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JEZ 776
EXPERIMENTAL ZOOLOGY 277:460–463 (1997)
Hypotonic Stress Induces Translocation of the
Osmolyte Channel Protein pICln in Embryonic
Skate (Raja eglanteria) Heart
Inflammatory Bowel Disease Center, Department of Medicine,
The University of Chicago, Chicago, Illinois 60637
Division of Biology and Medicine, Brown University, Providence,
Rhode Island 02912
Mote Marine Laboratories, Sarasota, Florida 34236
Volume expansion of cardiac cells from a wide variety of species stimulates the
efflux of the β-amino acid taurine through an osmolyte channel. Previous studies have suggested
that the osmolyte channel in epithelial cells is a swelling-activated anion channel (pICln). In skate
heart, a 37-kDa protein is present which is recognized by a specific antibody to a protein characterized in MDCK cells as pICln. This protein is present predominantly in the cytosol (only 10% in
the membrane fraction) of heart incubated under isotonic conditions. After transfer to hypotonic
medium (one-half osmolarity), the distribution of this protein is markedly altered and significant
amounts of the protein are found in the membrane fraction. After hypotonic exposure, the amount
of the protein in the membrane fraction rises to 38 ± 11% (range 18–53, n=3). The translocation to
the membrane fraction suggests that this protein may play a role in the taurine efflux in this
tissue stimulated by hypotonic stress. J. Exp. Zool. 277:460–463, 1997. © 1997 Wiley-Liss, Inc.
Under volume-expanded conditions, cardiac
cells, like a wide variety of other cells, demonstrate an efflux of osmolytes to accomplish a
regulatory volume decrease (RVD; for review
see Strange et al., ’96). A number of solutes participate in the RVD including the β-amino acid
taurine, polyols, methlamines, and electrolytes.
Cardiac cells of the skate, like those of many
species, accumulate millimolar concentrations
of the nonmetabolized amino acid taurine,
which is a major solute lost during the RVD
from this tissue (Goldstein et al., ’93). In the
chicken heart, volume-activated taurine efflux
is thought to occur through a swelling-activated
anion channel (Zhang et al., ’93) similar to
channels found in MDCK cells (Banderali and
Roy, ’92), lung cells (Kirk and Kirk, ’93), and
C6 glioma cells (Jackson and Strange, ’93).
Strange et al. (’96) have proposed that during RVD, osmolytes may be lost from epithelial
cells via a pathway involving the protein pICln.
Antibodies to this protein recognize a 37-Kda
protein (pICl n) in cells from a wide variety of
tissues and species (Krapivinsky et al., ’94) and
cloning techniques have identified this protein
in rabbit heart (Okada et al., ’95) and ciliary
epithelium (Anguita et al., ’95). This protein is
found in the cytosol and is not predicted to be
an integral membrane protein by any of the hydropathy algorithms used. Therefore, it is unclear how this protein may mediate the efflux
of solutes across the membrane. A model has
been proposed that upon such stimuli as hypotonic shock, the protein forms a dimer, perhaps
in a beta-barrel configuration, and that this
form inserts into the cell membrane and acts
like a channel or regulates a preexisting anion
channel (Buyse et al., ’96). Therefore, the aim
of the present studies was to test this model
and determine where this protein resides in the
skate cardiac cytosol under isotonic conditions
and if redistribution to the cell membrane occurs under hypotonic conditions.
Contract Grant sponsor: NSF; Contract Grant number IBN9505567; Contract Grant sponsor: NIH; Contract Grant number DK-38510, DK-42086; Contract Grant sponsor: Walt Disney
*Correspondence to: Leon Goldstein, Ph.D., Division of Biology and
Medicine, Brown University, Box G B311, Providence, RI 02912.
Received 9 October 1996; Revision accepted 18 December 1996
Distribution of pICln in embryonic
skate heart
Skate embryos for these studies were used 7–
11 weeks after oviposition with cardiac masses of
4–17 mg. Details of the surgical technique used
to obtain hearts were described previously (Goldstein et al., ’93). Hearts were incubated in isoosmotic elasmobranch incubation medium (940
EIM; 940 mosmoles/1, composition in mmol/l: 300
NaCl, 5 CaCl2, 5.2 KCl, 2.7 MgSO4, 15 Tris pH
7.4, 370 urea) or one-half osmolarity (460 EIM,
NaCl reduced to 100 and urea to 250 mM). Hearts
were placed individually into beakers containing
30 mL of either 940 EIM or 460 EIM. Hearts were
incubated for 1 h with constant, gentle oxygenation of the incubation medium. Following incubation, the hearts were frozen immediately on dry
ice. Frozen hearts were placed in 200 µl buffer
(composition in mmol/l: 10 Tris pH 7.4, 5 MgCl2,
1 phenylmethylsulfonyl fluoride, with 10 µg/ml
leupeptin, aprotinin, pepstatin, and tosylphenylchloroketone). Hearts were minced finely with a
razor blade and then homogenized by 30 strokes
with a tight-fitting glass homogenizer. Poorly broken material was pelleted (500 g for 2 min at 4°C).
The membrane fraction was resuspended in homogenization buffer and aliquots of both cytosol
and membrane fractions removed for protein
analysis using the bicinchononic acid procedure
(Smith et al., ’85). One-half volume of 3X Laemmli
stop solution (1% 2-mercaptoethanol, 0.5 M Tris
pH 6.8, 0.5% w/v SDS, 50% v/v glycerol) was added
to the remainder and samples were heated to 85°C
for 10 min and frozen.
For analysis of pICln 10 µg of protein was separated on 12.5% SDS-PAGE, and transferred to a
polyvinylidene fluoride membrane (Immobilon) using Towbin’s buffer (25 mM Tris, 192 mM glycine,
pH 8.8). Blots were blocked in 5% w/v nonfat dry
milk in Hanks buffered saline (HBS) with 0.2%
v/v Nonidet P40 (5% Blotto) for one hour. Blots
were incubated with affinity-purified polyclonal
anti-pICln antiserum (1 µg/ml) in HBS with 1%
w/v bovine serum albumin, overnight at 4°C. For
determination of separation of cytosol and membrane fractions, marker proteins were analyzed.
Na-K-ATPase was selected as the membrane
marker and hsp73/72 as the cytosol marker. Blots
were then washed in 5% Blotto and 1% Blotto
(milk reduced to 1% w/v) and then incubated in
peroxidase-conjugated goat anti-rabbit IgG or goat
anti-mouse IgG (1:4,000) for 60 min in 1% Blotto.
Blots were then washed in 1% Blotto and then in
HBS with Nonidet P40. pICln protein and the
marker proteins were detected using an enhanced
chemiluminescent system.
Antibody to pICln was a generous gift of Drs. D.
E. Clapham and G. B. Krapivinsky (Mayo Institute, Rochester, MN). Immobilon membrane was
obtained from Millipore (Medford, MA), peroxidase-conjugated goat anti-rabbit IgG and goat
anti-mouse from Southern Biotechnology (Birmingham, AL), anti-Na-K-ATPase from Upstate
Biotech (Lake Placid, NY), anti-hsp73/72 from Affinity Bioreagents (Boulder, CO), Nonidet P 40
from Amersham (Cleveland, OH), and chemiluminescent reagents from Pierce (Rockford, IL). All
other reagents were of the highest grade available and obtained from Fisher (Itasca, IL) or
Sigma Chemical (St. Louis, MO).
Identification of pICln in skate heart
Western blots were used to determine whether
pICln is found in skate heart and is similar in size
to the protein in MDCK cells. A specific polyclonal
antiserum recognizes a protein of similar molecular mass in both cell types (Fig. 1). The abundance
of the protein is likely lower in cardiac myocytes
since 10 µg of MDCK protein versus 200 µg of
skate heart protein were required to obtain similar Western blots. Although intact heart was used
in these experiments, cardiac myocytes compose
a majority of the cells in the tissue. Additionally,
as erythrocytes do not have pICln, these cells
would not interfere with the analysis (DavisAmaral et al., ’97).
Hypotonicity stimulates
redistribution of pICln
Under isotonic conditions, pICl n is predominantly in the cytosol of skate cardiac cells (Fig.
2). The amount of pICln in the cytosol and membrane fractions of skate hearts was analyzed
densitometrically using NIH IMAGE 1.54 software. In two of three cases, less than 10% of pICln
was in particulate fraction and in the other case,
this value was 19.8%. When the hearts are incubated in one-half hypotonic medium, the distribution of pICln changes dramatically (Fig. 2).
Large amounts of the protein redistribute so that
substantial amounts of pICln are found in the particulate fraction. This redistribution was observed
Fig. 2. Distribution of pICln under isotonic and hypotonic
conditions. Cell fractions were made as described in Materials and Methods and pICln in the fractions determined by
Western blotting. One-twentieth of the total protein in each
fraction was used to determine distribution of pICln. Chemiluminescent images shown represent distribution of pICln of
two hearts under hypotonic conditions and two different
hearts under isotonic conditions. M = membrane fraction. C
= cytosol fraction.
Fig. 1. Demonstration of pICln protein in skate heart.
MDCK (10 µg protein) was used since pICln is readily identified in these cells and 200 µg protein of skate heart homogenate was used. Western blots were developed with an
affinity-purified polyclonal antisera. Chemiluminescent image shown is representative of one of three different hearts.
in every heart used. The means (± S.E., n=3) for
particulate and cytosol distribution under isotonic
conditions were 10 ± 4% and 90 ± 4%, respectively,
and under hypotonic conditions these values were
38 ± 11% and 62 ± 10%. The values for the cytosolic percentage were significantly different at the
0.01 level by paired Student’s t test.
To confirm that cytosol and membrane fractions
were separated and that the distribution of
marker proteins did not change with hypotonic
stress, blots were analyzed using antibodies
against Na-K-ATPase as a membrane marker
and hsp73/72 as a cytosol marker. As shown in
Figure 3, clear separation of hearts into cytosol and membrane fractions was accomplished
since the marker proteins were found only in the
correct fractions in all three experiments. Hypotonic stress did not alter the distribution in any
experiment (n=3).
Fig. 3. Distribution of membrane (Na-K-ATPase) and cytosol (hsp73/72) markers under isotonic and hypotonic conditions. Cell fractions were made as described in Materials and
Methods and pICln in the fractions determined by Western
blotting. One-twentieth of the total protein in each fraction
was used to determine distribution of pICln and the marker
proteins. Chemiluminescent image shown is representative
of those in three separate experiments.
The regulation of cell volume within a narrow
range is vitally important to marine species which
may encounter a wide range of environmental osmolarities. The maintenance of cell volume by all
cells is an important function and all cells have
developed multiple pathways to accomplish this
action. Along with the efflux of electrolytes, numerous organic molecules are used to reduce cell
volume and accomplish the regulatory volume decrease. These organic molecules are of a wide variety of chemical groups. However, they share
certain characteristics: (1) they are all of a limited hydrated size, (2) they are all accumulated
by cells, and (3) they are generally not used as central metabolites in the cell. The β-amino acid taurine is one of these important organic osmolytes;
others include betaine, choline, and sorbitol.
pICln is a protein that mediates a hypotonicstimulated Cl current and is pivotal in Cl efflux
in hyptonically treated Xenopus oocytes (Paulmichl et al., ’92; Ackerman et al., ’94). This protein may be the one which is responsible for the
functions attributed to a volume-sensitive organic
anion channel (VSOAC) characterized in MDCK
and C6 cells (Strange et al., ’96). pICln appears to
be ubiquitous in tissues as diverse as brain, spleen,
intestine, and cardiac muscle all have this protein.
It also appears to be well conserved phylogenetically as a protein of the same molecular mass is
recognized in elasmobranchs and mammals.
In addition to the results reported here for cardiac cells, a recent preliminary report has shown
that pICln translocates from cytosol to membrane
fractions in other epithelial cells under hypotonic
stress (Laich et al., ’96). It appears therefore that
the phenomenon may be of general importance to
volume regulation in animal cells.
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sensitive chloride conductance regulatory protein, pICl n,
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pICl n protein forms homodimeric complexes in vitro.
Biochem. Biophys. Res. Commun., 218:822–827.
Davis-Amaral, E.M., M.W. Musch, and L. Goldstein (1997)
Chloride and taurine effluxes occur by different pathways
in skate erythrocytes. Am. J. Physiol., (in press).
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Meeting Abstracts, 24a.
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