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1 Department of Zoology and 2 College of Medicine, University of Florida, Gainesville, Florida 32611; and 3 Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Pendrin is an anion
exchanger in the cortical collecting duct of the mammalian nephron that
appears to mediate apical Cl
/HCO
/HCO
anion exchanger; vacuolar-proton-adenosinetriphosphatase; sodium-potassium-adenosinetriphosphatase; sodium chloride regulation; acid/base regulation; Dasyatis sabina
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PENDRIN is a recently
discovered protein in mammals encoded by the Pendred disease syndrome
(PDS) gene (9). Pendrin belongs to a diverse family of
anion exchangers (SLC26) that includes such transporters as the
sulfate-anion transporter (sat-1), diastrophic dysplasia sulfate
transporter (DTDST), and congenital chloride diarrhea chloride-base
exchanger (CLD) (10, 18, 30). This gene family is distinct
from the SLC4 sodium-independent anion exchanger (AE) family that
includes the well-studied AE-1 (band 3)
Cl/HCO/iodide exchanger (2, 15, 25). This
transporter has Cl/iodide,
Cl/HCO/OH, and Cl/formate
exchange activity when expressed in Xenopus oocytes and HEK-293 cells (28, 29, 31).
Recent studies have detected both pendrin mRNA (26,
31) and protein (26) in the cortical collecting
duct (CCD) of the mammalian nephron. Royaux et al. (26)
localized pendrin to the apical region of
vacuolar-proton-adenosinetriphosphatase (V-H-ATPase)-rich intercalated
cells that did not have basolateral AE-1 immunoreactivity. Furthermore,
Royaux et al. (26) demonstrated that isolated, perfused
CCD tubules from bicarbonate-loaded pendrin-knockout mice had a
decreased capacity for bicarbonate secretion compared with CCD tubules
from wild-type mice. Taken together, these data not only suggested that
intercalated cells with pendrin were bicarbonate-secreting cells but
also that pendrin may represent an apical
Cl/HCO
In cartilaginous fishes (elasmobranchs), the gills are the primary site
of acid/base-related ion transport (13). However, no study
has determined the identity or cellular location of a Cl/HCO/HCO
The Atlantic stingray is unique because it is one of the few
elasmobranch species with the physiological capability of living in
both freshwater and marine environments. In previous studies, we have
demonstrated that both Na+-K+-ATPase and
V-H-ATPase immunoreactivity are at relatively high levels in gills of
freshwater Atlantic stingrays and subsequently decrease when the
animals are acclimated to seawater (21, 22). Understanding
how salinity influences expression of a branchial anion exchanger is of
interest in the Atlantic stingray because it may reveal mechanisms this
species uses for acid/base and NaCl regulation in freshwater and marine
environments. For example, molecular, immunohistochemical, and
pharmacological evidence in freshwater bony fish (teleost) gills
suggests that an AE-1-like Cl/HCO/HCO/HCO
The goals of this study were to determine if pendrin immunoreactivity
is present in the gills of the Atlantic stingray, to identify the
specific cellular location of pendrin in the gill epithelium, and to
determine if branchial pendrin immunoreactivity is influenced by
environmental salinity. Our results present the first evidence of
pendrin immunoreactivity in a lower vertebrate and suggest that pendrin
or a pendrin-like Cl/HCO
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal collection and holding conditions. During the spring of 1999, 10 freshwater Atlantic stingrays were captured from the St. Johns River in Florida (Lake Jesup or Lake George), using trotlines baited with shrimp (see Ref. 23). These stingrays were held in two 379-liter freshwater closed-system tanks [5 animals/tank; <1 parts per thousand (ppt) salinity]. In addition, five marine Atlantic stingrays were captured via hook and line from Cedar Key, FL, transported to Gainesville, FL, and held in a 379-liter seawater closed-system tank (32 ppt salinity).
Five of the freshwater stingrays were left in freshwater (referred to as freshwater stingrays), while the other five were gradually acclimated to seawater as follows (referred to as seawater-acclimated stingrays). After 1 wk in freshwater, the salinity was raised to 16 ppt over 2 days (8 ppt per day). After 2 days in 16 ppt, the salinity was raised to 32 ppt seawater over 3 days. The animals remained in 32-ppt seawater for 1 wk before tissue samples were taken. The stingrays from Cedar Key, FL, remained in 32-ppt seawater for the entire period (referred to as marine stingrays). All animals were fed live grass shrimp (Palaemonetes sp.) every other day and were starved 48 h before tissue collection. All holding tanks were held in the same room, which contained a thermostat set at 25°C, with a 12:12-h light-dark photoperiod. Water temperature of the tanks ranged from 23 to 27°C during the experiment, and pH was adjusted as necessary to 8.2 using Malawi/Victoria Buffer (Seachem) for freshwater and Marine Buffer (Seachem) for seawater. The tanks were also equipped with biological filtration, which maintained ambient NH3 and NO2 levels below 1 part per million. Ion composition of the freshwater and seawater has been reported in a previous study (23).Collection of gill tissue. Animals were anesthetized in 4 liters of a 0.01% 3-aminobenzoic acid ethyl ester (MS-222, Sigma) solution made with tank water. Malawi/Victoria Buffer was added to the anesthetic of freshwater stingrays to offset acidification caused by the MS-222. Once anesthetized, animals were placed ventral side up in a slanted water bath with their gills immersed in the anesthetic.
To clear the gills of red blood cells, the animal was perfused with a 4°C marine elasmobranch Ringer solution (12). However, for freshwater stingrays, NaCl, urea, and trimethylamine-oxide concentrations in the Ringer were reduced to 200, 200, and 41 mM, respectively. The skin ventral to the heart and pericardium was removed, and 0.5-1.0 ml of blood was removed from the ventricle with a heparinized 25-gauge needle attached to a 1-ml syringe. An equal volume of heparinized Ringer solution was then injected into the ventricle and allowed to circulate for a few minutes. A cannula, connected to a perfusion bottle (1 m above animal), was inserted into the conus arteriosus and held by forceps. Once the perfusion was started, the sinus venosus was cut to relieve backpressure. The perfusion was continued until the gills appeared bleached and the fluid exiting the pericardial cavity was clear of blood (usually 3-5 min). Immediately after the perfusion, the animal was pithed, and the second left and right gill arches were removed and placed in an elasmobranch Ringer solution on ice. For immunohistochemistry, gill filaments were trimmed off the arches and placed in fixative (3% paraformaldehyde, 0.05% glutaraldehyde, and 0.05% picric acid in 10 mM PBS, pH 7.3) for 24 h at 4°C, then transferred to two changes of 75% ethanol for removal of fixative. Tissues were left in the second change of 75% ethanol until embedded. Additional filaments were snap-frozen in liquid nitrogen for immunoblot analysis and stored at
80°C until analyzed.
Antibodies.
The antibody used to detect pendrin was developed by Royaux et al.
(25) and is an affinity-purified rabbit polyclonal
antibody raised against amino acids 630-643 of human pendrin. This
sequence of amino acids is near the carboxy terminus of pendrin and is completely conserved among humans, rats, and mice (see Ref.
11). The antibody has been used to immunolabel pendrin in
HEK-293 cells transfected with pendrin cDNA and in rat thyroid tissue
(25). To detect Na+-K+-ATPase, a
mouse anti-chicken Na+-K+-ATPase 
-subunit
monoclonal antibody, a5, developed by Dr. D. Fambrough was obtained
from the Developmental Studies Hybridoma Bank under the auspices of the
National Institute of Child Health and Human Development and maintained
by The University of Iowa, Department of Biological Sciences (Iowa
City, IA). To detect V-H-ATPase, a rabbit anti-insect V-H-ATPase
B-subunit polyclonal antibody was kindly provided by Dr. W. Harvey,
Whitney Laboratory, University of Florida, with permission from Dr. S. Gill, University of California at Riverside. In previous studies
(21, 22), we have successfully used the latter two
antibodies to detect Na+-K+-ATPase and
V-H-ATPase in gills of the Atlantic stingray.
Immunoblot analysis of pendrin immunoreactivity.
Immunoblots were performed on polyvinylidene difluoride membranes
(PVDF; Bio-Rad) containing 20 µg of total gill membrane protein per
lane that were prepared for a previous study (21). Preparation of tissue for immunoblots was similar to Claiborne et al.
(4), with modifications. On a single day, gill filaments from a freshwater, seawater-acclimated, and marine stingray were prepared. First, the tissue was placed in ice-cold homogenization buffer (250 mM sucrose, 1 mM Na-EDTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 100 µg/ml PMSF, and 30 mM Tris) and was homogenized with a
motorized Tissue-tearor (Biospec Products) in a 4°C cold room on ice.
Homogenates were filtered through cheesecloth and centrifuged (3,000 g) for 5 min at 4°C to remove nuclei and debris. The
supernatant was then filtered through cheesecloth and centrifuged (50,000 g) for 30 min at 4°C to pellet membrane fractions.
The pellet was resuspended with a minimal volume of ice-cold
homogenization buffer, and then an equal volume of a modified Laemmli
sample buffer (16), without bromophenol blue and
-mercaptoethanol, was added to solubilize the proteins.
-mercaptoethanol were added to final concentrations of 0.01 and 2%, respectively.
A 20-µg sample of total gill membrane protein for one stingray in
each condition was loaded in triplicate and run on a 7.5% Tris · HCl precast polyacrylamide gel (Bio-Rad) for 1 h at
125 V. Proteins were then transferred to a PVDF membrane using a wet (20% methanol, Tris-glycine) transfer unit for 2.5 h at 90 V in a
4°C cold room with stirring. The protein preparation,
electrophoresis, and blotting were repeated for the remaining gill
samples, which resulted in a total of five PVDF membranes with each
containing, in triplicate, gill membrane protein from a single
freshwater, seawater-acclimated, and marine stingray.
Because these PVDF membranes were previously immunostained (see Refs.
21, 22), it was necessary to remove
antibodies that were bound to the membrane with a strip buffer (62 mM
Tris base, 2% sodium lauryl sulfate, 0.6% -mercaptoethanol, pH
6.7). Each PVDF membrane was first soaked in 100% methanol for 15 min
and then placed in the strip buffer for 30 min at 60°C. The PVDF was then placed in three washes of deionized H2O (5 min
each) to remove any residual -mercaptoethanol. The PVDF was blocked
with blocking buffer (Tris-buffered saline with 5% nonfat dry milk,
0.1% Tween-20, and 0.02% sodium azide, pH 7.4) for 1.5 h at
25°C and then incubated with the rabbit anti-human pendrin polyclonal
antibody (diluted 1:5,000 in blocking buffer) overnight at 4°C.
The PVDF was washed three times (15 min each) with Tris-buffered saline
containing 0.1% Tween-20 (TTBS, pH 7.4) and then incubated with an
alkaline-phosphatase-conjugated goat anti-rabbit IgG secondary antibody
(Bio-Rad; diluted 1:3,000 in blocking buffer) for 2 h at 25°C.
The PVDF was then washed three times (15 min each) with TTBS, and a
substrate solution (Bio-Rad Immun-Star ECL Kit) was applied to the PVDF
for 5 min at 25°C to generate a luminescent signal. Binding of
antibody was detected by exposing Hyperfilm-ECL imaging film (Amersham)
to the PVDF membrane. Negatives were digitized into TIFF files using a
UMAX flatbed scanner with transparency adapter. As a control, we
incubated stripped membranes with normal rabbit serum (Biogenex)
because preimmune serum from the rabbits that generated the anti-human
pendrin antibody was not available.
To quantify the relative abundance of pendrin immunoreactivity,
we measured the optical density (uncalibrated OD) of the immunopositive band in each animal using Scion Image version 4.02 (Scion). On a given
blot, optical density values were measured for each animal and then
standardized to the freshwater condition to calculate relative
abundance. Therefore, all relative abundance measurements of freshwater
gills were 1.0, and those of seawater-acclimated and marine gills were
a fraction of the freshwater value.
Immunohistochemical localization of pendrin immunoreactivity. The fixed gill tissue (stored in 75% ethanol) was dehydrated in an ethanol series and embedded into paraffin wax. Serial sections of gill tissue, parallel to the long axis of the filament, were cut at 6 µm and placed on poly-L-lysine-coated slides (3 sections per slide). Longitudinal sections of gill filaments provide cross-sectional orientations of gill lamellae, which appear as fingerlike projections, and interlamellar regions, which are basal to and between lamellae (see Fig. 3). Sections were deparaffinized in Hemo-De (Fisher Scientific), hydrated in an ethanol series, and washed in 10 mM PBS. A hydrophobic PAP-Pen (Electron Microscopy Sciences) was used to draw circles around the tissue sections, and then 3% H2O2 was placed on the sections for 30 min to inhibit endogenous peroxidase activity. Sections were also blocked with Biogenex Protein Block (BPB; normal goat serum with 1% bovine serum albumin, 0.09% NaN3, and 0.1% Tween-20) for 20 min. The polyclonal rabbit anti-human pendrin antibody (diluted 1:1,500 in BPB) was then incubated on the sections overnight at 4°C.
The antibody was rinsed off with PBS, and then the sections were soaked in PBS for 5 min. The sections were incubated with a biotinylated goat anti-rabbit IgG secondary antibody (Biogenex) and a horseradish peroxidase-labeled streptavidin solution (Biogenex) for 20 min each at 25°C (with a 5-min PBS wash after each incubation). Antibody binding was visualized by applying the chromagen 3,3'-diaminobenzidine tetrahydrochloride (DAB; Biogenex) to the sections for 5 min at 25°C. In each experiment, one section was exposed to normal rabbit serum or BPB in place of the anti-human pendrin antibody as a negative control, because preimmune serum from the rabbits that generated the anti-human pendrin antibody was not available.Colocalization of pendrin immunoreactivity with V-H-ATPase and Na+-K+-ATPase. To determine if pendrin immunoreactivity was expressed in V-H-ATPase-rich and/or Na+-K+-ATPase-rich cells, we used a double-labeling technique consisting of sequential immunolocalization procedures using two different chromagens. Tissue sections for double labeling were first deparaffinized, hydrated, blocked, and stained for pendrin as described above (except a 1:1,000 dilution of the anti-pendrin antibody was used when double labeling with V-H-ATPase). After treatment with the brown chromagen substrate (DAB), the slides were rinsed in deionized H2O for 10 min and blocked with BPB for 20 min. An antibody to Na+-K+-ATPase (monoclonal antibody a5 culture supernatant diluted 1:100 in BPB) or V-H-ATPase (polyclonal antibody serum diluted 1:5,000 in BPB) was then applied to the sections overnight at 4°C. Rinsing and developing were performed as described above, except a blue chromagen was used (Vector SG, Vector Laboratories).
Statistical analyses. Differences in pendrin immunoreactivity relative abundance measurements were detected using a Kruskal-Wallis nonparametric ANOVA, with a Kruskal-Wallis multiple comparisons test (6). All tests were two tailed, and differences were considered significant at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Immunoblot analysis of pendrin immunoreactivity.
In immunoblots of membrane proteins isolated from stingray gills, under
all salinities tested, the anti-human pendrin antibody bound to a
protein of ~144 kDa (Fig. 1). No
detectable signal was present when PVDF membranes were incubated with
normal rabbit serum instead of the primary antibody (data not shown).
Semiquantitative immunoblotting revealed that the relative abundance of
pendrin immunoreactivity was highest in gill membrane protein from
freshwater stingray gills (Fig. 2). In
gill membrane protein from seawater-acclimated stingrays, pendrin
immunoreactivity was diminished compared with freshwater gill protein
and was comparable to that from marine stingrays (Fig. 2).
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Immunohistochemistry of pendrin in stingray gills.
In freshwater stingrays, numerous pendrin-positive cells were found on
both gill lamellae and interlamellar regions (Fig. 3A). In seawater-acclimated
stingray gills, pendrin-positive cells were primarily detected on
interlamellar regions and were infrequently found on lamellae (Fig.
3B). In marine stingrays, pendrin-positive cells were found
exclusively on interlamellar regions, with no detectable
immunoreactivity on lamellae (Fig. 3C). No staining was
detected when normal rabbit serum or BPB was used instead of primary
antibody (data not shown).
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Colocalization of pendrin immunoreactivity with V-H-ATPase and
Na+-K+-ATPase.
Double labeling of gills for pendrin and V-H-ATPase immunoreactivity
revealed that the two transporters occurred in the same cells (Fig.
5). Regardless of environmental salinity,
all pendrin immunolabeling was in the apical region of cells that
stained for V-H-ATPase (Fig. 6). Double
labeling of gills for pendrin and Na+-K+-ATPase
immunoreactivity demonstrated that the two transporters occurred in
separate cells, regardless of environmental salinity (Figs. 7 and
8).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Our findings present the first evidence of a pendrin-like transporter in an ion-transporting tissue from any lower vertebrate. Immunoblotting with an anti-human pendrin antibody demonstrated the presence of an ~144-kDa protein in gill membrane enrichments from Atlantic stingrays (Fig. 1), a molecular mass that is slightly greater than the reported size of 95-115 kDa for pendrin in mammals (24, 25, 27, 31). The difference in size may indicate that the pendrin-like protein in elasmobranchs is composed of more amino acids and/or that the protein is heavily glycosylated in elasmobranchs compared with mammals. Pendrin has two potential glycosylation sites in mammals (9), but the protein has not been sequenced in elasmobranchs; therefore, the number of amino acids and potential glycosylation sites in the elasmobranch protein are unknown.
Semiquantitative immunoblotting revealed that pendrin immunoreactivity was most abundant in gill tissue from freshwater stingrays compared with seawater-acclimated and marine stingrays (Fig. 2). Our immunohistochemical findings demonstrated that the distribution of cells with detectable pendrin immunoreactivity was also influenced by environmental salinity. In freshwater stingray gills, pendrin-positive cells were found on both gill lamellae and interlamellar regions, whereas pendrin-positive cells were primarily found in the interlamellar regions of seawater-acclimated and marine stingray gills (Fig. 3). Furthermore, the intensity of the immunolabeling appeared to be stronger in pendrin-positive cells of freshwater stingray gills relative to seawater-acclimated and marine stingrays (Fig. 4). These salinity-related differences in cellular distribution and immunostaining intensity are consistent with the greater pendrin immunoreactivity detected in freshwater stingray gills by immunoblotting compared with gills of seawater-acclimated and marine stingrays (Fig. 2).
Immunohistochemistry also demonstrated that localization of pendrin
immunoreactivity within pendrin-positive cells occurred in the apical
region and that this subcellular localization was influenced by
environmental salinity. In freshwater stingray gills, pendrin-positive
cells had discrete, intense apical localization (Fig. 4A).
In seawater-acclimated and marine stingray gills, pendrin-positive cells lacked discrete apical staining and instead exhibited weaker, diffuse staining throughout the subapical cytoplasm (Fig. 4,
B and C, respectively). These observations are
similar to findings reported for transporters in other ion-secreting
epithelia that are trafficked between a cytoplasmic pool of vesicles
and the plasma membrane (7, 17, 34, 35). Therefore, in
addition to the greater relative abundance of pendrin immunoreactivity and number of pendrin-positive cells (see above), freshwater stingray gills may also have more pendrin-like transporters inserted into the
apical plasma membrane of pendrin-positive cells than do
seawater-acclimated and marine stingrays. This suggests the gill
epithelium of freshwater stingrays has a relatively high potential for
pendrin-like anion exchange activity, such as Cl/base exchange.
Greater protein expression and membrane insertion of a pendrin-like
exchanger in freshwater stingrays gills are consistent with the
physiological need for enhanced chloride uptake in freshwater environments to counteract the large diffusional and urinary losses of
chloride to the environment. Chloride uptake via a pendrin-like transporter would also provide a route for base secretion that could
contribute to acid/base regulation. In seawater-acclimated and marine
stingrays, lesser expression and apical membrane insertion of a
pendrin-like transporter may be adequate, because Cl/base
exchange would only be required for acid/base balance; chloride uptake
is no longer physiologically necessary in seawater environments and
would actually be opposite to the needs of NaCl homeostasis.
Double-labeling experiments clearly demonstrated that pendrin
immunoreactivity was exclusively found in the apical region of
V-H-ATPase-rich cells and not
Na+-K+-ATPase-rich cells (Figs. 5-8).
These results are similar to pendrin localization reported in the
mammalian CCD, where pendrin immunolabeling occurred in the apical
region of bicarbonate-secreting V-H-ATPase-rich intercalated cells that
were not immunoreactive for AE-1 (26). Because the pendrin
immunolabeling in the Atlantic stingray gill occurs in the apical
region of cells that are rich with basolateral V-H-ATPase, we suggest
that the V-H-ATPase-rich cells are analogous in function to type B
intercalated cells of the mammalian CCD and are a site of
pendrin-mediated apical Cl/HCO/HCO
It is important to note that our results do not rule out the
possibility that other anion exchangers exist in the elasmobranch gill
epithelium, such as an AE-1-like
Cl/HCO
Overall, the salinity-related differences in pendrin and V-H-ATPase
(see Ref. 22) immunoreactivity of the Atlantic stingray gill are consistent with the model of branchial NaCl and acid/base transport mechanisms we proposed in an earlier study (22).
We have incorporated our current results and those from a recent study
on Na+/H+ exchangers in elasmobranch gills
(8) to further develop our hypothetical model of ion
transport in the Atlantic stingray gill epithelium (Fig.
9).
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Perspectives
In summary, we have demonstrated that pendrin immunoreactivity is present in the gills of the Atlantic stingray, which is the first evidence of a pendrin-like transporter in any tissue from a lower vertebrate. Pendrin immunoreactivity is most abundant and most apical in the gills of freshwater stingrays compared with seawater-acclimated and marine stingrays and only occurs in the apical region of V-H-ATPase-rich cells, regardless of salinity. In conclusion, our findings suggest that a pendrin-like transporter may contribute to apical Cl/HCO| |
ACKNOWLEDGEMENTS |
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We thank the following people and organizations for input and/or assistance with this study: Dr. L. Chapman, K. Choe, K. Giesbrandt, Dr. L. Guillette, Jr., Dr. F. Nordlie, N. Reid, Dr. C. St. Mary, University of Florida Zoology Department, and the University Florida Seahorse Key Marine Laboratory.
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FOOTNOTES |
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This work was supported by Environmental Protection Agency-Science to Achieve Results Graduate Research Fellowship U-915419-01-0 (to P. M. Piermarini) and National Science Foundation Grant IBN-0089943 (to D. H. Evans).
Address for reprint requests and other correspondence: P. M. Piermarini, Dept. of Zoology, Univ. of Florida, 223 Bartram Hall, Box 118525, Gainesville, FL 32611 (E-mail: pmpierma{at}zoo.ufl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00178.2002
Received 21 March 2002; accepted in final form 24 June 2002.
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TOP
ABSTRACT
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RESULTS
DISCUSSION
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