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Neuroscience 136 (2005) 105–114

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EAD INDUCES INCREASED WATER PERMEABILITY IN ASTROCYTES

XPRESSING AQUAPORIN 4

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. GUNNARSON,a* G. AXEHULT,b G. BATURINA,c

. ZELENIN,a M. ZELENINAa,c AND A. APERIAa

Nordic Centre for Water Imbalance Related Disorders, Department ofoman and Child Health, Karolinska Institutet, Pediatric Unit, Re-

earch Laboratory, Q2:09 Astrid Lindgren Children’s Hospital, 171 76tockholm, Sweden

Department of Cell Physics, The Royal Institute of Technology, Stock-olm, Sweden

Laboratory of Physiological Genetics, Institute of Cytology and Ge-etics, Siberian Branch of Russian Academy of Sciences, Novosibirsk,ussia

bstract—The water channel aquaporin 4 (AQP4) is abun-antly expressed in astrocytes. There is now compelling ev-

dence that AQP4 may contribute to an unfavorable course inrain edema. Acute lead intoxication is a condition thatauses brain damage preceded by brain edema. Here weeport that lead increases AQP4 water permeability (Pf) instrocytes. A rat astrocyte cell line that does not expressquaporin 4 was transiently transfected with aquaporin 4agged with green fluorescent protein (GFP). Using confocalaser scanning microscopy we measured water permeabilityn these cells and in AQP4-negative cells located on the samelate. AQP4-expressing astrocytes had a three-fold higherater permeability than astrocytes not expressing AQP4.ead exposure induced a significant, 40%, increase in waterermeability in astrocytes expressing AQP4, but had noffect on Pf in astrocytes not expressing AQP4. The increasen water permeability persisted after lead washout, whilereatment with a lead chelator, meso-2,3-dimercaptosucciniccid, abolished the lead-induced increase in Pf. The effect ofead was attenuated in the presence of a calcium (Ca2�)/almodulin-dependent protein kinase II (CaMKII) inhibitor,ut not in the presence of a protein kinase C inhibitor. In cellsxpressing AQP4 where the consensus site for CaMKII phos-horylation was mutated, lead failed to increase water per-eability. Lead exposure also increased Pf in rat astroglial

ells in primary culture, which express endogenous AQP4.ead had no effect on Pf in astrocytes transfected with aqua-orin 3. In situ hybridization studies on rat brain after oral

ead intake for three days showed no change in distributionf AQP4 mRNA. It is suggested that lead-triggered stimula-ion of water transport in AQP4-expressing astrocytes mayontribute to the pathology of acute lead intoxication. © 2005

BRO. Published by Elsevier Ltd. All rights reserved.

Corresponding author. Tel: �46-8-517-77335; fax: �46-8-517-77328.-mail address: eli.gunnarson@kbh.ki.se (E. Gunnarson).bbreviations: AQP3, aquaporin 3; AQP4, aquaporin 4; AQP4 S111A,utated aquaporin 4 with Ser111 substituted to alanine; BIM, bisin-olylmaleimide; CaMKII, calcium (Ca2�)/calmodulin-dependent pro-ein kinase II; DMSA, meso-2,3-dimercaptosuccinic acid; DMEM, Dul-ecco’s Modified Eagle’s medium; FBS, fetal bovine serum; GFAP,lial fibrillary acidic protein; GFP, green fluorescent protein; PBS,

L

hosphate-buffered saline; PCR, polymerase chain reaction; Pf, waterermeability; PKC, protein kinase C.

306-4522/05$30.00�0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2005.07.027

105

ey words: glial cells, lead, toxicity, water channel, confocalicroscopy, primary culture.

erebral edema with cellular swelling is a common conse-uence of brain hypoxia, trauma and intoxication (Kimel-erg, 1995) and represents a risk for further morbidity andermanent brain damage. The cellular mechanisms in-olved in the development of brain edema are still to a

arge extent unknown.Aquaporins (AQPs) are water channels that allow rapid

smotically driven water transport across cell membranesPreston et al., 1992; Agre et al., 2002). Aquaporin 4AQP4) is the most abundantly expressed aquaporin withinhe brain (Jung et al., 1994; Nielsen et al., 1997). It isituated mainly in astrocytes, the cells commonly associ-ted with cell swelling during cerebral edema (Kimelberg,995). The physiological significance of brain AQP4 is notully understood. Studies of mice lacking AQP4, or withltered subcellular localization of AQP4, suggest that in theituation of brain edema, symptoms may actually be ag-ravated by the presence of AQP4 (Manley et al., 2000;ajda et al., 2002; Amiry-Moghaddam et al., 2003a, 2004).

Experimental acute lead intoxication has been showno be associated with astrocyte swelling (Lefauconnier etl., 1983; Selvin-Testa et al., 1997). Several divalent heavyetals are known to modulate aquaporin water permeabil-

ty (Preston et al., 1993; Niemietz and Tyerman, 2002;elenina et al., 2003). The effect of lead on AQP4 activityas however, to the best of our knowledge, not previouslyeen reported. Here we report that lead increase AQP4ater permeability (Pf) in an astrocyte cell line transiently

ransfected with AQP4 and in rat hippocampus astroglialells in primary culture. Further we present evidence thathe effect on AQP4 water permeability involves the (Ca2�)/almodulin-dependent protein kinase II (CaMKII) signalingathway. In a second part of the study, we investigated theffect of lead exposure on AQP4 mRNA expression in theat brain.

EXPERIMENTAL PROCEDURES

ell cultures

at astrocyte cell line (CTX TNA2, European Collection of Cellultures, Centre for Applied Microbiology & Research, Salisbury,iltshire, UK) was used in subpassages 3–7. The astrocytes were

rown on 40 mm coverslips (Bioptechs, Butler, PA, USA) in Dul-ecco’s Modified Eagle’s medium (DMEM) (Sigma-Aldrich, St.ouis, MO, USA) containing 0.5 U/ml penicillin and 50 �g/mltreptomycin supplemented with 10% fetal bovine serum (FBS)Gibco, Paisley, UK), 0.11 mg/ml sodium pyruvate, and 2 mM

-glutamine. On the second day of culture, the cells were trans-

ved.

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E. Gunnarson et al. / Neuroscience 136 (2005) 105–114106

ected with cDNA constructs encoding mouse AQP4 tagged withreen fluorescent protein (GFP) on the NH2-terminus or humanquaporin 3 (AQP3) tagged with GFP on the COOH-terminus (seeelow) using TransFast Transfection Reagent (Promega Corpo-ation, Madison, WI, USA) according to the manufacturer’s proto-ol. Experiments were performed on the fourth day of culture. Athe time of study the cells were subconfluent with a polygonalhape that is typical for cultured astrocytes with no neuronal cellsresent. Several transfection methods were tested in pilot studies.he TransFast method was found to yield the highest efficacy of

ransfection.To obtain astroglial cells in primary culture, rat hippocampi

ere dissected from pups at embryonic age of 18 days. Theemoved hippocampi were placed in 0.05% trypsin (Gibco) andncubated for 15 min at 37 °C. The cells were subsequentlyriturated and seeded onto 40 mm coverslips (Bioptechs) pre-oated with poly-L-lysine (1 mg/10 ml; Sigma-Aldrich). The cellsere grown in DMEM/F12 medium supplemented with 10% heat-

nactivated FBS (Gibco), 1.2 mg/ml glucose, 5 �g/ml insulinInvitrogen, Paisley, UK), 100 �g/ml transferrin (Invitrogen), 20 nMrogesterone (Sigma-Aldrich), 100 �M putrescine (Sigma-ldrich), 30 nM selenium dioxide (Sigma-Aldrich), 20 units-�g/mlenicillin/streptomycin (Sigma-Aldrich), and plated at a concentra-ion of 8–9�105 cells/coverslip. Cultures were maintained in anncubator with 5% CO2 at 37 °C and used after 21–30 days, athich time the cells were confluent. The vast majority of cells

�95%) were identified as astrocytes by immunostaining with anntibody against glial fibrillary acidic protein (GFAP). The cultureslso contained a few cells that were identified as neurons using

mmunostaining against microtubule-associated protein 2 (MAP2).

NA constructs

e used constructs encoding mouse AQP4.M23 tagged with GFPt the NH2-terminus (pGFP-AQP4.M23) and human AQP3 taggedith GFP at the COOH-terminus, described previously (Zeleninand Brismar, 2000; Zelenina et al., 2002, 2003). To clone mouseQP4 antisense probe (pAQP4as), 0.9 kb BamHI AQP4 cDNA

ragment from pGFP-AQP4.M23 was ligated into BamHI site ofBlueScript.SK�. To clone mouse AQP4 sense probe (pAQP4s),.9kb PstI–XbaI fragment from pAQP4as was ligated into PstI–XbaIof pBlueScript.KS�. pAQP4s and pAQP4as DNA samples wereydrolyzed by BssHII and used for sense and antisense probeynthesis, respectively. The structure of all constructs was ana-yzed using a BigDye Terminator Cycle Sequencing Ready Reac-ion kit (Applied Biosystems, Warrington, UK). The point mutationf Ser111 to alanine (S111A) in GFP-tagged AQP4 fusion proteinas generated using U.S.E. Mutagenesis KIT (Amersham Phar-acia Biotech AB, Uppsala, Sweden). The mutation and absencef other modifications were confirmed by sequence analysis of thehole insert.

easurement of water permeability

f was measured using a method that we described in detailreviously (Zelenina and Brismar, 2000; Zelenina et al., 2002). Byse of this method Pf in individual cells within cell monolayers cane determined and compared between cells that do and do notxpress GFP-labeled proteins. Briefly, coverslips with the trans-ected or primary cultured astrocytes were mounted in a closederfusion chamber (Focht Live Cell Chamber System, Butler, PA,SA) on the stage of a Zeiss 410 inverted laser scanning micro-cope. The chamber was filled with isoosmotic phosphate-buff-red saline (PBS) and scanned every 2 s with excitation at 488 nmnd emission collected at 515–525 nm. At the beginning of eachtudy, an image showing the distribution of GFP-tagged proteinsn the transfected cell line was recorded (Fig. 1A). An XZ recon-truction showed that GFP-AQP4 was evenly distributed in the

lasma membrane (Fig. 1B). Cells were loaded with the fluores- g

ent dye calcein (Molecular Probes Europe, Leiden, The Nether-ands) (Fig. 1C). Loaded cells were first exposed to 50 �M leadPb2�) acetate in isoosmotic PBS for 10 min. To measure Pf, cellsere initially perfused with isoosmotic PBS. The perfusate was

hen rapidly switched to a hypoosmotic PBS (150 mOsm). In theead studies, both perfusion solutions contained 50 �M lead ace-ate. The temperature was 10 °C throughout the measurements inrder to minimize the diffusional water movement through the lipidilayer of the plasma membrane. The obtained series of imagesere analyzed off-line by measuring the time course of the calceinuorescence in individual cells. The part of the obtained curvesecorded immediately after the solution switch was used for Pf

alculation. During this period (�10 s), the cell swelling is as-umed to be proportional to the permeability of the cellular mem-ranes to water. To calculate the Pf separately for cells that didnd did not express AQP4, the GFP and calcein images wereuperimposed (Fig. 1D). A curve was obtained for each individualell, showing changes in fluorescence intensity inside the celluring osmotic swelling (Fig. 1F). The Pf was calculated as pre-iously described (Zelenina and Brismar, 2000; Zelenina et al.,002). The initial region of the fluorescence curve was fitted withsingle exponential function and its time constant was used as aeasure of the rate of cell swelling. This value is relative byefinition and does not depend on the absolute values of fluores-ence intensity at the beginning of the cell swelling. Calcein waseported to be quenched by lead in solutions (Ueberfeld et al.,002). Due to the relative nature of the time constant used to calcu-

ate the Pf in our lead studies, this would not affect our measure-ents. We incubated the cells with lead acetate for 10 min before theeasurement. By the time of measurement, a new baseline of in-

ensity was reached from the point of view of calcein quenching. If aead uptake continued during the measurements, this would be com-ensated for in the same manner as for bleaching (Zelenina andrismar, 2000). Further, the tests performed on cells transfected withQP3 cells showed no change in Pf in the presence of lead, indicat-

ng that the increase in the time constant in the presence of lead is notue to a quenching of calcein by lead.

To test whether a chelator abolished the lead effect onf, 100 �M meso-2,3-dimercaptosuccinic acid (DMSA) (Sigma-ldrich) was applied to the cells. This chelator is commonly used

o examine the role of intracellular lead (Angle, 1993; Bernal et al.,997; Zhang et al., 2004). In a separate set of experiments wesed the CaMKII inhibitor KN-62 (Sigma-Aldrich) and protein ki-ase C (PKC) inhibitors bisindolylmaleimide (BIM) (Merck KGaA,armstadt, Germany) or calphostin (Merck) to explore the intra-ellular pathways of the lead effect.

For the astroglial cells in primary culture the time constant ofhe fitting of the initial part of the curve by a single exponentialunction was used directly as a measure of water permeability.

olymerase chain reaction (PCR)

otal RNA was extracted from cells using an RNeasy Total RNA KitQiagen, Hilden, Germany). Reverse transcription was performedollowed by PCR with specific primers to detect rat AQP4 mRNA. Therimers had the following sequence: rAQP4 3= GGC TTC CTT TAGCG ACG TTT GAG, rAQP4 5= TCC CTC TGC TTT GGA CTC AGCTT G. �-Actin was used as internal control. The PCR conditionsere as follows: 1) 7 min of denaturation at 95 °C; 2) 28 cycles of 30 sf denaturation at 94 °C, 45 s of annealing at 60 °C, and 1 min ofxtension at 72 °C for each cycle; and 3) a final extension step ofmin at 72 °C. PCR fragments were separated on agarose gel and

nalyzed using Gel Documentation System Fluor-S MultiImagerBio-Rad, Richmond, CA, USA).

n vivo studies

Animals. Studies were performed on 40 day old male Spra-

ue–Dawley rats (B&K Universal, Stockholm, Sweden) kept under

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E. Gunnarson et al. / Neuroscience 136 (2005) 105–114 107

tandard housing conditions with free access to food and water.ll animal experiments were approved by the local ethical com-ittee at Karolinska Institutet, Stockholm, Sweden and conformed

o international guidelines on the ethical use of animals. Effortsere made in order to minimize the number of animals in thexperiments and to avoid their suffering.

Animals were given lead acetate (Sigma-Aldrich) by gavagen a dose of 100 mg/kg body weight twice daily for three consec-tive days. New solutions of lead acetate were prepared daily tovoid precipitation. On the fourth day the animals were anesthe-ized, rapidly decapitated and tissues were immediately removed,rozen on dry ice, and stored at �80 °C until sectioning or analysisf lead content. Lead content in brains was analyzed by thenductive Coupled Plasma-Atomic Emission Spectrometry (ICP-ES) technique after wet digestion to confirm lead uptake andxposure levels. The detection level of the method was.0015 mg/kg wet weight.

A separate study was performed on 10 day old Sprague–awley rats. These animals received lead acetate in a dose of00 mg/kg intraperitoneally once daily for three days.

abeling of riboprobe and in situ hybridization

ll plasmids were linearized with appropriate restriction enzymes.5S-uridine-5prime-triphosphate (UTP)-labeled RNA antisense

ig. 1. Measurements of water permeability in AQP4-transfected astrocytn an astrocyte cell line transiently transfected with AQP4 tagged with Ghows that GFP-AQP4 was present at both apical and basal sides of themages are superimposed to allow identification of transfected cells. (E) Rat lung tissue homogenate (L) confirmed that the astrocyte cell line doesecordings from individual cells showing changes in calcein fluorescenceAQP4�) and astrocytes transfected with AQP4 (AQP4�). (G) AQP4�eans�S.E.M. from 373 untransfected (AQP4�) and 44 transfected (AQ

nd sense probes were transcribed in vitro using the appropriate c

emplate and phage T7 RNA polymerase, purified using Nensorbolumns (DuPont, Wilmington, DE, USA), and checked on dena-uring acrylamide gel.

To examine the expression of AQP4 mRNA in the brain, serialoronal sections (14 �m) of rat brain were prepared and stored at80 °C until use. Sections from lead-treated rats and control ratsere placed on the same slides in order to keep them in similaronditions during both hybridization and exposure to X-ray film.he in situ hybridization was performed as described (Zelenin etl., 2002).

ata presentation and analysis

ata are presented as means�S.E.M. Statistical analysis waserformed using Student’s t-test or, when appropriate, ANOVAollowed by Student-Newman-Keuls test for pairwise comparisonsMedCalc Software, Mariakerke, Belgium). A difference was con-idered statistically significant when P0.05.

RESULTS

cute effects of lead on water permeability ofransfected astrocytes

n this protocol studies were performed on an astrocyte

. The water permeability measurements were performed in individual cellsage showing the distribution of GFP-tagged AQP4. (B) The XZ image

The same cells after being loaded with calcein. (D) The GFP and calceinerformed on cell lysate from astrocyte cell line CTX-TNA2 (A) and fromss AQP4. �-Actin was used as an internal standard. (F) Representativefter hypoosmotic shock (300¡150 mOsm) by untransfected astrocytes

s had a significantly higher Pf compared with AQP4� cells. Values aretrocytes. * P0.001.

e cell lineFP. (A) Imcells. (C)T-PCR pnot expreemitted a

ell line derived from rat. The cell line had been trans-

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E. Gunnarson et al. / Neuroscience 136 (2005) 105–114108

ected with a GFP-AQP4 fusion protein. Cells that ex-ressed GFP-AQP4 (approximately 5%) were identifiedy their fluorescent signal. The signal was evenly dis-ributed in the plasma membrane, and the signal fromhe cytoplasm was very low (Fig. 1A, B). By superim-osing GFP images and calcein images (Fig. 1C, D), weould identify the cells that did or did not express AQP4AQP4� cells and AQP4� cells, respectively). To verifyhat the astrocyte cell line did not express endogenousQP4, a RT-PCR study was performed using total RNA

rom rat lung as a positive control. AQP4 mRNA couldot be detected in the astrocyte cell line (Fig. 1E). Timeourses of the calcein fluorescence intensity repre-entative for AQP4� and AQP4� astrocytes are shownn Fig. 1F. AQP4� astrocytes had a significantlyP0.001), approximately three-fold, higher Pf com-ared with AQP4� cells (Fig. 1G). The size of AQP4�nd AQP4� cells was similar, as judged from theiraximal circumference in the XY-projection.

To examine the effect of lead on water permeability,he astrocytes were incubated with or without 50 �M leadcetate for 10 min before Pf measurements. Lead had very

ittle effect on Pf in AQP4� astrocytes (3.8�0.1 �m/s,373, in vehicle-exposed and 3.4�0.1 �m/s, n445, in

ead-exposed AQP4� cells). In contrast, lead exposureesulted in a significantly (P0.001) higher Pf in AQP4�strocytes as compared with vehicle-exposed AQP4� as-

rocytes (16.3�0.6 �m/s, n59, and 11.7�0.6 �m/s, n44,

ig. 2. Lead increases water permeability in AQP4-expressing but notells) were exposed to vehicle or lead for 10 min before measuremenQP4� cells. * P0.001. (B) Dose-response curve of lead effect on

ontrols. (C) Astrocytes transfected with AQP3 (AQP3� cells) were exposedeans�S.E.M. from 13 vehicle-treated and nine lead-treated AQP3� cells. N

espectively) (Fig. 2A). To examine whether the lead effectas specific for AQP4, astrocytes transfected with AQP3

AQP3� cells) were studied. Lead did not change the Pf inQP3� cells (17.3�1.2 �m/s, n9, vs. 16.0�1.0 �m/s,13, in vehicle- and lead-exposed AQP3� cells, respec-ively) (Fig. 2C). Lead had no effect on water permeabilityn AQP3� cells (4.8�0.3 �m/s, n91, vs. 5.3�0.2 �m/s,102).

We then explored the dose-response relationship ofncreasing lead concentrations on the water permeability instrocytes. The dose-response effect of lead on AQP4 Pf

s shown in Fig. 2B. There was no effect of increasing leadoncentrations in AQP4� cells (data not shown).

echanisms for the lead effect in AQP4-expressingells

e next tested whether the observed lead-induced in-rease in water permeability of astrocytes expressingQP4 was reversed after removal of lead from the ex-

racellular medium. Cells were first exposed to lead for0 min as described above. Pf was then measured in theresence of lead. After a washout period of at least oneinute Pf was measured again, using lead-free solu-

ions. The water permeability of AQP4� cells in theresence of lead remained unchanged after the washouteriod. In contrast, application of the lead chelatorMSA for 10 min after lead exposure abolished the

expressing astrocytes. (A) Astrocytes transfected with AQP4 (AQP4�lues are means�S.E.M. from 44 vehicle-treated and 59 lead-treated

P4� astrocytes. Values are means�S.E.M. of increase in Pf in % of

in AQP3-t of Pf. VaPf in AQ

to vehicle or lead for 10 min before measurement of Pf. Values areS, not significant.

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E. Gunnarson et al. / Neuroscience 136 (2005) 105–114 109

ffect of lead on the AQP4� astrocyte water permeabil-ty (Fig. 3). The chelator did not have any effect on waterermeability of vehicle-treated AQP4� or AQP4� cellsr lead-exposed AQP4� cells (data not shown).

It has been suggested that lead may activate intra-ellular signaling pathways that include either CaMKIIWesterink et al., 2002; Suszkiw, 2004) or PKC (Marko-ac and Goldstein, 1988; Bressler et al., 1999). Whenells were preincubated for 10 min with an inhibitor ofaMKII, KN-62 (20 �M), the lead-induced increase inater permeability in AQP4� cells was abolished (Fig.A). The CaMKII inhibitor did not have any effect onater permeability of lead-exposed AQP4� cells. The

nhibitor alone had no effect on water permeability ofehicle-treated AQP4� or AQP4� cells (data nothown). AQP4 has a potential phosphorylation site foraMKII phosphorylation, Ser111, located in the first in-

racellular loop (Gunnarson et al., 2004). This aminocid residue was mutated to alanine to preclude phos-horylation. When expressed in the astrocyte cell line,he mutant AQP4 (AQP4 S111A) was localized in thelasma membrane. Cells expressing AQP4 S111A had.3-fold higher water permeability than neighboring cellshat did not express AQP4. Exposure to lead had noffect on Pf of cells expressing AQP4 S111A13.6�0.6 �m/s, n25, vs. 13.4�0.5 �m/s, n32, inehicle-exposed cells) (Fig. 4B).

In another protocol, cells were preincubated for 10 minith BIM (2�10�6 M) or calphostin (10�6 M), which both

nhibit the classical PKC isoforms. Neither inhibitor had anyffect on the lead-induced increase in water permeability of

ig. 3. Evidence for an intracellular effect of lead. Cells were either ey incubation with a chelator. Washout: Cells were exposed to lead foedium was replaced with a lead-free solution. Then Pf was meanchanged after the washout period. Chelator: Cells were first expos

f was then measured during hypoosmotic perfusion. Values are meay Student-Newman-Keuls test for pairwise comparisons was used. *

QP4� cells (data not shown). s

ffect of lead on Pf of astroglial cells in primaryulture

o test the physiological relevance of our findings wexamined whether the lead-induced increase in AQP4 wa-er permeability may also be present in astroglial cells inrimary culture. We used a primary culture from rat hip-ocampus. Astroglial cells were identified by their typicalhape, characterized by small extensions projecting from aelatively large cell body (Fig. 5A). To verify this, an immu-ocytochemistry study was performed after the Pf record-

ngs were completed. GFAP was used as a marker ofstroglial cells and labeled the majority of cells in theulture. The ratio between astroglial cells and neurons inhe culture was approximately 50:1. To examine if the cellsn primary culture expressed endogenous AQP4, a RT-CR study was performed. Strong AQP4 mRNA signalas detected after a 28 cycle-PCR, indicating that astro-lial cells in this type of primary culture express endoge-ous AQP4 (Fig. 5B). To study the effect of lead on waterermeability, the same procedure as for the astrocyte cell

ine was used. Representative time courses of calceinuorescence intensity observed in control and lead-ex-osed cells are shown in Fig. 5C. The water permeabilityas found to be significantly (P0.001) higher for thestroglial cells exposed to lead than for vehicle-exposedontrol cells (0.052�0.002, n58 and 0.033�0.002, n0, respectively) (Fig. 5D). Z-sectioning of primary culturedstrocytes as well as of AQP4� and AQP4� cell linestrocytes indicated that isoosmotic lead exposure had noffect on the volume and appearance of the cells (data not

o lead only, to lead followed by a washout period or to lead followedfollowed by a washout period when the lead-containing extracellular

ing lead-free solutions. Pf in lead-exposed AQP4� cells remainedd for 10 min, followed by addition of the chelator, DMSA, for 10 min.

. from 9 to 12 AQP4� cells. For statistical analysis ANOVA followed; NS, not significant.

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ong-term effects of lead on AQP4 expression inrain

he effect of lead exposure on the distribution of AQP4RNA in the brain was examined with in situ hybridizationn 10 and 40 day old rats. The results were similar in bothge groups and only results from the 40 day old rats arehown (Fig. 6). The lead treatment in this protocol resulted

n significantly elevated brain lead levels, 0.26�0.01 mg/kget weight, compared with the control brains, where leadas nondetectable. First, a semiquantitative PCR analysisf AQP4 expression in rat cerebellum and cerebrum hadeen performed. There was no detectable difference inQP4 mRNA abundance between lead-treated and control

ats (data not shown).AQP4 mRNA distribution detected by in situ hybridiza-

ion in control rats was in line with what has been reported

ig. 4. Inhibition of CaMKII and substitution of Ser111, a putative phosQP4� cells. (A) Cells were exposed to vehicle or lead for 10 min. Inin before lead and was present throughout the rest of the experimennalysis ANOVA followed by Student-Newman-Keuls test for pairwiseor CaMKII phosphorylation in AQP4, Ser111, was substituted to alaninxposed to vehicle or lead for 10 min. Values are means�S.E.M. fromot significant.

rom other groups (Venero et al., 1999). AQP4 mRNA fi

ignal was detected throughout the rostrocaudal axis of therain. Strong labeling was found in cerebellum (Fig. 6K),round the lateral ventricles (Fig. 6A), around the thirdentricle (Fig. 6C) and in the area of the paraventricularucleus (Fig. 6E). Signals were also detected in the hip-ocampal formation (Fig. 6E, G) and on the pial surfaceFig. 6G, I). Hybridization with the sense probe showed noetectable labeling (Fig. 6M). Treatment with lead acetateid not appear to have any effect on the distribution ofQP4 mRNA in the adult rat brain (Fig. 6A–L). The inten-ity of the signal was also similar.

DISCUSSION

he present study shows that lead specifically increasesater permeability in astrocytes expressing the waterhannel AQP4. This conclusion is based on the following

ion site for CaMKII, to alanine abolish lead-triggered increase of Pf inp of lead-exposed cells, the CaMKII inhibitor KN-62 was added for 10Values are means�S.E.M. from 10 to 24 AQP4� cells. For statisticalons was used. * P0.05; NS, not significant. (B) The consensus sitee mutated AQP4 was expressed in the astrocyte cell line. Cells werele-exposed and 25 lead-exposed cells expressing AQP4 S111A. NS,

phorylatone groutal time.comparise and th

ndings: Lead increased water permeability in an astrocyte

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E. Gunnarson et al. / Neuroscience 136 (2005) 105–114 111

ell line where cells had been transfected with AQP4. Nouch effect was observed in untransfected cells. Lead didot increase water permeability in cells that had beenransfected with AQP3. The observation that lead alsoncreased water permeability in rat astroglial cells in pri-

ary culture underlines the physiological significance ofur findings. It is highly unlikely that the lead-triggered

ncrease in permeability was due to a general toxic effect.ells not expressing AQP4, but cultured and studied under

dentical conditions, actually had a slightly lower waterermeability following lead exposure. The volume and ap-earance of cells did not change during the experimentaleriod. There was no leakage of calcein, indicating that thelasma membrane was intact.

It is well established that heavy metals can regulate ionhannels, either by gain or loss of function. Most mamma-

ian aquaporins, with the exception of AQP4 and AQP6,re inactivated by mercury. Mercury has no effect onQP4 and will activate AQP6 (Yasui et al., 1999; Nicchia etl., 2000). Recent studies suggest that several other heavyetals including nickel and copper (Zelenina et al., 2003,004), gold and silver (Niemietz and Tyerman, 2002) canodulate the activity of aquaporins. Lead is so far the onlyeavy metal shown to modulate the activity of AQP4. Leadas been reported to inhibit voltage-dependent Ca2�-hannels (Bernal et al., 1997; Peng et al., 2002), but the

ig. 5. Effect of lead on astroglial cells in primary culture. (A) Astrogliuorescent dye calcein. (B) RT-PCR performed on RNA extracted froepresentative recordings of calcein fluorescence emitted in single a

he presence of 50 �M lead or vehicle. (D) The water permeability, migher in the astroglial cells exposed to lead than for vehicle-expos

ead-exposed cells. * P0.001.

ffect of lead on water channels has not been reported i

reviously. Lead uptake has been found to be high instrocytes (Holtzman et al., 1984; Lindahl et al., 1999).he finding that the lead-induced increase in water per-eability persisted after lead washout and was abolishednly in the presence of a chelator strongly indicates that

ead was taken up by the astrocytes and acted within theell.

The molecular mechanisms by which lead affects wa-er permeability in AQP4-expressing astrocytes remain toe elucidated. It has been suggested that lead can activaten intracellular signaling pathway that involves CaMKIIWesterink et al., 2002; Suszkiw, 2004). Our results sup-ort this concept. Firstly, lead-triggered increase in AQP4ermeability was abolished in the presence of a CaMKII-

nhibitor. Secondly, mutation of the consensus site foraMKII completely abolished the lead response. It will ben important topic for future studies to explore the role ofhe Ca2�-calmodulin-dependent signaling for the regula-ion of AQP4 activity. It has also been suggested that leadan activate a signaling pathway that involves PKCMarkovac and Goldstein, 1988; Bressler et al., 1999). Weid, however, not find any effect of PKC inhibitors on the

ead response. We cannot exclude that lead also bindsirectly to AQP4, but at the present time relatively little isnown about amino acids that might coordinate lead. Sincehe function of AQP4 in astrocytes can be controlled by its

primary neuron/glia co-culture from rat hippocampus loaded with themary neuron/glia co-culture yielded a strong AQP4 mRNA signal. (C)ells in primary culture after hypoosmotic shock (300¡150 mOsm) inas the fluorescence intensity decay time constant, was significantlyValues are means�S.E.M. from 50 vehicle-exposed cells and 58

al cells inm the pristroglial c

easured

nteraction with other proteins, such as syntrophin (Neely

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E. Gunnarson et al. / Neuroscience 136 (2005) 105–114112

t al., 2001; Amiry-Moghaddam et al., 2003a), it is alsoossible that changes in protein–protein interactions con-ribute to the lead effect.

Investigations of AQP4 mRNA or AQP4 protein re-ponse to various impacts on the brain have revealedomewhat different results. In one study AQP4 mRNAxpression was found to be differentially upregulated inypertrophic astrocytes after lesions induced by injections

nto the rat brain (Vizuete et al., 1999), while in anothertudy AQP4 protein expression was found to be decreasedollowing cortical injury in rat brain (Kiening et al., 2002). In

recent publication, Papadopoulos and Verkman (2005)ound a strong upregulation of AQP4 protein in a model ofeningitis. In our study semiquantitative PCR and in situybridization studies suggested that there was no upregu-

ation of AQP4 mRNA in either 10 or 40 day old rats afterhree days of lead exposure. Furthermore, there was noign of AQP4 mRNA redistribution in the brain after leadxposure.

Emerging evidence suggests that the function of astro-yte AQP4 may have different impacts in physiological andathophysiological situations. Under physiological condi-

ions AQP4 has an important protective effect by facilitat-ng the potassium siphoning that occurs after neuronaltimulation (Nagelhus et al., 1999; Amiry-Moghaddam et

ig. 6. In situ hybridization analysis of AQP4 distribution in the brain inections hybridized with a 35S-antisense AQP4 riboprobe. Distributionreated with lead acetate for three consecutive days (B, D, F, H, J, L).RNA distribution or expression levels could be detected after lead tre

ormation; LV, lateral ventricle; Pa, paraventricular hypothalamic nucle

l., 2003b). There is also, however, compelling evidence d

hat the presence of AQP4 can be harmful in brain edema.QP4 knockout mice develop less brain edema after acuteater intoxication and have improved neurological out-ome after ischemic stroke (Manley et al., 2000). Alpha-yntrophin knockout mice, which lack the perivascular poolf AQP4 in astrocytes, have less pronounced brain edemaollowing transient cerebral ischemia than wild type miceAmiry-Moghaddam et al., 2003a). Removal or displace-ent of perivascular AQP4 is associated with a delayedevelopment of edema after water intoxication (Vajda etl., 2002; Amiry-Moghaddam et al., 2004). Acute lead

ntoxication is associated with brain edema (Pappas et al.,986; Perelman et al., 1993; Rojas-Marcos et al., 2002).aking the adverse effects of AQP4 in brain edema intoccount, our results may offer an explanation to some ofhe pathology of acute lead intoxication. Furthermore, con-idering the evidence regarding the harmful effects ofQP4, the development of an inhibitor of AQP4 should beeneficial for suppressing detrimental effects of events

eading to brain edema. Such an approach may prove toe useful in the situation of acute lead intoxication. Assum-

ng that the lead-induced activation of AQP4 water perme-bility is potentially hazardous, the lack of downregulationf AQP4 mRNA as a response to lead may prove to be

nd lead-treated rats. Autoradiograms of in situ hybridization of frontalmRNA in brain in 40 day old control rats (A, C, E, G, I, K) and rats

um (K, L) showed strong hybridization signal. No alterations in AQP4ybridization with sense probe showed no signal (M). Hi, hippocampal

third ventricle. Arrowheads in G–J indicate pial surface.

control aof AQP4Cerebell

isadvantageous in the situation of acute lead intoxication.

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E. Gunnarson et al. / Neuroscience 136 (2005) 105–114 113

cknowledgments—The authors would like to thank Louiseustafsson for excellent technical assistance. This study haseen supported by Swedish Research Council, CTRF, Märta ochunnar V. Philipsons Stiftelse, The Family Persson Foundation,tiftelsen Samariten, Sällskapet Barnavård, Russian Foundation

or Basic Research and the Nordic Center of Excellence Programn Molecular Medicine.

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(Accepted 6 July 2005)(Available online 3 October 2005)