Hypotonicity induced K+ and anion conductive pathways activation in eel intestinal epithelium
1 Department of Biological and Environmental Sciences and Technologies,
University of Lecce, Italy
2 Biochemistry Department, August Krogh Institute, 13 Universitetsparken,
Copenhagen, Denmark
* Author for correspondence (e-mail: trifone.schettino{at}unile.it)
Accepted 9 December 2004
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Summary |
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The eel intestinal epithelium responded to a hypotonic challenge with a biphasic decrease in the transepithelial voltage (Vte) and the short circuit current (Isc). This electrophysiological response correlated with a regulatory volume decrease (RVD) response, recorded by morphometrical measurement of the epithelium height. Changes in the transepithelial resistance were also observed following the hypotonicity exposure.
The electrogenic Vte and Isc responses to hypotonicity resulted from the activation of different K+ and anion conductive pathways on the apical and basolateral membranes of the epithelium: (a) iberiotoxin-sensitive K+ channels on the apical and basolateral membrane, (b) apamin-sensitive K+ channels mainly on the basolateral membrane, (c) DIDS-sensitive anion channels on the apical membrane. The functional integrity of the basal Cl- conductive pathway on the basolateral membrane is also required.
The electrophysiological response to hypotonic stress was completely abolished by Ca2+ removal from the Ringer perfusing solution, but was not affected by depletion of intracellular Ca2+ stores by thapsigargin.
Key words: eel, Anguilla anguilla, intestine, RVD, Ussing chamber, K+ channel, anion channel
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Introduction |
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For epithelial cells, like all the other cell types, alteration in cell
volume ultimately results in membrane damage and loss of the cellular
structural integrity; therefore, activation of `emergency' systems of rapid
cell volume regulation is fundamental in their physiology. The mechanisms for
the control of cell volume after osmotic stress are highly conserved and in
principle similar in cells from various tissues as well as between
evolutionary distant species (Gilles,
1988; Chamberlin and Strange,
1989
; Lang et al.,
1998
). Following a hypotonic cell swelling, they comprise the
release of osmolytes followed by loss of osmotically obliged water, termed
Regulatory Volume Decrease (RVD; Hoffmann
and Dunham, 1995
). A widely established strategy of electrolyte
transport regulation in RVD response in epithelial and non-epithelial cells is
the activation of K+ and Cl- efflux through independent
K+ and anion channels (Hoffmann
1978
; Busch and Maylie,
1993
; Deutsch and Chen,
1993
; Felipe et al.,
1993
; Hoffmann and Dunham,
1995
; Fürst et al.,
2000
; Hoffmann,
2000
; Niemeyer et al.,
2000
; Nilius et al.,
2000
; Strange et al.,
1996
; Okada, 1997
;
Valverde et al., 2000
).
Electroneutral K+-Cl- cotransporter is an alternative
system contributing to RVD in some cell types
(Lauf and Adragna, 2000
).
In the present study the physiological response to hypotonic stress was
investigated in a salt-transporting epithelium, the intestine of the
euryhaline teleost Anguilla anguilla. This tissue plays a key role in
the osmoregulation of the eel, especially in seawater, where the intestinal
epithelium carries out active salt absorption necessary in turn to replenish
passively lost water from the body, while excess salt is secreted by the gills
(Smith, 1930). The intestinal
epithelium is a useful model system for functional studies of epithelia that
perform near-isosmotic fluid absorption. The mechanisms accounting for ion and
water transport in the eel middle intestine have been characterized and
considerable information is also available on regulation of the transport (for
a review, see Schettino and Lionetto,
2003
). Briefly, it develops a transepithelial Cl-
absorption, measurable as transepithelial potential and short circuit current,
sustained by the operation of the luminal
Na+-K+-2Cl- cotransporter, in series with a
basolateral Cl- conductance and in parallel with an apical
K+ conductance. The Na-K-ATPase on the basolateral membrane, by
generating an inwardly directed electrochemical gradient for Na+,
provides the driving force for the
Na+-K+-2Cl- cotransporter and thus for the
active intracellular accumulation of Cl- (Trischitta et al.,
1992a
,b
).
This tissue is physiologically exposed to anisosmotic conditions, particularly
during the transfer of the animal from freshwater to seawater and vice
versa. As previously demonstrated (Lionetto et al.,
2001
,
2002
) it is sensitive to the
osmolarity of the extracellular medium, making this tissue a good
physiological model for epithelial cell volume regulation research.
To our knowledge this is one of the few works in which the ionic transport mechanisms that sustain the physiological response to hypotonic stress in the intestinal epithelium are examined not on cultured or isolated cells, but on a native tissue, and studied through an integrated analysis of the ion transport involved on the two plasma membranes.
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Materials and methods |
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All chemicals were reagent grade. Cacodylate buffer, glutaraldehyde, OsO4 and Epon 812 were purchased from Flukachemie GmbH (Buchs, Switzerland); all the other chemicals were purchased from Sigma (St Louis, MI, USA). Stock solutions of BAPTA-AM (50 mmol l-1 in DMSO), thapsigargin (1 mmol l-1 in DMSO), trifluoroperazione (10 mmol l-1 in distilled water), bumetanide (10 mmol l-1 in DMSO), apamin (100 µmol l-1 in distilled water) and iberiotoxin (100 µmol l-1 in distilled water) were prepared and kept at -20°C until use. Unless otherwise noted, solutions were freshly prepared.
Transepithelial electrophysiological measurements
The middle intestine of seawater-acclimated eels was removed, stripped of
longitudinal and circular layers using two pairs of fine forceps and mounted
vertically in a modified Ussing chamber (CHM6, World Precision Instruments,
Berlin, Germany) (membrane area: 0.6 cm2), where it was perfused on
both sides by isotonic teleost Ringer solution (NaCl 133 mmol l-1,
KCl 3.2 mmol l-1, NaHCO3 20 mmol l-1,
MgCl2 1.4 mmol l-1, CaCl2 2.5 mmol
l-1, KH2PO4 0.8 mmol l-1, glucose
20 mmol l-1; osmolarity: 315 mOsm kg-1).
The preparations were kept `open circuited' through the time course of the
experiments, except for a few seconds every 5 min for recording the
short-circuit current (Isc). Tissues were connected to an
automatic short-circuit current device (DVC-1000, World Precision Instruments)
by four Ag/AgCl electrodes (two voltage electrodes and two current electrodes)
that made contact with the bathing solutions via agar-Ringer filled
cartridges. Transepithelial voltage (Vte) was measured
with respect to the mucosal bath (grounded); Isc was
measured by passage of sufficient current through Ag/AgCl electrodes to reduce
the spontaneous Vte to zero automatically (resistance of
the chamber fluid was subtracted automatically). The Isc
is referred as negative when current flows across the tissue from the apical
membrane to the basolateral membrane. Transepithelial resistance
(Rte) was measured by pulsed current injection (33 µA
cm-2, 500 ms) through the tissue. This injected current produces a
voltage deflection (Vte) from which
Rte was calculated.
In the hypotonic stress experiments the osmolarity of the teleost Ringer solution was bilaterally decreased to 175 mOsm in two steps: first the concentration of NaCl was reduced to 66.5 mmol l-1 at unaltered osmolarity by mannitol replacement, then the hypotonic stress was applied by removing mannitol. This experimental maneuver was necessary to avoid the superimposing of diffusion potential in the unstirred layers, due to the NaCl reduction, on the hypotonic stress effect.
Drug treatments were performed by 1 h incubation of the tissue with the drug before removing mannitol. The same time protocol was respected in the corresponding control sample. In the drug test the drug was still present during the hypotonic stress application.
In hypotonic stress experiments Ringer osmolarity was decreased only once
in each experiment. The hypotonic stress response was quantified as
Vte and
Isc.
values were calculated as the differences between the Vte
and Isc values after hypotonic stress application and the
value before every 5 min during the time course of the exposure.
In all Ussing chamber experiments the temperature of the perfusing Ringer solution was kept constant at 18°C.
Morphometric analysis
Small segments of eel middle intestine, stripped from their outer layers,
mounted in the Ussing chamber for electrophysiological measurements and
perfused with Ringer solution (315 mOsm), were fixed before (control), after 5
min and after 45 min exposure to hyposmotic Ringer solution (160 mOsm),
respectively. Solutions utilized for fixation and postfixation were
respectively: (a) cacodylate buffer 0.1 mol l-1, pH 7.9, 315 mOsm
with mannitol; (b) cacodylate buffer 0.1 mol l-1, pH 7.9; (c) 2.5%
glutaraldehyde in cacodylate buffer a; (d) 2.5% glutaraldehyde in cacodylate
buffer b; (e) 1% OsO4 in cacodylate buffer a; (f) 1%
OsO4 in cacodylate buffer b. Fixation was performed as follows: the
epithelium segments were incubated for 36 h at 4°C in buffer c (control)
or buffer d (hypotonicity exposed segments). Thereafter, three washings (15
min each) in cacodylate buffer a (control) or b (hypotonicity exposed tissues)
were performed. Intestinal segments were then postfixed in 1% OsO4
in cacodylate buffer e (control) or f (hypotonicity exposed segments) and
embedded in Epon 812. Semithin (0.5 µm) sections were cut along planes
perpendicular to the luminal epithelium surface and stained with 1% Toluidine
Blue. Sections were placed on an optic microscope (Axiolab, Zeiss, Oberkochen,
Germany; objective utilized: 100x oil immersion), and the images
obtained from a video camera (Polaroid Digital Microscope Camera DMC 1, CCD;
1600x1200 pixels) were digitalized using NIH Image 1.62; each image was
calibrated with respect to overall magnification and in each specimen
epithelium thickness was measured in 80 points, where the plane of section
appeared to be exactly perpendicular to the basal membrane.
Plasma osmolarity
European eels fully adapted to seawater were transferred to freshwater.
Plasma samples were obtained immediately before the transfer and at regular
intervals thereafter up to 163 h. Plasma was obtained from blood collected
from the caudal vein of the eel by a heparinized syringe and immediately
centrifuged at 9000 g for 10 min (Beckman Microfuge® Lite
Centrifuge, Fullerton, CA, USA). The osmolarity of plasma samples was measured
by vapor pressure osmometer (5520, Delcon, Milano, Italy), also utilized for
measuring osmolarity of experimental solutions.
Statistics
Values are given as means ± S.E.M.
Statistical tests utilized to evaluate statistical significance of differences
were paired Student' t-test, one-way ANOVA test and Keuls-Newman
multiple comparison test, as indicated in legends to figures.
*P<0.05; **P<0.01.
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Results |
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This finding clearly indicates that in the eel, cells are physiologically exposed to hypotonic stress when the animal faces decreases in environmental osmolarity.
RVD response in eel intestinal epithelium
The effect of hypotonic stress on the volume of enterocytes in the native
tissue was studied by morphometrical analysis of the epithelium height as
described in Materials and methods. When the eel intestinal epithelium was
exposed to hypotonic stress (performed by decreasing Ringer osmolarity from
315 to 175 mOsm) the thickness of the epithelium showed a significant
(P<0.01) 18% increase after 5 min exposure (Figs
2A,B,
3), but decreased significantly
(P<0.01) and returned back to the initial value during the next 30
min(Figs 2A,C,
3). This finding suggests that
the eel intestinal epithelium exposed to hypotonic stress initially swells
because of the osmotic intake of water, but is able to regulate its volume in
a RVD response, even though the osmotic stress still persists.
|
|
In all the experiments the hypotonic stress applied was a 45% percentage
decrease in osmolarity of the perfusion solution; this represents a commonly
used hypotonic stress in volume regulation protocols
(Hoffmann, 1978;
Hazama and Okada, 1990
;
Diener et al., 1992
).
Electrophysiological response to hypotonic stress
In order to investigate the nature of the observed physiological response
to hypotonicity, functional studies were performed on the Ussing
chamber-mounted epithelium by transepithelial electrophysiological
measurements (transepithelial potential, short circuit current and
transepithelial resistance).
The exposure of seawater eel intestine to an hypotonic teleost Ringer produced a biphasic decrease in Vte and Isc, shown in Fig. 4: a first rapid transient phase, which lasted for about 5-10 min, followed by a more gradual and sustained one. There were also significant changes in Rte (Fig. 5), which rapidly increased soon after the hypotonic stress application, but tended to slowly decrease towards the initial value during the RVD phase.
|
|
Plasma membrane ionic transports involved
With the aim of investigating the ionic nature of the
Vte and Isc responses to hypotonic
stress, we first tested if the observed biphasic decreases in
Vte and Isc could be due to a
reduction in the rate of transepithelial Cl- absorption
via the luminal Na+-K+-2Cl+
cotransporter. To test this, 10 µmol l-1 bumetanide (specific
inhibitor of the Na+-K+-2Cl-) was added to
the luminal Ringer solution before decreasing external osmolarity
(Fig. 6A). Addition of
bumetanide in itself decreased both Vte and
Isc but did not have any significant effect on the
response to hypotonicity (neither Vte nor
Isc, not shown), suggesting that other membrane ion
transport mechanisms must be involved in the observed phenomenon.
|
By contrast, addition of the Na+-K+-ATPase inhibitor
ouabain (1 mmol l-1) to the serosal bathing solution
(Fig. 6B), by dissipating
K+ and Na+ gradients maintained by the pump operation
(Lew et al., 1979),
significantly decreased the hypotonicity induced Vte and
Isc response in both the first
(Fig. 6B) and the second phase
(data not shown). Since the decreases in Vte and
Isc are not secondary to a decrease in cotransport but are
dependent on an intact ionic gradient, we investigated whether the observed
changes are a result of a swelling activated K+ conductance,
K+ being the most abundant intracellular cation in eel enterocyte
(Marvão, 1994).
Cation channels
In basal conditions the main cation electrodiffusive pathway is a
Ba2+-sensitive K+ conductance on the apical membrane
that permits recycling of K+ into the lumen and contributes to the
operation of the luminal Na+-K+-2Cl-
transporter system (Trischitta et al.,
1992a; Marvão et al.,
1994
). Marvão et al.
(1994
), using conventional and
selective microelectrodes, also found a Ba2+-inhibitable
K+ conductance on the basolateral membrane.
In our experiments application of 2 mmol l-1 Ba2+
either on the luminal or the basolateral side of the epithelium had no effect
on the Vte and Isc (not shown)
responses to hypotonicity (Fig.
7A,B), suggesting that activation of other swelling-dependent ion
conductances, different from the basal ones, could be responsible for the
hypotonicity induced Vte and Isc
changes. Thus, the eventual involvement of other K+ channels, such
as Ca2+-activated K+ channels, important in cell volume
regulation in other cell types
(Fernandez-Fernandez et al.,
2002; Roman et al.,
2002
) were tested by utilizing iberiotoxin, a specific blocker of
the high conductance Ca2+-activated K+ channels (BK
channels; Galvez et al.,
1990
), and apamin, specific blocker of small conductance
Ca2+-activated K+ channels (SK2 and SK3;
Blatz and Magleby, 1986
;
Bond et al., 1998
).
|
In isotonic conditions, 0.1 µmol l-1 iberiotoxin (Ibtx) had no significant effect on the basal electrophysiological parameters Vte and Isc either on the serosal or mucosal membranes (Fig. 8A,B), but in hypotonic conditions, when applied on the serosal side, it decreased the hypotonicity induced depolarization of Vte in both the first and the second phases (Fig. 8C); when it was applied on the mucosal side it increased the hypotonicity induced depolarization of Vte in the second phase (Fig. 8D). These results might be explained by considering the `well type' potential profile of the enterocyte, where the K+ efflux through the basolateral membrane produces a depolarization of Vte (therefore its inhibition shows a decrease of the hypotonicity induced depolarization of Vte), while the K+ efflux through the apical membrane produces a hyperpolarization of Vte (therefore its inhibition shows an increase of the hypotonicity induced depolarization of Vte).
|
In isotonic conditions 1 µmol l-1 apamin had no effect on the basal electrophysiological parameters of either the basolateral or the mucosal membrane (Fig. 9A,B), but in hypotonic conditions, when applied on the serosal side, it decreased the hypotonicity induced depolarization of Vte only during a limited temporal window in the second phase (Fig. 9C). When apamin was applied on the mucosal side it slightly increased this depolarization in the second phase (Fig. 9D).
|
Effective volume regulation on each membrane requires a parallel efflux of
anions. Therefore, the involvement of a volume-activated anion channel was
tested using 0.5 mmol l-1 DIDS as an inhibitor. In isotonic
conditions mucosal DIDS had no effect on the basal electrophysiological
parameters Vte and Isc
(Fig. 10B), but in hypotonic
conditions, when applied on the mucosal side
(Fig. 10C), it significantly
decreased the hypotonicity induced depolarization either in the first or the
second phase. This result is consistent with the activation of anion channels
on the apical membrane during the hypotonic stress response. On the
basolateral (serosal) side DIDS significantly inhibited basal
electrophysiological parameters (Fig.
10A), because of its inhibition of basal Cl-
conductance (Bicho et al.,
1999). In hypotonicity conditions, when applied on the basolateral
side, DIDS also induced a significant decrease in the second phase of the
hypotonicity response (Fig.
10C). Similar results (not shown) were obtained with 0.5 mmol
l-1 NPPB, another inhibitor of basolateral Cl-
conductance.
|
Ca2+ dependence of the hypotonicity response
As reported in Fig. 11, 1 h
incubation of the tissue with BAPTA-AM (50 µmol l-1), a chelator
of intracellular Ca2+, significantly reduced the
Vte hypotonicity induced depolarization. Similar results
were obtained when the tissue was preincubated with 10 µmol l-1
trifluoroperazine, a calmodulin inhibitor
(Fig. 12).
|
|
Ca2+ removal from the Ringer solution nullified the
electrophysiological response both in the first and in the second phase
(Fig. 13B), on the contrary 1
µmol l-1 thapsigargin [a selective inhibitor of the
sarco(endo)plasmic reticulum type of Ca2+-ATPase leading to
depletion of the intracellular Ca2+ stores
(Tharstrup et al., 1990)] did
not elicit any significant effect (Fig.
13A).
|
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Discussion |
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In the present study we investigated the integrated ion transport response
activated by external hypotonicity in a native epithelium, the intestine of
the euryhaline teleost Anguilla anguilla, physiologically exposed to
hypotonic stress. When the eel moves from seawater to freshwater the intestine
is exposed to hypotonicity either on the basolateral side, because of the
dramatic decrease in the eel plasma osmolarity observed during the first 90 h
after transfer of the animal (Fig.
1), or the apical side, which can be exposed to a very diluted
medium because of the drinking behavior known in freshwater eels
(Maetz and Skadhauge,
1968).
Morphometrical analysis of the height of the epithelium revealed that the
eel intestine initially swelled following an hypotonic stress, but soon after,
in the following 45 min, it performed a regulatory volume decrease (RVD), as
do many other epithelia (for reviews, see
Hoffmann and Kolb, 1991;
Larsen and Spring, 1987
;
Macknight, 1991
;
Hoffmann and Ussing, 1992
).
The parallel monitoring of the transepithelial electrophysiological parameters
revealed that during the swelling phase and the RVD response there was a
biphasic decrease in Vte as well as in
Isc. There were also significant changes in
transepithelial resistance induced by hypotonicity exposure. In a leaky
epithelium such as the eel intestine the transepithelial resistance is
dominated by the resistance of the paracellular pathway, which in turn is due
to the resistance of the junctional complex in series with the lateral
intercellular space. Following a hypotonic stress the transepithelial
resistance showed an increase during the swelling phase, followed by a slow
recovery towards the initial value after 60 min exposure. These
Rte changes, similar to those observed for other
intestinal epithelia such as rat colonic epithelium
(Diener et al., 1992
) and rat
small intestine (Diener et al.,
1996
), are in the direction expected for narrowing of lateral
intercellular spaces during the swelling phase, followed by their slow
widening in the RVD phase. However, hypotonicity induced changes in junctional
resistance cannot be ruled out.
The use of specific ion transport inhibitors allowed us to clarify the ionic nature of the hypotonicity induced decrease in Vte and Isc.
Previous studies have shown that under basal conditions 80-90% of the eel
intestinal Vte and Isc is the result
of net transepithelial Cl- absorption, which is accomplished by the
Na+-K+-2Cl- cotransporter
(Trischitta et al., 1992a),
and that external hypertonicity increases Vte and
Isc by stimulation of the triporter
(Lionetto et al., 2001
). The
possibility that the observed decrease of Vte and
Isc induced by hypotonicity might be the result of an
inhibition of the luminal Na+-K+-2Cl-
cotransporter was ruled out by the observation that the decrease of
Vte and Isc induced by external
hypotonicity remained unaltered after luminal blockage by bumetanide of
Na+-K+-2Cl- triporter, suggesting that other
ion transport mechanisms could be involved.
The decrease of Vte and Isc induced by the hypotonic media reflects an electrogenic response of the tissue arising from the ion movements across the enterocyte cell membranes following their conductance changes. The Ba2+-sensitive basal K+ conductance is not involved, since the electrogenic response was unaffected by serosal or mucosal application of Ba2+. By contrast, the response was reduced by serosal application of iberiotoxin and apamin, specific inhibitors of high conductance (BK) and small conductance (SK2 and SK3) Ca2+-activated K+ channels, respectively, but was enhanced by the luminal application of iberiotoxin and slightly increased by luminal apamin. These results are consistent (as explained in the Results) with the hypotonicity induced activation of BK and SK Ca2+-activated K+ channels on the apical and the basolateral membranes. Furthermore, the iberiotoxin and apamin-sensitive components showed different time courses of activation: the iberiotoxin-sensitive pathway was rapidly activated in the first minutes of the response on the basolateral membrane, but on the mucosal membrane its main contribution was in the second phase of the response; the apamin-sensitive pathway contributed to the second phase of hypotonicity response on the serosal membrane, but across the apical membrane appeared to be minor, if present at all. The importance of K+ efflux in the hypotonic stress response is also suggested by ouabain experiments, where dissipation of the K+ gradient resulting from the pump inhibition significantly decreased the response.
The hypotonicity induced activation of high conductance
Ca2+-activated (BK) K+ channels is reported to occur in
other epithelial cell types such as human bronchial epithelial cell line
(Fernandez-Fernandez et al.,
2002), cultured proximal tubule cells
(Kawahara et al., 1991
) and
rat collecting tubules (Stoner and Morley,
1995
). In a diverse range of epithelial cells, from kidney
(Bolivar and Cereijido, 1987
;
Hirsch et al., 1993
), male
reproductive tract (Sohma et al.,
1994
), oviduct (James and
Okada, 1994
), and vestibulum (Takeuchi, 1992), high conductance
Ca2+-activated K+ channels have been detected on the
apical membrane. However, they have also been reported in the basolateral
domain of enterocytes (Burckhardt and
Gogelein, 1992
) and epithelial cells from acinar glands (Maruyama,
1983). In the present investigation, functional evidence of the hypotonicity
induced activation of these channels on both membranes is presented. Moreover,
the channels do not seem to contribute to ion transport processes under basal
conditions.
In our experimental model hypotonicity also seems to activate
apamin-sensitive K+ conductance, which is described in the
literature as small conductance Ca2+-sensitive K+
channels of the type SK2 and SK3 (Bond et
al., 1998), reported also to be involved in the RVD response after
swelling in human liver cell line (Roman
et al., 2002
).
To our knowledge this is the first evidence of functional involvement of
both high conductance and small conductance K+ channels in the
hypotonicity induced ionic response, although in human liver cell, Roman et
al. (2002) also suggested that
K+ channels additional to the SK2 channel were involved in RVD.
In eel intestinal epithelium the hypotonicity induced K+ channel
activation is paralleled by an anion channel activation on the apical
membrane, where DIDS, inhibitor of volume-activated anion channels
(Strange et al., 1996),
significantly reduced the Vte response. Activation of the
DIDS-sensitive conductance appears rapidly in the first minutes of the
Vte depolarization and is also maintained in the second
phase. DIDS applied on the mucosal side was effective only during the
hypotonic challenge and had no significant effect on basal
electrophysiological parameters, suggesting a specific role of this
conductance on the apical membrane in the hypotonic stress. On the serosal
side in isotonic conditions DIDS inhibited the basal electrophysiological
parameters, via inhibition of basal Cl- channels
(Bicho et al., 1999
) present on
the basolateral membrane. In hypotonic conditions basolateral DIDS also
induced a significant decrease in the second phase of the hypotonicity
response, similar to results obtained with basolateral K+ channel
blockers. Similar results were obtained using NPPB, another inhibitor of
basolateral Cl- conductance (data not shown), suggesting that the
hypotonicity induced activation of basolateral K+ conductance
requires the functional integrity of basal Cl- channels.
On the basis of these results we conclude that in the eel intestinal
epithelium a hypotonic stress activates separate K+ and anion
conductances, both on the basolateral and the apical membranes
(Fig. 14). The electrogenic
nature of the response can be explained by a K+ and anion
conductance increase in a ratio different from 1:1 on the two membranes. Since
the electrogenic response is a depolarization of Vte, we
suggest that on the basolateral membrane the increase in the K+
conductance, exceeds the increase in the Cl- conductance; by
contrast on the apical membrane, the increase in the anion conductance exceeds
the increase in the K+ conductance. It seems that the asymmetric
increase of cation (predominantly in the basolateral membrane) and anion
(predominantly in the luminal membrane) conductances in response to
hypotonicity are needed in order to balance cation and anion fluxes across the
respective membrane of the enterocyte. Before adding the hypotonic solution
the apical and basolateral membrane conductances are very different
(Trischitta et al., 1992b).
Together the various conductance changes on the two membranes result in the
measured potential difference depolarization across the epithelium (normal
polarity: serosal side negative) and thus also in the measured decrease in
Isc. The typical time course of the electrogenic response
could result from the sum of the different time courses of activation of these
separate conductive pathways on the two membranes.
|
Previously studies on Japanese eel have demonstrated the presence of
aquaporin 1 in the apical membrane of the enterocytes, and another epithelial
aquaporin isoform is supposed to be present on the basolateral membrane
(Aoki et al., 2003). Therefore,
it is possible to argue that in our experimental model KCl efflux on the
apical as well as on the basolateral membrane is followed by the osmotically
required loss of water through water-permeant aquaporins present on the two
membranes, thus accounting for the RVD response observed on morphometrical
analysis of the epithelium.
Role of calcium
An increase in intracellular Ca2+ concentration is important for
the hypotonicity induced decrease in Vte and
Isc, since chelation of intracellular Ca2+ by
BAPTA-AM produced a significant although not complete inhibition of the
response; comparison of the time courses of the effects of BAPTA-AM and
trifluoroperazine allows us to hypothesize that the role of intracellular
calcium in the hypotonicity response is mediated by a
Ca2+-calmodulin pathway. The lack of any effect of thapsigargin
ruled out the hypothesis that the release of intracellular Ca2+
stores is a Ca2+ signal in the eel intestinal hypotonicity
response. On the other hand, when Ca2+ was removed from bathing
solution the decrease in Vte and Isc
was completely abolished, clearly indicating a dependence of extracellular
Ca2+ in the hypotonicity response. We therefore suggest that a
swelling-activated influx of Ca2+ increases local cytosolic
Ca2+ levels, thereby activating BK and SK channels, both being
strongly dependent on intracellular Ca2+
(Vergara et al., 1998). With
respect to the increase in Cl- conductance, we cannot yet
distinguish whether we are dealing with Ca2+-activated
Cl- channels or swelling-activated Cl- channels, since
both can be inhibited by DIDS. Considering that some Cl- channels
are activated by extracellular Ca2+
(Uchida et al., 1995
;
Waldegger and Jentsch, 2000), it is possible to argue that in the eel
intestine extracellular calcium could also be essential for the activation of
hypotonicity activated anion channels.
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Acknowledgments |
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