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Transcript of Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes
The
The Veterinary Journal 168 (2004) 143–150
Veterinary Journalwww.elsevier.com/locate/tvjl
Aquaporin water channels AQP1 and AQP3, are expressedin equine articular chondrocytes
Ali Mobasheri a,*, Elisa Trujillo b, Susan Bell a, Stuart D. Carter a,Peter D. Clegg a, Pablo Mart�ın-Vasallo c, David Marples d
a Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UKb Servicio de Reumatologia, Hospital Universitario de Canarias, La Cuesta, Tenerife, Spain
c Departamento de Bioqu�ımica y Biolog�ıa Molecular, Universidad de La Laguna, La Laguna, Tenerife, Spaind School of Biomedical Sciences, University of Leeds, Leeds, UK
Accepted 1 August 2003
Abstract
Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular
cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells,
which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the
water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is
effectively influenced by the abundance of aquaporin (AQP) water channels. Recent studies have shown that several AQP water
channel isoforms are expressed in chondrocytes from Meckel�s cartilage, developing teeth and other orofacial tissues. The aim of the
present investigation was to determine if chondrocytes from equine articular cartilage express AQP water channels. Polyclonal
antibodies to AQP1, AQP2 and AQP3 were used in conjunction with immunohistochemistry, immunoblotting and quantitative flow
cytometry to determine if AQP1, AQP2 and AQP3 are expressed in equine articular chondrocytes. Our studies show that AQP1 and
AQP3 are expressed by chondrocytes in articular cartilage in situ and in isolated chondrocytes. We found no evidence for expression
of AQP2, the vasopressin-regulated water channel in chondrocytes. AQP1 and AQP3 may be involved in the transport of water and
small solutes and osmotically active metabolites across the chondrocyte plasma membrane during volume regulatory behaviour.
AQP1 may be involved in transporting metabolic water. AQP3 may participate in the transport of glycerol and structurally related
molecules.
� 2003 Elsevier Ltd. All rights reserved.
Keywords: Equine articular cartilage; Chondrocyte; AQP1; AQP3; Water permeability; Volume regulation; Immunohistochemistry; Flow cytometry
1. Introduction
Articular chondrocytes are specialized cells responsi-
ble for the synthesis and maintenance of cartilage matrix
(Buckwalter and Mankin, 1998). The extracellular ma-trix of cartilage consists of water (up to 70% of total
tissue weight), type II collagen, large aggregating pro-
teoglycans, small proteoglycans and smaller non-
collagenous matrix proteins. The cross-linked collagen
forms a meshwork in which negatively charged proteo-
* Corresponding author. Tel.: +44-151-794-4284; fax: +44-151-794-
4243.
E-mail address: [email protected] (A. Mobasheri).
1090-0233/$ - see front matter � 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2003.08.001
glycans are trapped attracting cations (mainly Naþ, Kþ
and Ca2þ) and osmotically obliged water which, in turn,
results in swelling of the proteoglycans, increasing the
tension within the collagen network. This swelling
mechanism provides the extracellular matrix with thefacility to resist tension and shear forces, thereby giving
cartilage its ability to resist compression under static and
dynamic mechanical load.
Chondrocytes are excellent sensors of ionic and os-
motic stimuli and respond to these signals in coordina-
tion with other environmental, hormonal and genetic
factors to regulate metabolic activity (Guilak, 2000;
Mobasheri et al., 2002). Although this metabolic regu-lation is essential to the health, structural integrity and
144 A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150
functional performance of articulating joints (Buckw-alter and Mankin, 1998), the mechanisms by which ionic
and osmotic changes modulate chondrocyte metabolism
are poorly understood (Guilak et al., 2002). Adaptation
to ionic and osmotic stimuli involves changes in the
physical properties of the chondrocyte plasma mem-
brane and the activity of channels and transporters
(Guilak et al., 2002; Mobasheri et al., 1998, 2002).
Chondrocytes have the capacity to regulate their volumein response to hyper and hypo-osmotic stress (Guilak
et al., 1995). This volume regulatory activity involves
mobilization of osmotically active solutes (osmolytes)
and water across the chondrocyte plasma membrane.
However, the molecular identity of plasma membrane
systems responsible for osmolyte and water transport in
chondrocytes has remained unknown.
Aquaporins (AQPs) are a family of water channelproteins related to the major intrinsic protein (MIP or
AQP0). Mammalian AQP proteins form water-perme-
able channels that provide the plasma membranes of red
cells, kidney proximal and collecting tubules and other
tissues with high permeability to water, thereby per-
mitting water to move in the direction of an osmotic
gradient. AQPs are expressed in diverse tissues (Borgnia
et al., 1999; Marples, 2001). Some members of the AQPgene family are permeable, not only to water, but also,
to small solutes such as glycerol and urea. The mam-
malian aquaporin family consists of ten members that
fall into two categories: the traditional aquaporins are
water channels and include AQP0, AQP1, AQP2,
AQP4, AQP5, AQP6 and AQP8; the unorthodox,
multifunctional aquaglyceroporins may transport water
in addition to small solutes. This latter category includesAQP3, AQP7 and AQP9 (Borgnia et al., 1999).
AQP1, originally known as CHIP28 (Agre et al.,
1987), or AQP-CHIP, is a homotetramer with six bilayer
spanning domains and is expressed in most tissues in the
mammalian body (Agre et al., 1993). AQP1 is a possible
candidate for disorders involving imbalance in ocular
fluid movement. AQP2 is a vasopressin-regulated renal
water channel and is highly expressed in the cortex andmedulla of kidney (Fushimi et al., 1994). AQP3 is highly
expressed in the kidney, colon, liver and small intestine,
where it has been proposed to transport water and small
solutes such as glycerol and urea (Ishibashi et al., 1994.
Ma et al., 1994).
We have previously hypothesized that water and
osmolyte channels are involved in cell volume regulation
and possibly also mechanotransduction in chondrocytes(Mobasheri et al., 1998, 2002; Trujillo et al., 2000). The
aim of this study was to determine if aquaporin water
channels are expressed by articular chondrocytes.
Polyclonal antibodies to AQP1, AQP2 and AQP3 were
used in conjunction with immunohistochemistry, Wes-
tern blotting and quantitative flow cytometry to com-
pare the relative expression levels of these three
aquaporins in sections of equine cartilage and in isolatedequine chondrocytes.
2. Materials and methods
2.1. Chemicals
Unless otherwise stated, all chemicals used in thisstudy were molecular biology grade and purchased from
Sigma/Aldrich and Sigma Biosciences. Materials for
immunohistochemistry were purchased from Vector
Laboratories and Dako Cytomation.
2.2. Tissues
Equine articular cartilage was obtained from thefemoropatellar and metacarpophalangeal joints of
horses at the Equine Division at the Philip Leverhulme
Large Animal Hospital, University of Liverpool. The
horses used for cartilage samples were euthanased for
clinical reasons and not for the purposes of the present
investigation. Full depth sections (8 lm thickness) of
articular cartilage from all zones (superficial, middle,
deep and calcified) were provided by the histopathologyservice of the Department of Veterinary Pathology,
University of Liverpool. Formalin-fixed paraffin-
embedded ovine kidney mounted on 3-aminopropyl-
triethoxysilane (APES) treated slides were used as a
positive control for AQP1, AQP2 and AQP3 expression.
2.3. Antibodies
Immunohistochemical studies were conducted using a
panel of polyclonal isoform-specific antibodies raised in
rabbits against rat AQP1, AQP2 and AQP3 (Chemicon
International). Polyclonal antibodies to rat AQP1 and
AQP2 developed in the laboratory of Dr. D. Marples
were also used in this study.
2.4. Immunohistochemistry
The DAKO EnVisionTM+ System, HRP (Dako
Cytomation) was used for visualisation of the antibody
staining. This is a two-step immunohistochemical
staining technique. This system is based on a horserad-
ish peroxidase (HRP) labelled polymer which is conju-
gated with secondary antibodies specific for rabbit. The
labelled polymer does not contain avidin or biotin andconsequently, non-specific staining resulting from en-
dogenous avidin–biotin activity is eliminated. Briefly,
full depth equine cartilage explants, including sub-
chondral bone, were removed and fixed for 2 h in 4%
paraformaldehyde in phosphate-buffered saline (PBS).
Explants were decalcified overnight in 10% EDTA in
PBS before paraffin embedding and sectioning (7 lm)
A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150 145
onto Vectabond coated slides (Vector Laboratories).The slides were de-waxed in xylene for 10 min at room
temperature (RT) before rehydration in a series of gra-
ded ethanol baths (100%, 70%, 50%). Wax boundaries
were drawn around the tissue sections using a paraffin
wax immunohistochemistry pen (DAKO). The endoge-
nous peroxidase activity in the cartilage sections was
quenched with by a one-hour incubation with 3% hy-
drogen peroxide solution in PBS (pH 7.4), containing 27mM KCl and 0.137 M NaCl. Sections were transferred
to a stainless steel humidified chamber and non-specific
binding blocked for 1 h using a 10% normal goat serum
(NGS) solution in PBS. The NGS was derived from a
donor herd of goats certified to have been pathogen free
at the time of bleeding and was essential for blocking
non-specific binding by the primary and secondary
antibodies.Full depth equine cartilage sections were incubated
overnight at 4 �C with the AQP1-3 antibodies diluted
1:200 in PBS containing 2% NGS. Subsequently, the
sections were rinsed 3� 15 min in PBS before incubation
with the secondary peroxidase-conjugated antibodies
(peroxidase-labelled polymer conjugated to goat anti-
rabbit immunoglobulins in Tris–HCl buffer containing
bovine serum albumin as a carrier protein) for a mini-mum of 1 h at RT. After rinsing in PBS (4� 15 min) the
peroxidase product was developed using DAKO Liquid
DAB+ Chromogen (3,30-diaminobenzidine chromogen
solution) which produces an insoluble brown coloured
product in the presence of the antibody–polymer com-
plex. Sections were counterstained with Mayer�s hae-
matoxylin, dehydrated in a series of ethanol baths
before being mounted in DPX and photographed usinga Nikon Microphot-FX microscope fitted with a Nikon
DXM1200 digital camera.
2.5. Equine chondrocyte isolation and FACS analysis
Equine chondrocytes were isolated by overnight di-
gestion of cartilage shavings in Dulbecco�s modified
Eagle�s medium (DMEM) supplemented with 5% fetalcalf serum, 1% antibiotic/antimycotic solution and
0.05% clostridial collagenase. Cell suspensions were
fixed for 10 min in 3.7% neutral buffered paraformal-
dehyde in PBS for 10 min at 37 �C. The fixed cells were
washed three times with PBS and incubated in 10%
NGS in PBS to quench non-specific binding sites.
Aliquots of 105 cells /100 lL PBS were placed into 1.5
mL Eppendorf tubes, spun down (2000 rpm, 5 min) andthe cell pellets were incubated with 100 lL aliquots of
AQP antibodies (AQP1, AQP2 and AQP3) at 4 �Covernight. Negative controls were incubated with 100
lL of a non-immune rabbit IgG (DAKO). Following
three washes with PBS, the cells were incubated with 100
lL FITC-conjugated goat anti-rabbit IgG diluted 1:100
(Sigma, UK) in PBS at 4 �C for 1 h in the dark. After
three further washes in PBS, the cells were resuspendedin 0.5 mL PBS and analyzed on a Becton–Dickinson
FACScan. Plot profiles and histograms were generated
using WinMDI flow cytometry software (http://facs.
scripps.edu/software.html). Statistical analysis was also
performed using WinMDI.
2.6. Western blotting with AQP1, AQP2 and AQP3
antibodies
Freshly isolated equine chondrocytes were washed
three times with serum-free DMEM and three times
with PBS. Total proteins were extracted with lysis buffer
(50 mM Tris–HCl, pH 7.2/150 mM NaCl/1% (v/v) Tri-
ton X-100/1 mM sodium orthovanadate/50 mM sodium
pyrophosphate/100 mM sodium fluoride/0.01% (v/v)
aprotinin/4 lg/mL pepstatin A/10 lg/mL leupeptin/1mM PMSF) on ice for 30 min. Insoluble material was
removed by centrifugation at 10 000g for 30 min. Ly-
sates were stored at )70 �C until use. For immuno-
blotting, equal amounts of total proteins were separated
on 12% polyacrylamide gels by SDS/PAGE under re-
ducing conditions. Proteins were transferred overnight
at 30 V onto nitrocellulose (Schleicher & Sch€ull) using a
mini transblot electrophoresis apparatus (Sigma).Membranes were blocked with 5% (w/v) skimmed-milk
powder in PBS/0.1% Tween 20 (blocking buffer) over-
night at 4 �C and incubated with primary antibodies to
AQP1 (1:500), AQP2 (1:500) and AQP3 (1:1000) diluted
in blocking buffer for 1 h at RT. After five washes in
blocking buffer, membranes were incubated with per-
oxidase-conjugated secondary antibody diluted in
blocking buffer for 30 min at RT. Membranes were fi-nally washed five times in blocking buffer, and devel-
oped using an Amersham Biosciences ECL enhanced
chemiluminescence kit.
3. Results
Immunohistochemical studies revealed high levels ofAQP1 expression in equine articular cartilage, particu-
larly in the deep and calcified zones of articular cartilage
(Fig. 1). The vasopressin-regulated AQP2 was not de-
tected in any of the equine articular cartilage zones ex-
amined (Fig. 1E–H). AQP3 was moderately expressed in
equine articular cartilage, particularly in the deep and
calcified zones (Fig. 1I–L). Full depth sections of equine
articular cartilage revealed that in addition to articularchondrocytes AQP1 is abundantly expressed in cells
bearing the morphological characteristics of osteocytes
in the subchondral bone (Fig. 1D). AQP3 was also ex-
pressed in low quantities in cells resembling osteocytes
in the subchondral bone (Fig. 1L).
In order to demonstrate that the polyclonal anti-
bodies were specific to the aquaporin isoform against
Fig. 1. Immunohistochemical localization of AQP1 and AQP3 in all zones of normal equine articular cartilage including superficial, middle, deep and
calcified cartilage. The cartilage was obtained from the normal femoropatellar joint of a 5-year-old animal. Although AQP1 expression was evident in
chondrocytes of all zones (brown peroxidase staining in A–D) it was most abundantly expressed in cells of the deep zone (C). AQP2 expression was
not detected in any of the equine cartilage zones (E–H). AQP3 expression was expressed in lower quantities compared to AQP1 but it was present in
large quantities in deep zone chondrocytes (K). Moderate AQP1 and AQP3 expression was evident in bone cells in the calcified zone and subchondral
bone (D, L). Bars indicate 10 lm.
146 A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150
which they were raised, we performed immunohisto-chemistry on formalin fixed, paraffin embedded sections
of normal ovine kidney. Due to the difficulties associ-
ated with obtaining fresh equine kidneys, we decided to
use fresh ovine kidneys as positive for AQP1, AQP2 and
AQP3 in the nephron. Fig. 2 shows expression of AQP1,
AQP2 and AQP3 in ovine renal cortex, medulla and
papilla. Abundant AQP1 immunostaining was observed
in the apical membranes of the proximal tubule and thedescending thin limb (as revealed by brown coloured
peroxidase staining in sites of AQP expression). AQP2
was expressed in the apical membrane domain of prin-
cipal cells in collecting ducts. AQP3 expression was
observed in lower quantities in basolateral membranes
of collecting ducts; labelling was mainly restricted to the
cortical, outer medullary, and inner medullary collecting
ducts.Quantitative FACS analysis demonstrated that iso-
lated equine chondrocytes express AQP1 and AQP3
(Fig. 3). Specific AQP1 fluorescence was detected
in 41.84% of the cells. AQP2 fluorescence was detected
in 1.13% of the cells (not shown). AQP3 was detected in
29.31% of the cells shown in the gated area (labelled R2
in Fig. 4A). These results confirm that antibodies to ratAQP1, AQP2 and AQP3 cross-react with equine AQPs.
Western blotting of freshly isolated equine articular
chondrocyte lysates was used to demonstrate the pres-
ence of AQP1 and AQP3 proteins. Fig. 4. shows the
Western blot data confirming AQP1 and AQP3 ex-
pression in equine chondrocytes. AQP2 expression was
not detected. We also confirmed the molecular weight of
AQP1 in equine chondrocytes; the main AQP1 bandwas approximately 28 kDa; the larger band in the AQP1
lane probably corresponds to the highly glycosylated
form of the AQP1 protein. AQP3 was detected as a
single protein migrating at around 28–30 kDa.
4. Discussion
Aquaporins belong to a large gene family of trans-
membrane proteins that form water permeable channels,
which may also be permeated by other small solutes.
Aquaporins are expressed in a variety of water trans-
porting epithelia and many other tissues where they fa-
cilitate water and solute transport across the cell
Fig. 2. Expression of AQP1, AQP2 and AQP3 in the ovine renal cortex, medulla and papilla. AQP1 was present in the apical (brush border)
membrane domain of proximal tubule segments (as indicated by brown peroxidase staining). AQP1 immunolabelling was also observed in the
squamous epithelium lining the descending thin limbs of the ovine loop of Henle which accounts for the high water permeability of this nephron
segment in the medulla (B) and to a lesser extent, the papilla (C). AQP1 expression was not detected in the thick ascending limb and medullary
collecting ducts. Expression of AQP2 was significantly lower than AQP1 and appeared to be particularly restricted to the apical membrane domain of
cells lining the distal nephron (cortical, medullary and papillary collecting ducts; D–F). AQP3 expression was more intense in the cortex and medulla
compared to the papilla. AQP3 expression was observed in basolateral membranes of the cortical and medullary collecting ducts (G, H).
A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150 147
membrane (Borgnia et al., 1999; Fushimi and Marumo,
1995; Nielsen and Agre, 1995). It is generally accepted
that AQP1 is the ubiquitously expressed, archetypal
water channel (Agre et al., 1993; Denker et al., 1998;
Nielsen et al., 1993; Sabol�ıc et al., 1992). AQP2 is avasopressin-regulated water channel expressed in renal
medullary collecting ducts (Fushimi et al., 1994; Nielsen
et al., 1993; Sabol�ıc et al., 1995). Unlike AQP1, which is
constitutively present in the plasma membrane, AQP2
abundance is regulated by vasopressin through short-
term exocytosis to the membrane and protein recycling
(Marples, 2001). AQP3 is a multifunctional channel
protein permeable to water, glycerol, urea and othersolutes (Ishibashi et al., 1994; Ma et al., 1994).
Although a great deal is known about the expression,
regulation and physiological function of aquaporins in
absorptive and secretory epithelia, thus far, there are
only three indirect reports of aquaporin expression in
skeletal cells. Early developmental studies in the rat have
demonstrated abundant AQP1 mRNA in the mesen-
chyme surrounding developing calcified bone (Bondy
et al., 1993). AQP1 has been found in the periosteum of
the guinea-pig inner ear (Stankovic et al., 1995). In ad-dition AQP1 and AQP3 have been identified in devel-
oping human teeth and selected orofacial tissues (Wang
et al., 2003). There are no reports of aquaporins in
articular chondrocytes from articular cartilage of load-
bearing joints despite the potential importance of aqu-
aporins in volume regulatory chondrocyte responses
load-induced to ionic and osmotic changes in their ex-
tracellular environment.Chondrocytes have been shown to be particularly
sensitive to their physicochemical environment (Guilak,
2000, 1995; Guilak et al., 2002). In response to osmotic
stresses that are often secondary to mechanical com-
pression of cartilage, chondrocytes re-organize their
Fig. 3. FACS analysis of isolated equine chondrocytes probed with polyclonal antibodies to AQP1, AQP3 and secondary FITC-conjugated anti-
rabbit IgG. (A) Scatter plot of starting chondrocyte population based on cell size and granularity. (B) Background fluorescence from control
chondrocytes (incubated with FITC–conjugated secondary IgG only). (C) Scatter plot of chondrocytes probed with AQP1 antibodies. (D) Specific
fluorescence from cells incubated with antibodies to AQP1. (E) Scatter plot of chondrocytes probed with AQP3 antibodies. (F) Specific fluorescence
from cells incubated with antibodies to AQP3. AQP1 and AQP3 were expressed in 41.84% and 29.31% of the cells, respectively. The peaks shown
under the M1 bar in D and F represents cells exhibiting a specific fluorescence signal above the pre-set threshold limit shown under the M1 bar in B.
Antibodies to AQP2 did not result in any specific staining or fluorescence signal (results not shown).
148 A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150
cytoskeleton and activate plasma membrane transport
mechanisms to regulate normal cell volume. This regu-lation is essential for the maintenance of the extracel-
lular matrix since the metabolic activity of chondrocytes
is influenced by alterations in the osmotic environment
of cartilage tissue (Guilak et al., 2002). The transport of
water across the chondrocyte plasma membrane con-
stitutes an important component of volume regulatory
activities during recovery from swelling and shrinkage.
In view of this potentially important function and the
apparent lack of published information, in this study,
we examined the expression of three aquaporins (AQP1,AQP2 and AQP3) in sections of articular cartilage and
freshly isolated chondrocytes and showed that AQP1
and AQP3 are present in chondrocytes.
The immunohistochemical, FACS and Western blot
analyses presented in this paper strongly indicate that
AQP1 and AQP3 are expressed in equine articular car-
tilage and isolated equine chondrocytes. The immuno-
histochemistry data presented clearly show plasma
Fig. 4. Western blot analysis of AQP1 and AQP3 expression in equine
articular chondrocytes. The Western blot data confirm the presence of
AQP1 and AQP3 but reveal the absence of AQP2 expression in equine
chondrocytes. The molecular weight of AQP1 in equine chondrocytes
was confirmed to be approximately 28 kDa; larger bands in the AQP1
lane may correspond to the highly glycosylated form of the AQP1
protein. The AQP3 protein was detected as a single protein of 28–30
kDa.
A. Mobasheri et al. / The Veterinary Journal 168 (2004) 143–150 149
membrane AQP1 and AQP3 expression in chondro-
cytes. Chondrocytes in the deep zone of articular carti-
lage exhibited the strongest AQP1 and AQP3 expression
levels. These studies confirm our preliminary observa-
tions in human and porcine articular cartilage (Trujillo
et al., 2000) and support a role for an aquaporin (AQP1)and an aquaglyceroporin (AQP3) in water and small
osmolyte transport across the plasma membranes of
these cells, respectively. AQP1 may be responsible for
conferring water permeability to chondrocytes and may
play an important role in allowing chondrocytes to re-
spond to changes in their ionic and osmotic environ-
ment. AQP3 may be involved in the transport of water,
glycerol and structurally related small osmolytes acrossthe chondrocyte membrane. Recent studies in peritoneal
mesothelial cells indicate AQP1 expression is up-regu-
lated by osmotic agents such as glucose (Lai et al., 2001).
In addition, studies in cultured human keratinocytes
show that osmotic stress up-regulates AQP3 gene ex-
pression (Sugiyama et al., 2001). Thus, AQP1, AQP3
and other AQPs may enable chrondrocytes to respond
to changes in their osmotic environment by regulatingcell volume.
In conclusion, we have shown that AQP1 and AQP3
are expressed in equine articular chrondrocytes. These
water channel proteins are likely to play a critical role in
the regulation of intracellular homeostasis in chron-
drocytes and may be important in allowing chondro-
cytes to respond to changes in their ionic and osmotic
environment by volume regulatory behaviour. Thepresence of AQP1 in equine chondrocytes supports a
role for AQP1 in water transport in across the chon-
drocyte membrane. The water being transported across
the chondrocyte membrane is likely to be metabolic or
extracellular matrix water. AQP3 may be involved in
water and solute transport in equine chondrocytes.
Based on the data presented, we propose a novel hy-
pothesis that extracellular matrix properties may influ-ence the chondrocyte�s internal environment and vice
versa and expression of sodium and water transporters
is a manifestation of such physiological adaptation. It is
possible that the expression of AQPs is altered when
chondrocytes are removed from their surrounding ex-tracellular matrix. It is also possible that mechanical and
osmotic pressure and consequent effects on the osmotic
environment of the cells will result in alterations in
chondrocyte AQPs. Future studies will focus on the
expression of AQP1, AQP3 and other members of the
AQP gene family in pathologies of articular cartilage
and their sensitivity to mercurial and stilbene derived
compounds capable of interfering with AQP function.
Acknowledgements
Supported by grants from the University of Liverpool
Research Development Fund (AM), the Medical Re-
search Council (DM) and Programa Sectorial de Pro-
mocion General del Conocimiento, Ministerio deEducacion y Cultura, Spain (ET, P M-V). The authors
wish to thank Dr. Alan Verkman (Cardiovascular Re-
search Institute, University of California at San Fran-
cisco) for helpful suggestions.
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