Vol. 281, Issue 3, G816-G822, September 2001
Spontaneous water secretion in T84 cells: effects of
STa enterotoxin, bumetanide, VIP, forskolin, and A-23187
Roxana
Toriano1,
Arlinet
Kierbel1,
Marco Antonio
Ramirez2,
Gerhard
Malnic2, and
Mario
Parisi1
1 Laboratorio de Biomembranas, Departamento de
Fisiología, Facultad de Medicina, Universidad de Buenos Aires,
1453 Buenos Aires, Argentina; and 2 Departamento de Fisiologia e
Biofísica, Instituto de Ciências Biomédicas,
Universidade São Paulo, 05508 São Paulo, Brazil
 |
ABSTRACT |
The regulated
Cl
secretory apparatus of T84 cells responds to several
pharmacological agents via different second messengers (Ca2+, cAMP, cGMP). However, information about
water movements in T84 cells has not been available. In the absence of
osmotic or chemical gradient, we observed a net secretory
transepithelial volume flux (Jw = 
0.16 ± 0.02 µl · min1 · cm2) in
parallel with moderate short-circuit current values
(Isc = 1.55 ± 0.23 µA/cm2). The secretory Jw
reversibly reverted to an absorptive value when A-23187 was added to
the serosal bath. Vasoactive intestinal polypeptide increased
Isc, but, unexpectedly,
Jw was not affected. Bumetanide, an inhibitor of
basolateral Na+-K+-2Cl
cotransporter, completely blocked secretory Jw
with no change in Isc. Conversely, serosal
forskolin increased Isc, but
Jw switched from secretory to absorptive values.
Escherichia coli heat-stable enterotoxin increased secretory
Jw and Isc. No difference
between the absorptive and secretory unidirectional Cl
fluxes was observed in basal conditions, but after STa stimulation, a
significant net secretory Cl flux developed. We conclude
that, under these conditions, the presence of secretory or absorptive
Jw values cannot be shown by
Isc and ion flux studies. Furthermore, RT-PCR
experiments indicate that aquaporins were not expressed in T84 cells.
The molecular pathway for water secretion appears to be transcellular,
moving through the lipid bilayer or, as recently proposed, through
water-solute cotransporters.
water-ion permeability; chloride secretion; aquaporins; water-solute cotransport; heat-stable enterotoxin; vasoactive
intestinal peptide
| |
INTRODUCTION |
REGULATION OF THE
BALANCE of electrolytes and their secretion mechanisms have been
extensively studied in T84 cells, a human colonic tumor cell line that
maintains vectorial electrolyte transport (11).
This cell line secretes Cl in response to a variety of
secretagogues, the effects of which are mediated by cAMP- or
Ca2+-related mechanisms (6, 10, 12, 26, 42).
Furthermore, in the case of Escherichia coli heat-stable
enterotoxin (STa), the activation of the STa receptor guanylate cyclase
(35) stimulates Cl secretion via cGMP
accumulation (20, 37, 39). Despite these results and other
reports on ion secretion in this cell line (19), no
information on water and ion coupling has been obtained.
Vectorial fluid transport in epithelia can be associated with a
transepithelial hydrostatic pressure gradient, with a transepithelial osmotic gradient, or in their absence, with ionic transport (31, 32). These water movements can occur among or across the
epithelial cells, and in the latter case, the accepted water pathway is
the lipid bilayer itself (18) or specific water channels,
the aquaporins (1). Characterization and cloning of
epithelial water channels have provided important information on this
subject (25, 30, 43). Alternatively, solute-water
cotransport, a controversial mechanism conceptually different from
those previously mentioned, has been proposed (29, 44,
45).
Functional studies can give further insight into the water pathways in
"water channel-containing" and in "water channel-lacking" epithelial cells. The characteristics of transepithelial water permeability in two renal cell lines, cultured on a permeable support,
were previously described by our laboratory: RCCD1 cells devoid of
known aquaporins (5) and LLC-PK1 cells transfected with
AQP2 (41).
The aim of this study was to correlate water fluxes with the
simultaneous determination of electrical parameters and
Cl fluxes in T84 monolayers. In addition, the influence
of different pharmacological agents [A-23187, bumetanide, vasoactive
intestinal polypeptide (VIP), forskolin, and STa] was studied.
Minute-by-minute recordings of the transepithelial net water fluxes
(Jw) were associated, under different
experimental conditions, with the measurement of the transepithelial
potential difference (
VT) and resistance (RT), short-circuit current
(Isc), and transepithelial and unidirectional 36Cl fluxes. In other experimental series,
the presence of aquaporins was explored in RT-PCR experiments. Under
the experimental conditions described here, water secretion did not
always correlate with parallel changes in Isc.
This indicates that direct measurements of water flow are necessary to
define the bulk absorptive or bulk secretory characteristics of an
epithelial barrier.
| |
MATERIALS AND METHODS |
Cell culture.
T84 cells, obtained from the American Type Culture Collection
(Rockville, MD), were grown as monolayers in 1:1 mixture of DMEM and
Ham's F-12 medium supplemented with 14 mM NaHCO3, 3.2 mM
glutamine, 10 U/ml penicillin-streptomycin, 15 mM Na-HEPES (pH 7.4),
and 5% fetal bovine serum (GIBCO BRL) in a 5% CO2
atmosphere at 37°C. For these experiments, cells between
passages 60 and 78 were seeded on Transwell
holders (1-2 × 106 cells/Transwell on
3-µm-pore Nucleopore filters, 4.5-cm2 surface area;
Corning-Costar) and cultured for 10-12 days.
Solutions.
For the experiments, cells were bathed on either side with minimum
medium containing 1:1 DMEM-Ham's F12 (GIBCO BRL-Life Technologies) and
14 mM NaHCO3 plus 15 mM Na-HEPES, pH 7.4 when bubbled with 5% CO2-95% O2.
Measurement of water fluxes.
To perform water flux measurements, the Transwell holders, their bottom
covered with the confluent cell layer, were directly inserted between
two-barrel Lucite hemichambers so as to define two independent
compartments, as previously described (41). One of the
compartments (serosal) was open to the atmosphere, whereas the other
compartment (mucosal) was hermetically sealed. A positive hydrostatic
pressure gradient (4.5 cmH2O) was continuously applied to
the mucosal bath. The closed chamber was connected with a
small-diameter polyethylene tube to the net water measurement system,
where Jw was recorded every minute, as described
elsewhere (Ref 15; see Fig. 1). Briefly,
the position of a liquid meniscus inside a capillary tube was
photoelectrically detected. Displacements to the right or to the left
were proportional to the amount of water moving across the tissue
layer. The system sensitivity was 50 nl. The data were computed in
microliters per minute per square centimeter. The serosal bath was
continuously bubbled with the appropriate
CO2-O2 mixture to maintain the pH of the medium
at 7.4 ± 0.1 (37°C). Additional details of the method used are
provided in previous reports from our laboratory (5, 15,
41).
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of the automatic device to
measure and register net water fluxes across epithelia. It is based on
the detection of a liquid meniscus position inside a capillary tube
using an electrooptical device. Displacement to the right or to the
left is proportional to the amount of water absorbed or secreted by the
epithelial layer. The data are digitalized and converted to a
volumetric number (µl/min), and a graphic representation is given on
the monitor screen. Positive values indicate absorptive fluxes, whereas
negative values represent secretory fluxes. A, thermostated
chamber (m, mucosal side; s, serosal side); B, Transwell
holder; C, plastic tube with physiological solution;
D, second chamber; E, folded and impermeable
plastic membrane; F, plastic tube with photoopaque solution;
G, glass capillary; H, detection system;
I, device-computer connection. The water cycling system to
maintain a constant temperature and the gas port to bubble the tissue
with CO2-O2 are not shown.
|
|
In pharmacological studies, T84 cells were cultured in six-holder
clusters. To minimize random fluctuations, three holders were routinely
taken as controls and the rest were tested with the agents. These
agents (2 × 106 M A-23187, 105 M
bumetanide, 108 M VIP, and 105 M forskolin)
were added to the serosal bath in different experiments.
In STa experiments, confluent monolayers developed on Transwell holders
were preincubated for 1 h in experimental medium and then exposed
to STa (2.5 × 107 M in the apical bath) for 1 h. VT and RT values
were monitored to test STa action (36) before the holder
was mounted in the experimental chamber.
Electrophysiological studies.
VT and Isc were
continuously recorded with an automatic voltage-clamp system
(Physiological Instruments) and Navycite (ME2AG4) electrodes.
RT was initially measured with a Millicell-ERS
electric resistance system (Millipore), and it was estimated during the experiments every 90 s from current deflections in response to a 1 mV/s pulse.
Unidirectional 36Cl fluxes.
Once the holders were inserted in the experimental chamber,
36Cl was added to the mucosal or the serosal bath (1 µCi/ml). Samples (1 ml) were taken from the basolateral or apical
baths every 5 min. Serosa-to-mucosa, mucosa-to-serosa, and net fluxes
were calculated, taking into account corrections for sampling dilution
and back fluxes.
RT-PCR studies.
Total RNA from kidney (positive control) or T84 cells was isolated
using the SV total RNA isolation system (Promega). Reverse transcription was performed on 2 µg of total RNA using the
SuperScript Preamplification System for first-strand cDNA synthesis
(GIBCO BRL). RNAs were placed in 50 µl of RT reaction buffer
containing 1× PCR buffer, 0.5 µg of oligo(dT) primer, 0.1 mg/ml BSA,
10 mM dithiothreitol (DTT), 2.5 mM MgCl2, and 10 U/µl
RNAsin. The reaction was heated for 3 min at 80°C and cooled to
45°C. A PCR buffer solution (25 µl) containing 1× PCR buffer, 0.1 mg/ml BSA, 10 mM DTT, 2.5 mM MgCl2, 400 µM dNTP, and 100 U of SuperScript II RT was added to half of the reaction. Control
experiments in absence of the enzyme SuperScript II RT were performed
on the other 25 µl of the reaction. RT reaction was carried out for
1 h at 45°C and stopped by heating for 2 min at 95°C.
For PCR experiments, AQP-degenerated oligonucleotide primers were
designed corresponding to the most highly conserved sequences surrounding the NPA motifs in the aquaporins (33): sense
primers, WC1-up (5'-STB GGN CAY RTB AGY GGN GCN CA-3') and WC2-up
(5'-GGG ATC CGC HCA YNT NAA YCC HGY NGT NAC-3'); antisense primers,
WC1-dw (5'-GCD GRN SCV ARD GAN CGN GCN GG-3') and WC2-dw (5'-CGG AAT TCG DGC DGG RTT NAT NSH NSM NCC-3'). The reverse-transcribed RNA was
amplified by 40 cycles of PCR (1 min at 94°C, 1 min at 52°C, 1 min
at 72°C) using 100 pmol of WC1-up and WC1-dw; the products were
reamplified using 100 pmol of WC2-up and WC2-dw for 35 cycles with the
same protocol. Internal positive control was included in each
experiment by using 
-actin specific primers (sense: 5'-CGG AAC CGC
TCA TTG CC-3'; antisense: 5'-ACC CAC ACT GTG CCC ATC TA-3').
Statistics.
Data are presented as means ± SE. Statistical significance was
determined using the paired or unpaired t-test, and a
P value <0.05 was considered significant.
| |
RESULTS |
T84 cells spontaneously show a secretory flux: effects of A-23187.
T84 cells were cultured on permeable filters for 10-12 days before
they were mounted in the flux measurement chamber. A significant and
sustained spontaneous net secretory Jw (from
basolateral to apical side at 37°C) was observed in the absence of
any osmotic or chemical gradient and even against a hydrostatic
gradient (4.5 cm of water) present in our measurement system (Table
1; negative Jw
values in Table 1 and in text indicate secretory fluxes, whereas positive values represent absorptive fluxes). Table 1 shows that this
secretory Jw was associated with high
RT and rather moderate VT and Isc
values. Figure 2 shows that
addition of a calcium ionophore, A-23187 (2 × 106
M), to the serosal bath induced a reversible switch from a secretory to
an absorptive Jw.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Mean curve showing the evolution of water flux
(Jw) in T84 cells. Experimental values are
means ± SE of 5 experiments. A-23187, the calcium ionophore
(2 × 106 M) was added to the serosal bath; Wash,
the pharmacological agent was washed, replacing the serosal medium with
a fresh experimental medium. Negative values indicate secretory fluxes
and positive values absorptive fluxes.
|
|
Effect of bumetanide, VIP, and forskolin on Isc and
water movements in T84 cells.
It has been proposed that Cl secretion in colonic and in
T84 cells may involve Cl entry at the basolateral
membrane through a Na+-K+-2Cl
cotransporter and Cl exit at the apical membrane through
a Cl conductance (28), presumably the cystic
fibrosis transmembrane regulator (CFTR) (38). Figure
3 shows that serosal addition of
bumetanide (105 M), a specific blocker of the
Na+-K+-2Cl cotransporter,
significantly decreased water secretion without parallel changes in
nonstimulated Isc, whereas no significant change
in transepithelial resistance was observed. On the other hand, Fig.
4 shows that the addition of VIP
(108 M) resulted, as previously reported
(12), in an increase in Isc and a
30% reduction in RT. We observed no
simultaneous change in net water secretion. Together, the bumetanide
and VIP results show that changes in fluid secretion are not always
paralleled by changes in Isc and vice versa.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Mean curves (n = 5) showing the
simultaneous evolution of water fluxes (Jw,
 ) and short-circuit current
(Isc,  ) in T84 cell monolayers
in the absence of osmotic or chemical gradients. At the arrow,
105 M bumetanide was added to the serosal bath.
Inset shows the effect of bumetanide on transepithelial
resistance (RT) during the experiments, in which
no significant reduction was observed. Experimental
RT value is mean ± SE as percentage of
basal value.
|
|
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Time course of Jw
() and Isc () in
T84 cells: effect of vasoactive intestinal polypeptide (VIP).
Jw and Isc were
simultaneously measured under basal conditions (absence of osmotic or
chemical gradients) and in the presence of drug (108 M,
arrow) in the serosal bath. Experimental values are means ± SE of
8 experiments. Inset shows the RT
values (% of basal) before and after drug addition. In the presence of
VIP, RT was reduced 30%.
|
|
The effects of forskolin on T84 cells are presented in Fig.
5. As expected, a strong stimulation of
Isc was observed whereas the spontaneous
secretory Jw shifted to an absorptive value.
Although RT was reduced 70%, the
RT value after forskolin (740 ± 127 
· cm2; n = 5) was still
elevated for a cellular monolayer.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Simultaneous recording of time course changes in
Jw () and
Isc () in T84 cell line induced
by serosal forskolin (105 M, arrow). Data are means ± SE of 5 experiments. Inset shows the
RT values (% of basal) before and after drug
addition. In the presence of forskolin, RT was
reduced 67%.
|
|
Effects of STa on Jw, Isc, and
Cl secretion in T84 cells.
It was reported previously that STa stimulates cGMP production and
Cl secretion in T84 cells (20). We have now
compared water secretion, Isc, and
Cl secretion in T84 epithelia before and after this
enterotoxin action. The results obtained are summarized in Table 1. As
expected, an increase in Isc and
Cl secretion was observed together with an enhancement in
water secretion. Nevertheless, the ratio between the before-STa and after STa-values did not present the same order of magnitude for Jw, Isc, and
Cl secretion.
RT-PCR results.
To determine whether aquaporins are expressed in T84 cells, RT-PCR
experiments were performed using degenerated aquaporin primers
(33); the results obtained showed that although AQP mRNAs were detected in the kidney tissue (positive control),
no aquaporin transcripts were amplified in T84 cells (Fig.
6).
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 6.
RT-PCR experiments performed using degenerated primers for
aquaporins (AQPs) in mRNAs from kidney (positive control) and T84
cells. -Actin was used as an internal control. Assays were carried
out in presence (+) or absence () of RT enzyme. Specific bands can be
observed in all the positive controls, but transcripts were absent in
T84 cells. MW, molecular weight.
|
|
| |
DISCUSSION |
This study focuses on water secretion in T84 cells and their
modification under the influence of different stimuli such as STa. When
grown on permeable support, these cells exhibit high RT and develop significant Cl
secretion in response to environmental changes (2, 6, 8, 10, 12,
13, 26, 42). These observations support the notion that the T84
cell line expresses characteristics of colonic secreting cells
(9).
Water secretion in T84 cells: effects of A-23187, bumetanide, VIP,
and forskolin.
The human intestinal cell line T84 has been extensively utilized as a
model to explore electrogenic Cl secretion, where water
movement has been assumed as passive, and coupled with
Cl exit (9). The results
presented in this paper indicate that this is not always the case. Our
experiments show that fluid secretion in T84 cells occurred in the
absence of any chemical or osmotic gradient, features suggesting
energy-requiring mechanisms for the transport of fluid. A remarkable
observation was that the spontaneous secretory
Jw observed in this high-resistance
epithelium was associated neither with an important
Isc nor with a significant Cl exit
(Table 1).
Under physiological conditions, intestinal epithelial cells are
exposed, in different circumstances, to relative high-pressure gradients. Our experimental system implies the presence of a moderate hydrostatic pressure applied on the mucosal surface (4.5 cmH2O). Water secretion was observed, in T84 monolayers and
in basal conditions, even against this pressure gradient. In previous
reports, water movements across mammalian intestinal epithelia were
studied with the same experimental device and no secretory response was
observed associated with the applied hydrostatic pressure gradients
(4, 16, 17). As for cell lines cultured on permeable
supports, no correlation between water secretion and the applied
hydrostatic pressure gradients was observed in Caco-2 and LCC-PK1
monolayers (32, 41).
Tai et al. (40) reported that A-23187 reversibly decreased
tight junction resistance in T84 epithelial monolayers. Our results showing a reversible switch from a secretory to an absorptive Jw can thus be easily understood: in the
presence of a mucosal hydrostatic pressure, tight junctions opening
would induce a paracellular water movement that would disappear rapidly
when the ionophore was removed from the serosal bath. If an associated
water secretory response exists, it would be masked by the significant
paracellular absorptive movement.
The Na+-K+-2Cl cotransporter has
been immunolocalized to the basolateral domain in T84 cells
(9), and the current consensus model indicates that net
Cl secretion involves Cl entry into the
cell through this cotransporter. Previous studies (10, 11,
12) showed that, under the action of different stimuli,
Isc was caused by electrogenic Cl
exit whereas isotonic water secretion would be associated with the
ionic flux. This mechanism cannot explain the observed secretory Jw under basal conditions. If the basal
Jw observed here was associated with a pure
electrogenic Cl secretion, the expected
Isc would be ~30
µA · cm2, and the expected associated net flux of
Cl would be ~200 × 104
µEq · min1 · cm2.
Nevertheless, the inhibitory effects of bumetanide (Fig. 3), an agent
that blocks the Na+-K+-2Cl
cotransporter, on the observed Jw suggest
specific coupling of Cl and water in at least part of the
secretion reported here. The lack of effect of this drug on the basal
Isc strengthens the view that the observed
spontaneous water secretion was coupled to a nonelectrogenic process.
When intracellular pH regulation in this cell line was studied,
evidence was obtained for the presence of a Na+-independent
Cl/HCO
exchanger, for the presence of a Na+-HCO cotransport system, and also for the presence of a Na+/H+ exchanger
(34). Our results strongly suggest that
HCO secretion would be present in basal conditions
in T84 cells. A straightforward interpretation of the bumetanide
results would then be a secondary inhibition of HCO secretion associated with the reduction in Na+ and
Cl entry into the cell. In summary, to understand the
significant basal secretory Jw observed in the
absence of net Cl secretion and associated with a
moderate Isc, we can propose two working
hypotheses: 1) a very significant nonelectrogenic HCO secretion is associated with an isotonic
secretory flow; or 2) basal secretion is hypotonic.
Interestingly enough, the presence of a significant spontaneous
secretor flux in RCCD1 cells was reported recently, although it was
associated in this case with relatively higher
Isc values (5). The observed water
secretion could be, in this case, abolished by ion substitution, low
temperature, and other inhibitors of transport pathway.
The interpretation of VIP effects showed in Fig. 4 is complex. If an
isotonic and electrogenic Cl secretion was present at the
beginning of the observed increase in Isc, the
spontaneous secretory Jw observed would also
increase up to a value duplicating the basal flow (from ~0.15 to 0.30 µl · min1 · cm2).
Nevertheless, if VIP effects reduced paracellular hydraulic resistance
(electrical resistance was in fact reduced), it would be possible that
a pressure-driven flow could compensate the increase in the secretor
flux. Future experiments must clarify this point.
The effect of forskolin (Fig. 5) can be straightforwardly interpreted
as a pure absorptive effect, both increasing Isc
and inducing water reabsorption. Interestingly,
Jw and Isc values were reasonable in the expected range, if a monovalent cation (Na+?) were electrogenically and isotonically transported.
Results from bumetanide, VIP, and forskolin experiments depicted in
Figs. 3-5 led us to conclude that Isc and
Jw can be differently associated in different
experimental situations. Consequently, Isc
values per se could not be considered as coupled to a "secretory" or "absorptive" volume flux.
Water and ion coupling in T84 cells: effect of STa toxin.
STa effects on water secretion in T84 cells can be straightforwardly
interpreted as associated with Cl secretion. This is
strongly supported by results presented in Table 1. However, the same
results show that secretory Cl and water fluxes can be
partially dissociated in these cells. The most striking information was
obtained when changes in Isc, Jw, and Cl secretion were compared.
First, we may compare the increases in Cl net secretion
and Isc induced by the action of STa. The
observed data indicate that, as previously proposed, the STa-induced
increase in Isc was associated with a "net"
Cl exit. The cellular mechanism is likely to involve
uptake of Cl across the basolateral membrane via the
Na+-K+-2Cl cotransporter and
diffusion out of the cell across the apical membrane through CFTR
and/or other Cl channels. Previous studies demonstrated
that the CFTR channel is expressed in T84 cells (see Ref.
7). However, the amount of secreted water was higher than
that expected from the observed Cl secretion. It can then
be concluded that the nonelectrogenic secretion of another anion
(HCO?) is involved, as in the basal secretion phenomenon.
Water pathways in water secretion.
The water pathway(s) involved in secretion mechanisms remains unclear.
Water can move across epithelial barriers through either a
transcellular or a paracellular route. It was recently demonstrated in
MDCK cells that little water, if any, permeates the paracellular pathway during isotonic reabsorption (22). We can accept
that a similar situation occurs during isotonic secretion. This would be especially reasonable in T84 cells forming a high-resistance epithelial barrier. We can conclude that water moves transcellularly in
the spontaneous secretory flux reported here.
Water could cross cell membranes by two different pathways, the lipid
bilayer or specific water channels (aquaporins) (1, 14,
31). Since the presence of AQP1, AQP3, AQP4, and AQP8 in the
colon was reported (23-25, 27), the first candidates
to explain water movements in T84 were water channels. Our RT-PCR studies (Fig. 6) indicate the absence of aquaporin mRNAs in T84 cells,
in accordance with the observation that, in general, epithelial cell
lines in culture are devoid of aquaporins. For instance, in LLC-PK1
cells, a hydrosmotic response to vasopressin was only reconstituted
after AQP2 transfection (21, 41). A lack of AQP
expression in a high-resistance epithelial cell line (3, 5) derived from rat cortical collecting duct (RCCD1) was also observed recently. Nevertheless, significant ionic-associated water
transfers were observed (5). A third hypothetical
mechanism (ion-water cotransport proteins) has been proposed to explain the water flux associated with the K+-Cl,
Na+-glucose, and Na+-dicarboxylate
cotransporters (29, 44, 45).
Despite the lack of aquaporin RNA transcription, significant water
fluxes were observed in T84 cells. The water pathway was probably
transcellular. The results presented here can be understood if we
accept that, in all cases, solute transport induces a "local osmosis
process" driving water across the lipid bilayer of the epithelial
cells. To explain water secretion we would need to propose the buildup
of an osmotic gradient in the mucosal unstirred layer. Alternatively,
water could be moving, as previously mentioned, by a cotransport
mechanism (29, 44, 45). Further experimental evidence is
necessary to clarify this point.
In summary, 1) net water movements were, for the first time,
measured in T84 formed epithelia showing a spontaneous water secretion
not associated with a Cl net exit; 2) basal as
well as pharmacologically induced water movements (absorptive or
secretory) were coupled to different electrogenic and nonelectrogenic
ionic transfers; 3) STa-induced fluid secretion cannot be
explained by an isotonic Cl exit alone; and 4)
relevant water secretory fluxes were measured in T84 cells in the
absence of aquaporin messenger RNAs.
| |
ACKNOWLEDGEMENTS |
This work was supported by grants from Concejo Nacional de
Investigaciones Científicas y Técnicas (CONICET,
Argentina); Universidad de Buenos Aires, Argentina; Fondo Nacional para
la Ciencia y la Técnica (FONCYT, Argentina); Fundação
de Amparo à Pesquisa, São Paulo (FAPESP, Brazil); and
Conselho Nacional de Desenvolvimento Cientifico e Tecnológico
(CNPq, Brazil).
| |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Parisi, Lab. Biomembranas, CC 128, suc 53B, (1453) Buenos Aires, Argentina (E-mail: parisi{at}mail.retina.ar).
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.
Received 22 January 2001; accepted in final form 11 May 2001.
| |
REFERENCES |
1.
Agre, P,
Sasaki S,
and
Chrispeels MJ.
Aquaporins: a family of water channel proteins.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F461-F468,
1993[Free Full Text].
2.
Benya, RV,
Marrero JA,
Ostrovskiy DA,
Koutsouris A,
and
Hecht G.
Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl secretion.
Am J Physiol Gastrointest Liver Physiol
276:
G64-G72,
1999[Abstract/Free Full Text].
3.
Blot-Chabaud, M,
Laplace M,
Cluzeaud F,
Capurro C,
Cassingena R,
Vandewalle A,
Farman N,
and
Bonvalet JP.
Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance.
Kidney Int
60:
367-376,
1996.
4.
Capurro, C,
and
Parisi M.
Water handling in the rat jejunum: effects of acidification of the medium.
Pflügers Arch
421:
17-21,
1992[ISI][Medline].
5.
Capurro, C,
Rivarola V,
Kierbel A,
Escoubet B,
Farman N,
Blot-Chabaud M,
and
Parisi M.
Vasopressin regulates water flow in a rat cortical collecting duct cell line not containing known aquaporins.
J Membr Biol
179:
63-70,
2001[ISI][Medline].
6.
Cartwright, CA,
McRoberts J,
Mandel KG,
and
Dharmsathaphorn K.
Synergistic action of cyclic AMP and calcium mediated chloride secretion in a colonic epithelial cell line.
J Clin Invest
76:
1837-1842,
1985.
7.
Chao, AC,
de Sauvage FJ,
Dong YJ,
Wagner JA,
Goeddel DV,
and
Gardner P.
Activation of intestinal CFTR Cl channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.
EMBO J
13:
1065-1072,
1994[ISI][Medline].
8.
Cliff, WH,
and
Frizzell RA.
Separate Cl conductances activated by cAMP and Ca2+ in Cl secreting epithelial cells.
Proc Natl Acad Sci USA
87:
4956-4960,
1990[Abstract/Free Full Text].
9.
D'Andrea, L,
Lytle C,
Matthews JB,
Hofman P,
Forbush B, III,
and
Madara JL.
Na:K:2Cl cotransporter (NKCC) of intestinal epithelial cells.
J Biol Chem
271:
28969-28976,
1996[Abstract/Free Full Text].
10.
Dharmsathaphorn, K,
and
Pandol SJ.
Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.
J Clin Invest
77:
348-354,
1986.
11.
Dharmsathaphorn, K,
McRoberts J,
Mandel KG,
Tisdale LD,
and
Masui H.
A human colonic tumor cell line that maintains vectorial electrolyte transport.
Am J Physiol Gastrointest Liver Physiol
246:
G204-G208,
1984[Abstract/Free Full Text].
12.
Dharmsathaphorn, K,
McRoberts J,
Masui H,
and
Mandel KG.
Vasoactive intestinal polypeptide-induced chloride secretion by a colonic epithelial cell line. Direct participation of a basolaterally localized Na+,K+,Cl cotransport system.
J Clin Invest
75:
462-471,
1985.
13.
Dho, S,
Stewart K,
and
Foskett JK.
Purinergic receptor activation of Cl secretion in T84 cells.
Am J Physiol Gastrointest Liver Physiol
262:
G67-G74,
1992.
14.
Diamond, JM.
Osmotic water flow in leaky epithelia.
J Membr Biol
51:
195-216,
1979[ISI][Medline].
15.
Dorr, R,
Kierbel A,
Vera J,
and
Parisi M.
A new data-acquisition system for the measurement of the net water flux across epithelia.
Comput Methods Programs Biomed
53:
9-14,
1997[ISI][Medline].
16.
Escobar, E,
Galindo F,
and
Parisi M.
Water handling in the human distal colon in vitro: role of Na+, Cl and HCO3.
Biochim Biophys Acta
1027:
257-263,
1990[Medline].
17.
Escobar, E,
Ibarra C,
Todisco E,
and
Parisi M.
Water and ion handling in the rat cecum.
Am J Physiol Gastrointest Liver Physiol
259:
G786-G791,
1990[Abstract/Free Full Text].
18.
Finkelstein, A.
Water Movements Through Lipid Bilayers, Pores and Plasma Membranes. Theory and Reality. New York: Wiley, 1987.
19.
Greger, R.
The membrane transporters regulating epithelial NaCl secretion.
Pflügers Arch
432:
579-588,
1996[ISI][Medline].
20.
Huott, PA,
Liu W,
McRoberts JA,
Giannella RA,
and
Dharmsathaphorn K.
Mechanism of action of Escherichia coli heat-stable enterotoxin in a human colonic cell line.
J Clin Invest
82:
514-523,
1988.
21.
Katsura, T,
Verbavatz JM,
Farinas J,
Ma T,
Ausiello DA,
Verkman AS,
and
Brown D.
Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.
Proc Natl Acad Sci USA
92:
7212-7216,
1995[Abstract/Free Full Text].
22.
Kovbasnjuc, O,
Leader J,
Weinstein A,
and
Spring K.
Water does not flow across the tight junctions of MDCK cell epithelium.
Proc Natl Acad Sci USA
95:
6526-6530,
1998[Abstract/Free Full Text].
23.
Koyama, N,
Ishibashi K,
Kuwahara M,
Inase N,
Ichioka M,
Sasaki S,
and
Marumo F.
Cloning and functional expression of human aquaporin 8 cDNA and analysis of its gene.
Genomics
54:
169-172,
1998[ISI][Medline].
24.
Koyama, N,
Yamamoto T,
Tani T,
Nihri K,
Kondo D,
Funaki H,
Yaoita E,
Kawasaki K,
Sato N,
Hatakeyama K,
and
Kihara I.
Expression and localization of aquaporins in rat gastrointestinal tract.
Am J Physiol Cell Physiol
276:
C621-C627,
1999[Abstract/Free Full Text].
25.
Ma, T,
and
Verkman AS.
Aquaporin water channels in gastrointestinal physiology.
J Physiol (Lond)
517:
317-325,
1999[Abstract/Free Full Text].
26.
Mandel, KG,
McRoberts JA,
Beuerlein G,
Foster ES,
and
Dharmsathaphorn K.
Ba2+ inhibition of VIP and A23187 stimulated Cl secretion by T84 cells monolayers.
Am J Physiol Cell Physiol
250:
C486-C494,
1986[Abstract/Free Full Text].
27.
Matsuzaki, T,
Suzuki T,
Koyama H,
Tanaka S,
and
Takata K.
Water channel protein AQP3 is present in epithelia exposed to the environment of possible water loss.
J Histochem Cytochem
47:
1275-1286,
1999[Abstract/Free Full Text].
28.
Matthews, JB,
Awtrey CS,
Thompson R,
Hung T,
Tally KJ,
and
Madara JL.
Na+-K+-2Cl cotransport and Cl secretion evoked by heat-stable enterotoxin is microfilament dependent in T84 cells.
Am J Physiol Gastrointest Liver Physiol
265:
G370-G378,
1993[Abstract/Free Full Text].
29.
Meinild, AK,
Loo DDF,
Pajor AM,
Zeuthen T,
and
Wright EM.
Water transport by the renal Na+-dicarboxylate cotransporter.
Am J Physiol Renal Physiol
278:
F777-F783,
2000[Abstract/Free Full Text].
30.
Nielsen, S,
and
Agre P.
The aquaporin family of water channels in kidney.
Kidney Int
48:
1057-1068,
1995[ISI][Medline].
31.
Parisi, M,
Amodeo G,
Capurro C,
Dorr R,
Ford P,
and
Toriano R.
Biophysical properties of epithelial water channels.
Biophys Chem
68:
255-263,
1997.
32.
Parisi, M,
Escobar E,
Huet C,
Ripoche P,
Louvard D,
and
Bourguet J.
Water handling in Caco-2 cells: effects of acidification of the medium.
Pflügers Arch
423:
1-6,
1993[ISI][Medline].
33.
Raina, S,
Preston GM,
Guggino WB,
and
Agre P.
Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues.
J Biol Chem
270:
1908-1912,
1995[Abstract/Free Full Text].
34.
Ramirez, MA,
Toriano R,
Parisi M,
and
Malnic G.
Control of cell pH in the T84 colon cell line.
J Membr Biol
177:
149-157,
2000[ISI][Medline].
35.
Rao, MC.
Toxins which activate guanylate cyclase: heat-stable enterotoxins.
Ciba Found Symp
112:
74-93,
1985[Medline].
36.
Rao, MC,
Guandalini S,
Smith PL,
and
Field M.
Mode of action of heat-stable Escherichia coli enterotoxin. Tissue and subcellular specificities and role of cyclic GMP.
Biochim Biophys Acta
632:
35-46,
1980[Medline].
37.
Schulz, S,
Green CK,
Yuen PST,
and
Garbers DL.
Guanylyl cyclase is a heat-stable enterotoxin receptor.
Cell
63:
941-948,
1990[ISI][Medline].
38.
Sheppard, DN,
and
Welsh MJ.
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J Gen Physiol
100:
573-91,
1992[Abstract/Free Full Text].
39.
Singh, S,
Singh GS,
Heim JM,
and
Gerzer R.
Isolation and expression of a guanylate cyclase-coupled heat-stable enterotoxin receptor cDNA from a human colonic cell line.
Biochem Biophys Res Commun
179:
1455-1463,
1991[ISI][Medline].
40.
Tai, Y,
Flick J,
Levine S,
Madara J,
Sharp G,
and
Donowitz M.
Regulation of tight junction resistance in T84 monolayers by elevation in intracellular Ca2+: a protein kinase C effect.
J Membr Biol
149:
71-79,
1996[ISI][Medline].
41.
Toriano, R,
Ford P,
Rivarola V,
Tamarappoo BK,
Verkman AS,
and
Parisi M.
Reconstitution of a regulated transepithelial water pathway in cells transfected with AQP2 and an AQP1/AQP2 hybrid containing the AQP2-C terminus.
J Membr Biol
161:
141-149,
1998[ISI][Medline].
42.
Weymer, A,
Huott P,
Liu W,
McRoberts J,
and
Dharmsathaphorn K.
Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line.
J Clin Invest
76:
1828-1836,
1985.
43.
Yamamoto, T,
and
Sasaki S.
Aquaporins in the kidney: emerging new aspects.
Kidney Int
54:
1041-1051,
1999.
44.
Zeuthen, T.
Cotransport of K+, Cl and H2O by membrane proteins from choroid plexus epithelium of Necturus maculosus.
J Physiol (Lond)
478:
203-219,
1994[ISI][Medline].
45.
Zeuthen, T,
Meinild AK,
Klaerke DA,
Loo DD,
Wright EM,
Belhage B,
and
Litman T.
Water transport by the Na+/glucose cotransporter under isotonic conditions.
Biol Cell
89:
307-312,
1997[Medline].
Am J Physiol Gastrointest Liver Physiol 281(3):G816-G822
0193-1857/01 $5.00
Copyright © 2001 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
