusted to pH 7.4), or in the presence of

25

inhibitors as described above. The pHi was determined by obtaining the ratio of emission


12

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wavelength at 535 nm for excitation wavelengths of 490 nm and 440 nm and a calibration

2

curve as described previously16 from the beginning (after the pre-incubation period) to the end

3

of the 3 min Li+ exposure.

4 5

RNA interference for NHE-1 knockdown

6

The sequence 5’-GGAGUUUGCCAACUACGAA-3’ was used to decrease NHE1 expression

7

and a non-specific sequence 5’-AUUGUGAGACUCAGACCA-3’ was used as a negative

8

control. Briefly, for each well on the 24-well plate, 30 µM of the NHE1-siRNA

9

oligonucleotide or the negative control oligonucleotide in 50 µL of Opti-MEM and 1.5 µL of

10

Lipofectamine 2000 in 50 µL of Opti-MEM were preincubated for 5 min and then mixed

11

together and incubated for an additional 25 min at room temperature. After the addition of

12

400 µL of Opti-MEM, the entire mixture was added to the well and the cells were further

13

cultivated for an additional 2 days. The mRNA levels of NHE1 were determined by q-PCR.

14

Then the cells were incubated with 0.1 mM Li+ for 3 min to study the effect of siRNA-NHE1

15

on the uptake of Li+.

16

Li+ permeability through HBLEC monolayers

17

Effect of different incubation buffers on [14C]-sucrose permeability through HBLEC model

18

Sucrose was used to evaluate paracellular permeability since this is a well-established method

19

for in vitro BBB models. BBB HBLECs permeability was measured according to the method

20

described by Dehouck et al.17 Briefly, filters containing cultured HBLECs were transferred in

21

wells (12-wells plate) with 1.5 mL Ringer HEPES buffer named RH buffer prepared with

22

150.6 mM NaCl, 5.2 mM KCl, 6 mM NaCO3, 2.2 mM CaCl2, 0.2 mM MgSO4, 5 mM HEPES

23

and 2.8 mM D-Glucose containing 0.1 or 1 mM Li+ without or with different Na+ transport

24

inhibitors (0.1 mM DMA, 0.1 mM DIDS, 10 µM bumetanide, 30 µM S0859 or 50 µM

25

zoniporide), or by replacing the RH buffer by a RH buffer without Na+ (RH-Na+) prepared


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Page 14 of 43

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with 155.8 mM NMDG-Cl-, 6 mM KCl, 6 mM NaCO3, 2.2 mM CaCl2, 0.2 mM MgSO4, 5

2

mM HEPES and 2.8 mM D-Glucose containing 1 mM Li+. 0.5 mL of the same buffers as in

3

the lower compartment containing 50 µM of [14C]-sucrose were added to the upper

4

compartment. At different time points (10, 15, 20 and 25 min), filters were placed in a new

5

well with 1.5 mL of the same RH buffer. An aliquot of 200 µL from the lower compartment at

6

each time point and an aliquot of 20 µL from the initial solution were collected and used for

7

[14C]-sucrose quantification (Tri-Carb 2100TR, PerkinElmer). The endothelial permeability

8

coefficient (Pe) of [14C]-sucrose was calculated. Briefly, the average volume cleared is plotted

9

versus time, and the slope is estimated by linear regression. Both insert permeability (PSf, for

10

insert only coated with matrigel™) and insert HBLECs permeability (PSt, for insert with

11

matrigel™ and cells) were taken into consideration, according to the following formula:

12

1/PSe = 1/PSt – 1/PSf

13

The permeability value for the endothelial monolayer was then divided by the surface area of

14

the porous membrane of the insert (membrane surface 1.12 cm2) to obtain the endothelial

15

permeability coefficient (Pe) of the molecule (in cm.min-1).

16

Effects of Na+ transporter inhibitors on the Li+ permeability through HBLEC monolayers

17

Inhibitors were evaluated for effects on permeability of Li+ through HBLEC cells. Briefly, 0.5

18

mL and 1.5 mL of RH buffer containing different concentration of inhibitors (0.1 mM DMA,

19

0.1 mM DIDS, 10 µM bumetanide, 30 µM S0859 or 50 µM zoniporide) or RH buffer without

20

Na+ were incubated in the upper compartment and lower compartment of the filters containing

21

HBLECs cells, respectively. Then LiCl was added to the upper compartment to achieve a

22

final concentration of Li+ at 1 mM. At different time points (10, 15, 20 and 25 min), filters

23

were placed in a new well with 1.5 mL of the same RH buffer. An aliquot of 200 µL from the

24

lower compartment at each time point was collected and the quantification of Li+ was


14

Page 15 of 43 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


1

performed by ICP-MS. The endothelial permeability coefficient (Pe) of Li+ was calculated

2

according to the formula mentioned above.

3 4

Li+ and Na+ assays by ICP-MS

5

Inductively-coupled plasma mass spectrometry (ICP-MS) measurements were performed with

6

a 7700 Series (Elan DRCe, Perkin Elmer) equipped with a third generation Octopole Reaction

7

System (ORS3) using helium gas to determine Li+ concentrations in cell lysates, brain

8

mineralized tissue samples, and buffers. A peristaltic pump from tubes arranged on a CETAC

9

ASX-500 Series auto-sampler (CETAC Technologies, Omaha, NE, USA) pumped the sample

10

solutions. 150 µL of samples were diluted with 450 µL of 0.1 M HNO3. The mineralized

11

brain tissue was diluted with 8 mL of 0.1 M HNO3. The calibration curve was firstly diluted

12

by the same matrix of the corresponding samples: blank cell lysates, brain and buffer solution,

13

then further diluted by 0.1M HNO3. The lowest limit of quantification for Li+ was 0.6 nM. In

14

some experiments the intracellular Na+ concentration was measured in hCMEC/D3 cell

15

lysates after exposure with the KH (control) or KH lacking Na+ (Na+-free KH) buffer. Briefly,

16

hCMEC/D3 cells cultured in 6-well (n=3 per condition, in triplicate) with culture medium

17

were washed 2 times with KH buffer and then incubated with KH or Na+-free KH for 8 min

18

in total (5 min of pre-incubation followed by the 3 min incubation with Li+) at 37°C. After

19

incubation with buffer of interest, cells were washed 2 times with Na+-free KH on ice at 4°C,

20

buffer was removed, and cells were scraped and lysed with three successive freeze thawing

21

cycles and intracellular Na+ content was determined by ICP-MS.

22 23

Statistical analysis

24

Data are expressed as mean value ± SEM (standard error of the mean) except for in situ rat

25

brain perfusion as mean ± SD (standard deviation). Statistical analysis was performed using


15


Page 16 of 43

1

ANOVA with Dunnett a posteriori test to compare different groups with the control. P value

2

< 0.05 was considered statistically significant.


16

Page 17 of 43 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


1

Results

2

In situ rat brain perfusion

3

The brain Li+ Kin measured in control rat was 0.88 ± 0.20 µL.s-1.g-1 (n = 6). BBB permeability

4

of Li+ was low when its extraction ratio at 2% was compared to that of the highly lipophilic

5

passive diffusion molecule diazepam (Kin = 42.3 µL.s-1.g-1) set at 100%. Brain perfusion with

6

Na+-free perfusate (NMDG-Cl) resulted in a significant 2.45-fold (p

Sodium Transporters Are Involved in Lithium Influx ... - ACS Publications

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