Localization and characterization of phenamil-sensitive Na+ influx in isolated rainbow trout gill epithelial cells
1 Dept of Biology, Okanagan University College, Kelowna, British Columbia,
VIV 1V7, Canada
2 Dept of Biological Sciences, University of Alberta, Edmonton, Alberta, T5G
2E9, Canada
* Author for correspondence (e-mail: sdreid{at}ouc.bc.ca)
Accepted 28 October 2002
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Summary |
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Key words: mitochondria-rich cells, MR, chloride cells, CC, pavement cells, PVC, peanut lectin agglutinin, PNA, transport, fish, Na+ influx, density gradient
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Introduction |
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The primary goal of this study was to isolate pharmacologically and
characterize the mechanism and site of Na+ uptake in isolated
populations of gill epithelial cells. A pharmacological hallmark for eNaC
activity is sensitivity to the 5'-substituted amiloride derivative
phenamil (Kleyman and Cragoe,
1988; Alvarez et al.,
2000
). Percoll density-gradient separation, combined with peanut
lectin agglutinin (PNA) binding and magnetic bead separation, was used to
separate dispersed fish gill cells into sub-populations. We identify the
PNA- MR cell fraction (
-MR cell) as the site of
acid-activated phenamil and bafilomycin-sensitive Na+ uptake in
fish gill.
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Materials and methods |
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Isolation and fractionation of gill epithelial cells
Isolation and fractionation of gill epithelial cells was completed using a
modification of techniques developed by Goss et al.
(2001) and Galvez et al.
(2002
). Trout (210-325 g) were
removed from the aquarium and killed by cephalic blow. The entire branchial
basket was removed and rinsed quickly in dechlorinated tapwater to remove
congealed external blood and mucus. Individual arches were isolated, lightly
blotted and placed in a 50 ml polyethylene conical centrifuge tube (Falcon)
containing approximately 20 ml ice-cold Cortland's saline: 143 mmol
l-1 NaCl, 5 mmol l-1 KCl, 1.5 mmol l-1
CaCl2, 1 mmol l-1 MgSO4, 5 mmol
l-1 NaHCO3, 5 mmol l-1
NaH2PO4, 5 mmol l-1 glucose, pH 7.8;
osmolarity, 290 mosmol l-1). The isolated arches were washed with
Cortland's saline (4°C, 3x5 min), then re-suspended in 10 ml of 0.2
mg ml-1 collagenase (Sigma Chemical Co., St Louis, MO, USA) and
incubated for 20 min at 18°C with vigorous agitation (300 revs
min-1). After digestion, 10 ml of ice-cold phosphate-buffered
saline (PBS; 137 mmol l-1 NaCl, 2.7 mmol l-1 KCl, 4.3
mmol l-1 Na2HPO4, 1.4 mmol l-1
NaH2PO4) was added to slow the collagenase digestion,
and individual arches were gently scraped with a glass slide to remove the
remaining gill epithelium. The resulting cell suspension was passed through a
series of nylon meshes (gravity, 254 µm; vacuum, 94 µm) to remove large
debris and isolate single cells. The resultant cell suspension was then
transferred to a 50 ml centrifuge tube, diluted to a final volume of
approximately 45 ml with PBS and centrifuged (500 g, 10 min,
4°C). The cell pellet was resuspended in 40 ml PBS and the centrifugation
and resuspension were repeated. To remove contaminating red blood cells
(RBCs), the cell pellet was resuspended in 7.5-10 ml of erythrocyte lysis
buffer (154 mmol l-1 NH4Cl) for exactly 1 min. This
technique is based on a standard technique for removing contaminating RBCs
during white cell isolation. The cell lysis procedure is based on osmotically
shocking suspended cells and using differences in rate of swelling to
specifically eliminate RBCs. Those cell types that swell rapidly (RBCs) will
burst, while those with a greater compliment of ion transporters and the
ability to generate a regulatory volume decrease (RVD) response (other cells)
gain volume at a less hazardous rate. Exactly 1 min after resuspension in
lysis buffer, the suspension was diluted to 45 ml with PBS (4°C) and
centrifuged (500 g, 5 min, 4°C). The resultant cell pellet
was washed (2x20 ml) and placed on ice for at least 45 min to allow the
cells to recover. The cells were then centrifuged and resuspended in a final
volume of 2 ml PBS. This procedure removed >90% of contaminating RBCs with
little appreciable loss in other cell numbers. To separate the cells into
various sub-populations, a modification of Galvez et al.
(2002
) was used. The cell
suspension was layered over a discontinuous Percoll density gradient (1.03 g
ml-1, 1.05 g ml-1 or 1.09 g ml-1 Percoll in
PBS) in 15 ml conical centrifuge tubes (Falcon). The cell suspension was then
centrifuged (45 min, 2000 g, 4°C) and the bands from each
interface collected, washed twice in PBS and used for analysis as
required.
Magnetic cell separation
A previous paper (Goss et al.,
2001) has demonstrated that the 1.06-1.09 g ml-1
interface of the Percoll gradient is made up of MR cells. However, within this
fraction, there are two distinct populations: cells that bind the lectin PNA
and those that do not. To separate these two fractions, we took advantage of
this differential staining using a magnetic bead separation technique, as
recently developed by Galvez et al.
(2002
). The addition of the
erythrocyte-lysis technique slightly altered the migration pattern in these
experiments such that the Percoll density gradient was altered to isolate MR
cells from a 1.05-1.09 g ml-1 interface. Cells from the 1.05-1.09 g
ml-1 interface were resuspended in either 1 ml or 2 ml of 40 µg
ml-1 PNA-FITC (fluorescein isothiocyanate) conjugate (20 min,
4°C with continuous mixing). As FITC is light sensitive, these and the
following procedures were performed under minimal light conditions. After
incubation, unbound PNA-FITC was removed by centrifugation (2x500
g, 5 min) and the cells resuspended in 1-2 ml of anti-FITC
microbeads (10 µl microbead stock per ml PBS; Mitylnei Biotech, Auburn, CA,
USA) and incubated (20 min, 4°C with continuous mixing). Unbound anti-FITC
microbeads were removed by centrifugation (1x500 g, 5
min) and the cells resuspended in 1 ml or 2 ml PBS and applied to a rinsed
positive-selection (LS+) separation column placed in a magnetic field. 30
µm pre-filters were attached to the top of the separation column. Following
passage of the cell suspension through the column, the column was rinsed
(3x3 ml degassed PBS) and the 9 ml cell suspension collected. As these
cells were not bound by the PNAFITC conjugate, we refer to this
fraction as PNA-negative (PNA-). Cells that bound with PNA remained
on the column until removed from the magnetic field. Once the column was
removed from the magnetic field, 5 ml of degassed PBS was moved through the
column using the plunger provided and the fraction was collected in a 15 ml
conical centrifuge tube. This fraction is termed the PNA-positive
(PNA+) fraction. The PNA- and PNA+ fractions
were centrifuged and the pellets resuspended in PBS. Cell concentrations were
determined using duplicate 15 µl aliquots of suspension and directly
counting cells with a hemocytometer. Cell viability was regularly assessed by
Trypan blue exclusion. Concentrations were adjusted to that desired by further
diluting (additional PBS) or concentrating (centrifugation and removal of
supernatant) the cell suspension.
Na+ influx experiments
Accumulation of Na+ by isolated trout gill epithelial cells was
based on the uptake of 22Na+ by a fixed number of cells
for a fixed period of time. The cell number and flux time were determined
based on initial time course experiments using crude suspensions of gill
epithelial cells from a two-step (1.03-1.09 g ml-1) discontinuous
Percoll gradient.
To measure 22Na+ uptake in cells from each fraction, aliquots were centrifuged (1000 g, 5 min) and the pellet resuspended in 1 ml flux solution (pH 7.8, 285 mosmol l-1, 153 mmol l-1 N-methyl-D-glucamine (NMDG)-Cl, 1 mmol l-1 KCl, 1.8 mmol l-1 CaCl2, 1.1 mmol l-1 MgCl2, 5 mmol l-1 Hepes, 15 mmol l-1 NaCl) containing the desired combinations of inhibitors. Once re-suspended, 37 kBq 22Na+ (1 µl 22Na+ stock; NEN, Boston, MA, USA) was added to the wall of the centrifuge tube above the cell suspension and the suspension was mixed. After exactly 1 min, 200 µl (x3) of the cell suspension was removed and placed onto pre-wetted individual glass microfiber filter circles (Whatman GF/C, 2.4 mm diameter, 0.2 µm pore size) supported by small pieces of plastic mesh, contained within the barrel of 20 ml syringes connected to a vacuum line. The incubation solution was immediately removed by vacuum and the trapped cells rinsed (4x5 ml) with 154 mmol l-1 NaCl saline. Preliminary tests of adding an aliquot of 22Na+ in the absence of cells demonstrated that 2x5 ml rinses of the glass microfiber filters were sufficient to reduce 22Na+ activity to background levels. This filter-vacuum system allowed for rapid (<10 s) washing of the cells and the removal of free 22Na+. In parallel, two 100 µl aliquots of incubation saline were dispensed into individual 7 ml scintillation vials for later determination of specific activity. Washed filter disks were placed in 7 ml scintillation vials, and 5 ml scintillation cocktail (ACS; Amersham, Baie d'Urfe, QC, Canada) was added. 22Na+ activity (c.p.m.) was determined (Beckman LC6200) and corrected for background activity (pre-washed filters in 5 ml scintillation cocktail).
To determine the mechanism contributing to overall Na+ uptake in
the isolated cell populations, the flux saline for all conditions contained 50
µmol l-1 ouabain to remove nonspecific Na+ efflux
from cells due to the action of the Na+/K+-ATPase, 20
µmol l-1 bumetanide to block the
Na+/K+/2Cl- co-transporter and 50 µmol
l-1 HOE-694 to block the endogenous basolateral
Na+/H+-exchanger (NHE). Amiloride and other amiloride
analogues could not be used to block the NHE due to their efficacy at also
blocking eNaC family members. HOE-694 (10 µmol l-1) is a
selective NHE-1 inhibitor without effect on eNaC in mammalian systems
(Scholtz et al., 1993;
Woll et al., 1993
;
Le Grand et al., 1996
;
Loh et al., 1996
). We
demonstrate that HOE-694 is also effective in inhibiting NHE activity in
epithelial cells isolated for trout gills.
Intracellular acidification and activation of the H+ excretion/Na+ uptake mechanism in the isolated cells was accomplished by replacement of NaCl with Na-propionate, which kept the extracellular [Na+] constant at 15 mmol l-1 with no resulting changes in external specific activity (SA). Internal SA (based on a lowest predicted internal [Na+] of 2 mmol l-1) did not exceed 5% of external SA, so correction for backflux was not performed. The involvement of H+-ATPase in 22Na+ influx was determined by prior (1 min) incubation of the cells with bafilomycin (10 nmol l-1). The involvement of the purported fish gill eNaC homologue in 22Na+ uptake was determined by prior (1 min) incubation of the cells with the Na+ channel blocker phenamil (10 µmol l-1).
Calculations
Na+ influx (JinNa; measured in
nmol Na+ 106 cells-1) was calculated using
the following equation:
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Na+ influx rate was calculated using equation 1 divided by the duration of the incubation and expressed as nmol Na+ min-1 106 cells-1.
Measurement of pHi in isolated MR cells
Cells were monitored for changes in intracellular pH (pHi) using the
indicator dye BCECF-AM. Cells isolated from the Percoll gradient were placed
in PBS containing BCECF-AM (5.0 µmol l-1) for 45 min at 5°C.
Cells were then washed, resuspended in PBS and placed on a cover slip and
allowed to adhere for 15 min prior to examination. The cover slip was mounted
in an imaging perfusion chamber and placed on an inverted microscope (Nikon
Eclipse TM-300). Cell pHi was monitored by ratiometric imaging (measuring
emission at 540 nm after excitation at 495 nm or 440 nm) of small populations
of cells (3-5 cells per experiment) at 15 s intervals. The fluorescence
microscope was equipped with epiillumination via a xenon arc lamp
(Lambda LS, Sutter Instruments, Novato, CA, USA). Cells were alternatively
excited at 495±5 nm and 440±10 nm using a dichroic 515 nm cut-on
filter, and emitted light was measured using a 540±25 nm filter. Cells
were visualized using Plan-Fluor objectives at 40x (0.6 N.A. air) or
100x (1.3 N.A. oil) magnification and images were digitally captured on
a 12-bit CCD camera (Cooke SensiCam, Kelheim, Germany). Dye-incubated cells
were imaged in the presence or absence of HOE-694 (50 µmol l-1).
In situ calibration of pHi at the end of each experiment was
performed by perfusing through Hepes-buffered solutions of various pHi (pH
7.8, 7.5, 7.2, 6.9) in the continuous presence of nigericin (5 µmol
l-1) and monitoring the fluorescence ratio
(f495/f440). The ratios were then used to construct a
calibration curve to calculate pHi during the experimental run.
Chemicals
All drugs used were dissolved in dimethylsulfoxide (DMSO) and diluted to
the required concentration with appropriate saline. The final concentration of
DMSO was 0.01% in all 22Na+ exposure salines.
Bafilomycin A1 from Streptomyces griseus, bumetanide and ouabain were
purchased from Sigma Chemical Co., phenamil (phenamil methanesulfate) was
purchased from Research Biochemicals International (Natick, MA, USA), and
HOE-694 (3-methylsulphonyl-4-peperidinobenzoyl, guanidine hydrochloride) was
generously provided by Dr Lang of Hoeschst, Germany.
Statistical analysis
Difference between treatments was analyzed using two-way analysis of
variance (ANOVA) followed by Fisher's least significant difference (LSD)
multiple means comparison test (JMP 3.1.6, SAS Institute Inc.). In all cases,
the level of significance was set at P<0.05.
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Results |
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Using a total gill cell population isolated from a 1.03-1.09 g ml-1 two-step discontinuous Percoll gradient, we established the methodology for measurement of unidirectional 22Na+ uptake. Preliminary experiments revealed measurable linear uptake of 22Na+ for up to 1 min. We therefore employed a 1-min flux period for all measurements of 22Na+ uptake. To measure Na+ influx in isolated cells, we used bumetanide (20 µmol l-1) as an antagonist of the Na+/K+/2Cl- co-transporter (NKCC) and ouabain (100 µmol l-1) as an antagonist for the Na+/K+-ATPase; this reduced the background activity of these Na+ transporters. However, to measure influx through an Na+ channel, inhibition of the gill cell Na+/H+-exchanger (NHE) was also necessary. We were unable to use effective concentrations of amiloride or any of the amiloride derivatives to specifically block the NHE due to the confounding inhibitory effect of these compounds on the epithelial Na+ channel (eNaC) at concentrations effective against the basolateral NHE. To resolve this problem, we used the NHE inhibitor HOE-694, a relatively new NHE-1-specific inhibitor (Ki approximately 1 µmol l-1) in other systems with no known inhibitory properties on the Na+ channel (Sholtz et al., 1993). To ensure that HOE-694 was effective in inhibiting Na+ uptake in fish gill cells, two approaches were used. First, 22Na+ influx was determined in the presence and absence of a saturating concentration of 50 µmol l-1 HOE-694 (Fig. 2). In these experiments, control cells were found to have an Na+ influx rate of 21±0.34 nmol min-1 106 cells-1 in the absence of HOE-694, while in the presence of saturating concentrations of HOE-694, Na+ influx rates were significantly reduced by 50% to 10.4±1.25 nmol min-1 106 cells-1 (Fig. 2). Second, we measured pHi by live cell imaging and examined the rate of recovery from addition of 10 mmol l-1 proprionic acid to the bath in the presence and absence of HOE-694. While addition of proprionic acid resulted in similar reductions in pHi, the rate of recovery (dpHi/dt) was reduced by 40% (from 0.731 pH units min-1 to 0.41 pH units min-1) in cells exposed to 50 µmol l-1 HOE-694 (Table 1). Higher concentrations (100 µmol l-1) of HOE-694 did not further reduce dpHi/dt. These results suggest that 50 µmol l-1 HOE-694, an NHE inhibitor, was effective at eliminating the NHE-mediated Na+ influx in a suspension of mixed trout gill epithelial cells.
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In gills, the transport rate for Na+ under control conditions is approximately 200-300 µmol kg-1 h-1. This Na+ uptake is driven by the constant production of metabolic acids in the body of the fish, requiring the gill ion-transporting cells to excrete H+ in exchange for Na+. However, in unstimulated cells isolated and maintained in a pH-buffered media, we hypothesized that the Na+ channel/H+ excretion would be relatively quiescent, and uptake through the Na+ channel would only become apparent during an event such as intracellular acidification and increased activity of the V-type H+-ATPase. If the uptake was present under acid-stimulated conditions and was sensitive to phenamil, this would indicate the presence of a phenamil-sensitive epithelial Na+ channel. To examine if acidification could increase the rate of 22Na+ uptake, we measured uptake on isolated gill cells under control and acid-stimulated conditions. To induce an intracellular acidosis, 10 mmol l-1 proprionic acid was again added to the media approximately 1 min prior to flux measurement ([Na+] was kept constant). Addition of proprionic acid resulted in a rapid decrease in pHi of approximately 0.2 pH units (see Table 1) and a significant 43% increase in Na+ influx from 10.5±1.25 nmol min-1 106 cells-1 to 15.0±1.54 nmol min-1 106 cells-1 (Fig. 2). If the noted increase in 22Na+ influx were attributable to an increase in influx through an Na+ channel, the pharmacological Na+ channel antagonist phenamil, which is ineffective against the NHEs at the concentration used (10 µmol l-1), would prevent the increase. Fig. 2 shows that the increase in Na+ uptake noted during acidification was completely inhibited by the presence of the Na+ channel blocker phenamil.
The above experiments established that gill cells possessed an
acid-stimulated, phenamil-sensitive increase in Na+ uptake.
However, these experiments were conducted on a gill cell population isolated
from a two-step gradient. Using a three-step Percoll density gradient
separation as previously described (Goss
et al., 2001; Wong and Chan,
1999
), we were able to separate non-MR PVCs (1.03-1.05 g
ml-1 interface) from MR cells (1.05-1.09 g ml-1
interface). It was found that the erythrocyte-lysis procedure used in
isolating the MR cells tended to reduce the density of the MR cells by
approximately 0.01 g ml-1, resulting in MR cells migrating to a
slightly less dense interface of the Percoll gradient. To obtain total yields,
distribution patterns and similar populations of cells to that of previous
studies (Galvez et al., 2002
;
Goss et al., 2001
), the
density layer that separated PVCs from MR cells was reduced from 1.06 g
ml-1 to 1.05 g ml-1. We confirmed the appropriateness of
these changes through independent testing of cell sub-population counts and
mitochondria content (mitochondria fluorescence using the labeling dye
Mitotracker).
This separation allowed us to further define the site of phenamil-sensitive Na+ uptake. Measurement of Na+ uptake in 1.03-1.05 g ml-1-isolated PVCs under control-unstimulated conditions was 13.7±0.93 nmol min-1 106 cells-1. However, cells from this fraction demonstrated no proprionate-stimulated increase in Na+ influx. Furthermore, application of phenamil or the V-type H+-ATPase inhibitor bafilomycin (2 µmol l-1) was without effect in either the unstimulated or the proprionic acid-stimulated cells (Fig. 3).
|
When Na+ uptake was measured in the 1.05-1.09 g ml-1-isolated MR cells (Fig. 4), control rate was approximately 6-fold higher (68.3±7.32 nmol min-1 106 cells-1) than in the 1.03-1.05 g ml-1 fraction (Fig. 3). The basal level of Na+ uptake was unaffected by phenamil treatment. However, addition of bafilomycin resulted in a significant decrease in Na+ influx, although this reduction was not further potentiated by the addition of phenamil (Fig. 4). Addition of proprionic acid to the cells resulted in a 40% increase in Na+ influx from 68.3±7.32 nmol min-1 106 cells-1 to 96.0±11.0 nmol min-1 106 cells-1 (Fig. 4). This increase was entirely phenamil-sensitive, as addition of phenamil returned the Na+ influx to unstimulated values. Bafilomycin, which reduced basal Na+ uptake in unstimulated cells, also reduced Na+ uptake by a similar amount in proprionic acid-stimulated cells. This reduction was, again, not further potentiated by phenamil, suggesting that a general reduction in Na+ uptake during bafilomycin treatment is not related to transport through the Na+ channel.
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Once we had demonstrated that there was a phenamil-sensitive component of acid-stimulated Na+ uptake in MR cells, we used the magnetic bead separation technique to further isolate the phenamil-sensitive activity to a specific MR cell sub-type. Using this technique, we were able to isolate MR PNA+ cells from MR PNA- cells. The control rate of Na+ uptake in the PNA+ gill cells was similar to that found in the total MR gill cell fraction. Under control-unstimulated conditions, there was no significant phenamil sensitivity. However, bafilomycin reduced Na+ uptake by almost 65% in PNA+ cells, while addition of phenamil in the presence of bafilomycin did not result in a further reduction in Na+ uptake. Intracellular acidification with proprionic acid did not increase the rate of Na+ uptake in PNA+ cells nor did addition of phenamil affect the rate of uptake under acid-stimulated conditions. As was seen in the unstimulated cells, bafilomycin significantly reduced the basal rate of Na+ uptake in proprionic acid-stimulated cells, but this was not further reduced by phenamil (Fig. 5).
|
The PNA- fraction of the MR cell layer represents the cells allowed to pass through the magnetic separation column while it was still in the magnet. We tested the rate of Na+ uptake in these cells as well under both unstimulated and acid-stimulated conditions (Fig. 6). The control rate of Na+ uptake in the PNA- gill cells was similar to that found in both the MR gill cell fraction and the PNA+ fraction. Additionally, similar to the PNA+ cells, there was no significant phenamil sensitivity under control-unstimulated conditions. Bafilomycin induced similar reductions in Na+ uptake (55%), while the addition of phenamil and bafilomycin did not result in a further reduction in Na+ uptake over that of bafilomycin-treated cells. Most significantly, however, unlike the PNA+ cells, intracellular acidification of PNA- cells with proprionic acid produced a significant increase in Na+ uptake that was completely blocked by phenamil, indicating the presence of phenamil-sensitive Na+ uptake in PNA- cells (Fig. 6).
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Discussion |
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One of the primary objectives of the present study was to isolate the
location of the epithelial Na+ channel (eNaC) that has been
purported to be electrically coupled to a V-type ATPase and to be responsible
for branchial Na+ uptake in freshwater fish. The presence of an
Na+ channel was confirmed following the identification of a
phenamil-inhibitable acid-induced Na+ influx in only the cells that
we have previously termed MR PVCs (Galvez
et al., 2002).
The freshwater fish gill shares a number of common functional and
morphological features with the inner medullary collecting duct (IMCD) of the
mammalian kidney. Both epithelia function in ion regulation and acid/base
regulation (Madsen and Tisher,
1984,
1986
) and, importantly, have
similar morphological and physiological features as well
(Goss et al., 1997
). The
correlate of the respiratory PVC is termed the principal cell in the IMCD,
while the MR cells of the IMCD are classified as intercalated cells (IC).
Importantly, the latter have been subdivided into two functionally distinct
cell types,
and ß. The
-type (acid-excreting) IC is
characterized as not binding PNA (LeHir et
al., 1982
), having apical H+-ATPase and basolateral
Cl-/HCO3- exchange (Brown et al.,
1988a
,b
),
while the ß-type (base-secreting) IC is characterized as being able to
bind PNA, having apical Cl-/HCO3- exchange
and basolateral H+-ATPase
(Stetson and Steinmetz, 1985
),
i.e. a reversed polarity to that of the
-type cell. Given the
morphological and physiological similarities, we suggest that the
PNA- MR cells are termed
-MR cells, to match the
physiological characterization and nomenclature of the analogous
-IC
found in the IMCD, while the PNA+ MR cells should also be renamed
to match the similar PNA-binding characteristics found in the ß-IC found
in the IMCD. We will use the terms
-MR cells and ß-MR cell
throughout the remainder of the manuscript.
A significant concern with our methodology is the lack of proper
orientation present in the cells when suspended in solution. MR cells exist as
polarized cells with an apical and serosal solution. Ideally, our experiments
would have been performed on cells cultured so as to allow for direct
re-orientation of the MR cells with independent access to apical and basal
surfaces. When a mixed suspension of gill epithelial cells are plated, the
respiratory cells or PVCs will grow well in culture, while the MR cells will
not, at least in a single seed protocol
(Pärt and Wood, 1996).
More recent publications have demonstrated successful growth of undefined MR
cells in culture. However, their experimental evidence indicates that these
preparations do not transport Na+ or Cl- in a proper or
similar manner to the fish gill and therefore cannot, as yet, serve as a
surrogate model for the fish gill culture
(Fletcher et al., 2000
;
Kelly and Wood, 2001
). As we
were interested in characterizing the transport properties of each of the cell
subtypes, our only choice was to complete these experiments in vitro
using dispersed cells. We are currently trying (without success as yet) to
incorporate gill epithelial cell culture and our sub-type separation into a
viable model. Once this goal is achieved, it would be exciting to revisit
these experiments using cells with the appropriate polarization and more
environmentally relevant exposure media.
To isolate the movement of Na+ through the Na+
channel, we inhibited other routes of Na+ using ouabain, an
Na+/K+-ATPase inhibitor, bumetanide, a
Na+/K+/2Cl- co-transporter inhibitor and
HOE-694. HOE-694 is an Na+/H+ exchange (NHE) inhibitor
that has been shown to be specific for NHE isoform 1 (NHE-1) in a variety of
vertebrate tissues and cells (Scholtz et
al., 1993; Woll et al.,
1993
; Le Grand et al.,
1996
; Loh et al.,
1996
; Bleich et al.,
1998
). Half-maximal inhibition of NHE activity is 10 µmol
l-1 and 20 µmol l-1 in fibroblasts
(Woll et al., 1993
) and
isolated rat atria (Le Grand et al.,
1996
), respectively. The present study represents the first
application of HOE-694 in the study of ion transport in branchial epithelium
of freshwater fish. Our findings suggest that HOE-694 is an effective NHE
inhibitor in trout gill epithelial cells. In this study, 50 µmol
l-1 HOE-694 was found to inhibit Na+ influx by almost
50% in a suspension of gill epithelial cells that contain PVCs, CCs and MR
PVCs. The remaining movement of Na+ could be due to the activity of
other Na+-dependent transporters. Possible transporters include
members of the Na+/HCO3- co-transport family
or the Na+/Ca2+-exchanger (NCX) family. While there is
only nominal HCO3- present in the bathing solution, it
is likely that the effect of NBC would be minimized, but it cannot be
discounted. NCX activity has been demonstrated to exist in CCs (Flik et al.,
1985
,
1995
;
Flik and Verbost, 1993
;
Li et al., 1997
), while the
existence of an NCX in the other cell types remains to be investigated.
The localization of the acid-stimulated Na+ channel was based on
the use of phenamil, an eNaC blocker. Phenamil was effective in reducing
acid-stimulated Na+ influx but only in -MR (PNA-)
cells, suggesting that the route of Na+ entry is via an
eNaC-type Na+ channel. These data do not support the existence of
acid-sensing ion channels (ASICs) in gill epithelium. ASICs have been found in
mammalian neuronal tissue (Bassilana et
al., 1997
; Lingueglia et al.,
1997
). However, ASIC Na+ channels experience a decrease
in Na+ movement when exposed to reductions in pHi
(Zeiske et al., 1999
), an
opposite response to that noted in the present study. We suggest an
approximately 0.2 pH unit drop in pHi induced by proprionic acid-simulated
V-type H+-ATPase activity and an increased inward movement of
Na+ through the Na+ channel to maintain charge
balance.
Bafilomycin was extremely effective in reducing Na+ influx under
basal and acid-stimulated conditions, whether alone or in combination with
phenamil. Fenwick et al.
(1999) reported that the
inhibitory effect of bafilomycin on the rate of Na+ uptake was
dose-dependent and seen at concentrations as low as 0.01 µmol
l-1. 10 µmol l-1 bafilomycin reduced whole-body
Na+ influx by up to 90% and 70% in freshwater tilapia and carp,
respectively (Fenwick et al.,
1999
). These data provide support for previous suggestions that
Na+ uptake in freshwater fish is associated with a proton-motive
force created by a proton pump and provide indirect evidence for the major
significance of this mechanism in the branchial uptake of Na+ by
freshwater fish. We found that bafilomycin (2 µmol l-1) reduced
Na+ uptake by 30-60% in our MR cells but not at all in the PVCs.
The bafilomycin sensitivity was not significantly different between the
PNA+ and PNA- MR cells, suggesting that both CCs and MR
PVCs possess H+-ATPase. Caution must be exercised, however, with
the use of bafilomycin and interpretation of net or unidirectional
Na+ movements, as bafilomycin inhibited the basal mechanisms of
Na+ influx probably via a change in membrane potential.
Preliminary evidence from our lab using the membrane potential dye bis-oxynol
demonstrates that addition of bafilomycin results in depolarization of the
cell membrane potential. Evidence for a role for H+-ATPases in
setting the membrane potential has resulted in a number of recent papers
(Harvey et al., 1998
;
Wieczorak et al., 1999; Beyenbach,
2001
) and it has been conclusively demonstrated for at least three
types of MR cells with high plasma membrane H+-ATPase activity. In
osteoclasts (Mattsson et al.,
1993
), frog skin MR cells
(Ehrenfeld and Klein, 1997
),
MR cells of insect Malpighian tubules
(Beyenbach et al., 2000
) and
even plant transporting cells (Sze et al.,
1999
), bafilomycin treatment significantly depolarizes these
cells. This depolarization would reduce net driving forces for any
Na+ transport dependent on the electrochemical gradient and reduce
overall Na+ fluxes independently.
The ß-MR cells (PNA+) were found to be approximately 1% of
the total cells collected, while the -MR cells (PNA-)
represented approximately 6% of the cells collected from the gill. Pärt
and Wood (1996
) reported that
bafilomycin had no effect on the pHi of primary cultured gill PVCs. However,
as no MR cells were present in their study, this agrees with our present
findings and those of others that V-type H+-ATPases are present
only in MR cells and not in respiratory PVCs
(Sullivan et al., 1995
;
Galvez et al., 2002
). Only
-MR cells experienced increased rates of Na+ influx in
response to increased availability of intracellular H+ and only
this portion of the flux was phenamil-sensitive. We conclude that the
-MR cells are the main contributors to branchial Na+ uptake
in freshwater fish. Our identification of the Na+
channel-containing cell type will allow for future studies to focus on
elucidating the molecular identity and physiological characterization of this
transport.
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Acknowledgments |
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References |
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