special communication
Regulation of sodium transport in M-1 cells
NAZIH L. NAKHOUL, KATHLEEN S. HERING-SMITH,
CECILIA T. GAMBALA, AND L. LEE HAMM
Departments of Medicine, Section of Nephrology, and Physiology, Tulane University School
of Medicine, and Veterans Affairs Medical Center, New Orleans, Louisiana 70112
aldosterone; dexamethasone; arginine vasopressin; aprotinin; collecting duct cells
THE MAMMALIAN CORTICAL collecting duct (CCD) plays a
major role in regulating renal Na1 absorption and as
such is important in controlling total body Na1 homeostasis. The general model of Na1 reabsorption in CCD
involves coordinated Na1 entry across the apical membrane through Na1 channels and Na1 exit across the
basolateral membrane by Na-K-ATPase (39). Previous
studies of isolated perfused CCD and model culture
epithelia (such as A6 cells) have yielded invaluable
information about the membrane properties and transport mechanisms involved in Na1 regulation in this
segment. These studies indicate that many hormones
[e.g., adrenal steroids, arginine vasopressin (AVP),
phorbol esters, bradykinin, and endothelin] contribute
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to regulation of Na1 transport in CCD (5, 6, 39). Among
these, aldosterone (Aldo) is the main stimulant of Na1
reabsorption in the mammalian collecting duct.
Administration of Aldo stimulates Na1 reabsorption
by augmenting Na1 entry across the apical Na1 channels and by subsequently activating basolateral Na-KATPase (35, 39). Because of the importance of this
process for Na1 balance, the cellular and molecular
mechanisms of hormonal regulation of Na1 transport
remain under intense study using a variety of experimental preparations. Cell culture models provide many
advantages to address issues that are difficult to study
in vivo. However, most cell culture studies of the effects
of mineralocorticoids have used the A6 amphibian cell
line rather than mammalian cells (16). M-1 cells, a
mammalian cell line derived from microdissected CCD
of a mouse transgenic for the early region of SV40 virus,
maintain many characteristics of CCD and were used
for this study. Although M-1 cells have been utilized
since their development for a variety of studies, particularly addressing epithelial Na1 channels, the hormonal
regulation of transepithelial transport in these cells
has not been fully characterized. Therefore, the purpose of the present studies was to determine the acute
and chronic hormonal regulation of Na1 transport in
these mammalian cells.
The present study was designed to address several
important issues. First, we characterized the transepithelial transport characteristics of these cells and the
conditions under which M-1 cells in serum-free media
can be used to study transport. Second, we characterized the differential effects of Aldo and dexamethasone
(Dex) on transport. We were interested in comparing
the effects of both hormones, the time course during
which an effect on transport developed, and the possibility that their actions overlap. Third, we investigated
the effects of other hormones that acutely modulate
Na1 transport in other CCD preparations. These included epidermal growth factor (EGF), phorbol esters
[phorbol myristate acetate (PMA)], and AVP. Finally,
we investigated whether these mammalian cells contain an endogenous protease activity as has been
recently reported in A6 cells.
METHODS
Cell culture. The M-1 cell line was originally derived by
Stoos et al. (47) from a mouse transgenic for the early region
of simian virus 40 [strain Tg(SV40)Bri/7]. The M-1 cells used
0363-6127/98 $5.00 Copyright r 1998 the American Physiological Society
Downloaded from http://ajprenal.physiology.org/ by 10.220.33.4 on June 18, 2017
Nakhoul, Nazih L., Kathleen S. Hering-Smith, Cecilia
T. Gambala, and L. Lee Hamm. Regulation of sodium
transport in M-1 cells. Am. J. Physiol. 275 (Renal Physiol. 44):
F998–F1007, 1998.—The M-1 cell line, derived from the
mouse cortical collecting duct (CCD), is being used as a
mammalian model of the CCD to study Na1 transport. The
present studies aimed to further define the role of various
hormones in affecting Na1 transport in M-1 cells grown in
defined media. M-1 cells on permeable support, in serum-free
media, developed amiloride-sensitive current 4–5 days after
seeding. As expected for the involvement of epithelial Na1
channels, a-, b-, and g-subunits of the epithelial Na1 channel
were identified by RT-PCR. Either dexamethasone (Dex,
10–100 nM) or aldosterone (Aldo, 1026 –1027 M) for 24 h
stimulated transport. Cells grown in the presence of Aldo and
Dex had higher transport than with Dex alone. Spironolactone added to Dex media decreased transport. The acute
effects of hormones reported to inhibit Na1 transport in CCD
were also examined. Epidermal growth factor, phorbol esters,
and increased intracellular Ca21 with thapsigargin did not
alter transport. Arginine vasopressin caused a transient
increase in transport (probably Cl2 secretion), which was not
amiloride sensitive. Also, the protease inhibitor aprotinin
decreased Na1 transport; in aprotinin-treated cells, trypsin
stimulated transport. This study demonstrates that adrenal
steroids (Dex . Aldo) stimulate Na1 transport in M-1 cells. At
least part of this response may represent activation of
mineralocorticoid receptors based on an additive effect of Dex
and Aldo, as well as inhibition by spironolactone. Responses
to immediate-acting hormones is limited. However, an endogenous protease activity, which activates Na1 transport, is
present in these cells.
HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
repeated measurements could be obtained. Vte was determined with the basolateral chamber as reference. Rte was
obtained by injecting current across the epithelium for a
period of ,1 s. The Ieq was calculated as the ratio of Vte to Rte
(corrected for the resistance of the fluid). The distance
between the two electrodes was constant in all experiments.
Ieq was normalized, by dividing Ieq by the surface area (4.52
cm2 ) of active membrane. Using this method, we were able to
monitor Vte and Rte on a daily basis to verify formation of
confluent monolayers without contaminating the cultures.
Multiple measurements on the same Transwell are not
possible using the Ussing chamber. To study the acute effects
of hormones, measurements using EVOM were obtained from
Transwells placed in a converted incubator maintained at
37°C and at 5% CO2. After a period of equilibration (1–2 h) in
the incubator, multiple and periodic measurements of Vte and
Rte were obtained once every 3 or 5 min. Once baseline
readings had stabilized, the experimental agent (see below)
was added to the appropriate compartment(s) in three Transwells at a time, while vehicle was added to another set of
three Transwells whose Vte and Rte closely matched those of
the hormone-treated ones. In this manner, the effects could be
compared in a paired fashion while eliminating wide variations in transport that would inevitably occur as the cultures
grow in time.
Measurements of intracellular Ca21. Changes in [Ca21]i
were determined from fluorescence measurements of fura 2
trapped intracellularly. Cells grown on coverslips were washed
twice with mammalian Ringer solution and then incubated at
37°C with a solution containing 5 µM of the acetoxymethyl
ester of fura 2 (fura 2-AM; Molecular Probes, Eugene, OR).
The fura 2-AM, a precursor of the dye fura 2, was added from
a 1 mM stock solution in DMSO. Adequate cell loading was
obtained by incubating the cells for ,60 min. Cells were then
washed twice with control solution and then transferred to a
special chamber where the coverslips formed the bottom of
the chamber. The Ringer solution was HCO2
3 free (HEPES
buffered) with a pH of 7.4 at 37°C, and the cells were
continuously superfused with this solution. Using a PTI
system (PTI, Princeton, NJ), we determined [Ca21]i values by
alternating the excitation wavelength between 340 and 380
nm and measuring the emission signal at 510 nm. Change in
fluorescence ratio (R340/380 ) obtained in this manner is proportional to changes in [Ca21]i. Data points were recorded at 12
points/s. Approximately 15–20 cells were in the field of
measurement.
Reverse transcription-polymerase chain reaction. We also
used RT-PCR to verify the presence of the three subunits of
the apical Na1 channel. mRNA was isolated from M-1 cells by
affinity chromatography on oligo(dT)-cellulose with the MicroFast Track kit (Invitrogen). The RT-PCR reactions were
performed using the Invitrogen cDNA Cycle Kit. First-strand
cDNA was synthesized from M-1 cell mRNA using random
primers and avian myeloblastosis virus reverse transcriptase. Promega Taq DNA polymerase was used for PCR. Initial
melting was at 94°C for 5 min, and then 30 cycles of the
following were run: 1) melting at 94°C for 1 min, 2) annealing
at 55°C for 2 min, and 3) extension at 72°C for 2 min. Final
extension was 72°C for 8 min. RT-PCR products were visualized by ultraviolet light using ethidium bromide staining
after 1% agarose gel electrophoresis.
Primers were designed based on the published sequences of
the rat and human a-, b-, and g-subunits of the apical
amiloride-sensitive Na1 channel, because the mouse sequence has not been published (7, 9, 30, 31). Primers were
selected on the basis of areas of identity (or near) between the
rat and human sequences. For a-subunit, sense and antisense
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in this study were obtained from Drs. Geza Fejes-Toth, B.
Stoos, and J. Garvin and belonged to the same strain as the
ones originally described. Cells were used from passages
11–17 with no obvious changes in electrical properties among
different passages. The cells were initially cultured in plastic
flasks (Costar, Corning, NY) and grown to confluence in a
humidified incubator at 37°C and 5% CO2. The cultures were
initially maintained in a defined medium consisting of equal
amounts of Ham’s F-12 and low-glucose DMEM (Sigma),
supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50
mg/ml streptomycin, 5% fetal bovine serum, growth promoting factors (transferrin, insulin, sodium selenite, 6.25 mg/ml
each), and 100 nM Dex. Media were changed every 2 days.
After cells reached confluence, usually in ,7 days, they were
passaged by trypsinization and plated onto semipermeable
membranes for electrophysiological studies or on coverslips
for intracellular Ca21 ([Ca21]i ) measurements. For transepithelial measurements, cells were seeded on semipermeable
membranes, 24 mm in diameter (Transwells, Costar). The
seeding density was 2.5 3 105 cells/Transwell and the active
surface area was 4.52 cm2. On the third day after seeding, the
culture medium was changed to the identical media described
above except without serum. Cells were maintained in this
media (Dex) until formation of confluent monolayers. Cells
seeded on coverslips for optical measurements were treated
in exactly the same way and were usually studied 5–8 days
after seeding. Cell monolayers were confirmed to be confluent
by development of adequate transepithelial resistance (Rte )
and voltage (Vte ), which were monitored daily. At this point,
the media was changed again to contain Aldo (1 µM), or Dex
(100 nM), or vehicle (ethanol). Electrical monitoring of Vte and
Rte was maintained daily, and media changes were kept at
2-day intervals.
Electrophysiological transepithelial measurements. Measurements of Vte, Rte, and short circuit current (Ieq) were
obtained by two methods. In the first method, the Transwells
with the M-1 cells forming a monolayer were mounted in a
Ussing-type chamber, especially designed to accept Transwells (EVC-4000; WPI, Sarasota, FL). A Transwell containing
no cultured cells filled with media or mammalian Ringer
solution was used to null junction potentials and to compensate for the resistance of the system. The confluent monolayer
of M-1 cells separated the apical and basolateral compartments. The two compartments were filled with continuously
circulating symmetrical solutions which contained 25 mM
bicarbonate, and were continuously bubbled with 5% CO2 (pH
7.4). The whole apparatus was jacketed by warm air and
maintained at a constant temperature of 37°C. Two lowresistance Ag:AgCl electrodes made contact with the two
chambers through Ringer-Agar bridges placed in the solution
lines and were used to measure Vte. Two other Ag:AgCl
electrodes were placed very close on either side of the tissue
through 3 M KCl-agar bridges and were used to inject a
current across the epithelial monolayer. The monolayer culture was maintained in an essentially open-circuit condition
and Vte was continuously measured in this manner. During
periodic intervals (every 5 min), Vte was clamped at 0 mV
(short-circuit condition), and Ieq was measured. Rte was
calculated from the ratio of Vte to Ieq.
In the second method, Vte was measured by means of a set
of two Ag:AgCl electrodes (STX electrodes, WPI) using an
ohm/volt meter (EVOM, WPI). The voltage readings were
nulled to zero when Vte was measured across a Transwell with
no cultured cells. Transepithelial measurements on confluent
M-1 monolayers were performed while the Transwells sat
undisturbed in the culture plates. Measurements were made
under sterile conditions in a laminar flow hood and thus
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HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
RESULTS
Electrical transepithelial parameters: baseline values. Our initial experiments defined the time course
during which M-1 cells develop transport properties
that are characteristic of the CCD. To do this, daily
measurements of Vte, Rte, and Ieq were taken. Measurements were conducted either before changing media or
at least 3 h thereafter. All Transwells were measured
daily in the laminar flow hood using the STX-2 electrodes and the EVOM ohm/volt meter. Each (one)
culture plate, with six Transwells, was removed from
the incubator individually, and measurements were
completed within 3–5 min. This short time interval is
necessary to prevent drift in measurements due to
changes in temperature and pH of the cells. Measurements over a longer period of time (.10 min) displayed
gradual instability, particularly in Rte, which usually
increased slowly. Concurrently, daily experiments were
also conducted on at least two Transwells using the
Ussing chamber, as outlined above. Measurements
from both techniques could thus be compared.
Four days after seeding (1 day after serum removal),
cells developed apical-negative Vte ranging from 5 to 25
mV and Rte ranging from 200 to 800 V · cm2. The
presence of Dex proved to be crucial, because the
transepithelial readings consistently collapsed in its
absence. Subsequent daily readings showed that adequate measurements are maintained for the next 4–6
days with optimal values usually obtained 1–2 days
after removal of serum. In 45 cultures, the average
peak values were as follows: Vte 5 24.5 6 1.6 mV, Rte 5
2,795 6 163 V · cm2, and Ieq 5 8.7 6 0.5 µA/cm2. In
cultures in which serum was not removed, the measure-
ments were not significantly different. High Vte and Rte
are characteristic properties of the mammalian CCD.
When measured in the Ussing chamber, the time
course for the development of Vte and Ieq was essentially
the same as that obtained from EVOM readings. However, two important observations were apparent. The
yield of successful experiments in the Ussing chamber
was very low since stable readings could often not be
obtained. Usually, this was caused by a continuous
downward drift in Rte that eventually led to a progressive rise in Ieq to an open reading; this occurred after a
short (,10 min) lag time during which Ieq remained
stable. On the other hand, when stable measurements
were possible, these readings were usually a fraction
(30–50%) of the measurements obtained using the
EVOM. Although one would expect that the environment of cells in the Ussing chamber would be better
controlled with a more stable pH and temperature,
EVOM experiments were apparently less detrimental
to the cells. These observations suggested that these
cells are relatively fragile and very sensitive to physical
perturbations, as would occur when solutions are circulating in the Ussing chamber. In support of this,
transepithelial measurements (using EVOM) immediately after changing the media yielded much lower
values of Vte and Ieq compared with immediately before
media change. In other experiments, using EVOM, we
found out that even careful removal of the bathing
media, followed by returning it back to the respective
compartment, substantially diminished Vte and Ieq.
These effects were transient, and cells usually fully
recovered if left undisturbed for 2–3 h. These effects
may indicate a loss of monolayer integrity (presumably
by affecting tight junctions) and diminished transport
in response to even minimal perturbations. These
results rendered the use of the Ussing chamber for
studying transepithelial transport in these cells extremely difficult. Consequently, we performed most
experiments using the EVOM on Transwells while still
in the culture plates, in a converted cell culture incubator maintained at 37°C and 5% CO2. Measurements
conducted in this fashion were consistent and reliable.
Effect of amiloride. Na1 transport in M-1 cells, like
the CCD, presumably involves luminal Na1 uptake
through Na1 channels. To verify that apical Na1 channels are responsible for the development of Ieq and Vte in
M-1 cells, amiloride was added to the apical compartment. In 22 experiments, addition of 10 µM amiloride
rapidly depolarized Vte from 20.4 6 2.2 to 4.7 6 0.8 mV
and increased Rte from 2,812 6 131 to 3,977 6 271
V · cm2, and the Ieq decreased from 8.0 6 1 to 1.2 6 0.2
µA/cm2 (P , 0.001).
Effects of Aldo and/or Dex on transepithelial measurements. The next series of experiments was performed to
determine how M-1 cells respond to treatment with
glucocorticoids and/or mineralocorticoids. Three factors
were determined: 1) the degree of stimulation of Ieq and
Vte by Aldo and Dex, 2) the time course of steroiddependent stimulation of Na1 transport, and 3) whether
the effects of these hormones were additive.
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primers were ACA ACA CCA CCA TCC ACG and GCC ACC
ATC ATC CAT AAA G, designed to yield a 913-bp product
(between nucleotides 347 and 1260 of the rat sequence). For
b-subunit, sense and antisense primers were CCT ACA AGG
AGC TGC TAG TGT G and GAA GTG CCT TCT CTG TCA
TG, designed to yield a 785-bp product (between nucleotides
134 and 919 of the rat sequence). For g-subunit, sense and
antisense primers were CTC GTC TTC TCT TTC TAC AC and
GCA GAA TAG CTC ATG TTG, designed to yield a 541-bp
product (between nucleotides 318 and 859 of the rat sequence).
Solutions and chemicals. In most cases cells were maintained in culture media as previously described, and steroids
or other hormones were directly added to the solution. The
control bathing solution for the Ussing chamber experiments
contained (in mM) 5 KCl, 1 MgSO4, 1.2 CaCl2, 5 alanine, 10
sodium acetate, 8.3 glucose, 2 sodium phosphate, and sufficient NaCl to adjust the osmolality to 300 6 5 mosmol/kgH2O.
The solution was buffered with 25 mM HCO2
3 and continuously bubbled with 5% CO2 to yield a final pH of 7.4 at 37°C.
The dye loading solution was HCO2
3 free and contained 10
mM HEPES as the main buffer. All hormones and other
chemicals used in this study were purchased from Sigma
Chemical (St. Louis, MO) unless specified otherwise.
Statistics. All results are reported as means 6 SE. Comparing independent sets of data, unpaired t-tests were used to
determine significance. In experiments where hormones were
added to cells that served as their own control, paired t-tests
were used. In all cases, P , 0.05 was considered to be
significant; n is the number of experiments performed.
HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
Fig. 1. Stimulation of equivalent short-circuit current (Ieq, left) and
transepithelial voltage (Vte, right) by dexamethasone (Dex) or aldosterone (Aldo). Aldo or Dex significantly increased Ieq and Vte in
comparison to nontreated cells (‘‘None’’). Stimulation by Dex was
highest, and the differences among all groups were statistically
significant (P , 0.05, n 5 11). * Significantly different from None
group.
Fig. 2. Effect of Aldo on Ieq and Vte in presence of Dex. Stimulation of
Ieq (left) in presence of both Aldo and Dex was higher than stimulation
by Dex alone (P , 0.05, n 5 9). Increase in Vte (right) was not
statistically significant. * Significantly different from Dex group.
We also checked the effect of Aldo when stimulation
by Dex was less than maximal by lowering the concentration of Dex to 10 nM (1/10th usual Dex). As shown in
Fig. 3, Ieq in cells treated with 1 µM Aldo plus 10 nM
Dex (5.7 6 0.3 µA/cm2 ) was higher (n 5 15, P , 0.01)
than that in 10 nM Dex only (4.6 6 0.3 µA/cm2 ). The
difference in Vte was not statistically significant (15.2 6
1.1 vs. 13.2 6 0.8 mV). These results are qualitatively
similar to the previous experiments with 100 nM Dex
and still showed an additive effect of Aldo to that of Dex.
However, Ieq was significantly higher (9.7 6 0.8 µA/cm2 )
in the group with Dex at 100 nM concentration. These
data clearly demonstrate an additional effect of Aldo to
that of Dex.
To further determine whether the stimulatory effect
of Dex was purely through binding to the glucocorticoid
receptor, we examined the effect of spironolactone, a
mineralocorticoid antagonist, on Ieq and Vte in Dextreated M-1 cells. As shown in Fig. 4, spironolactone (10
µM) in the presence of 100 nM Dex significantly
decreased Ieq from 8.3 6 0.6 (Dex) to 5.8 6 0.6 µA/cm2
Fig. 3. Effect of Aldo on Ieq (left) and Vte (right) in presence of 10 nM
Dex. Stimulation of Ieq was greater in presence of Aldo and 10 nM Dex
than with 10 nM Dex alone (P , 0.01, n 5 15). * Significantly different
from Dex group.
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Upon removal of serum from the culture medium on
the third day after seeding, M-1 cells were divided into
three groups. One group had 100 nM Dex continuously
present. In another group, Aldo (1 µM) but not Dex was
added. The third group had no steroids (‘‘None’’ group)
in the culture medium but did contain vehicle (ethanol). Transwells in each group were matched to have
comparable initial readings of Vte and Rte. Ieq was
stimulated in both the Dex and the Aldo groups.
Maximal stimulation appeared at ,24 h, after which a
gradual decrease in Ieq was observed; therefore all
subsequent data refer to the 24 h time points. The
vehicle control group (i.e., was not treated with any
steroids) did not show any stimulation of Ieq and had
the lowest readings. Although both glucocorticoids and
mineralocorticoids significantly stimulated Ieq and Vte,
stimulation by Dex was always greater than that by
Aldo. Figure 1 shows the stimulatory effects of steroids
on Ieq after 24 h of treatment with the respective
hormones. In Dex-treated cells, Ieq (9.2 6 0.9 µA/cm2,
n 5 11) and Vte (22.4 6 2.5 mV) were respectively
higher than those in Aldo (6.0 6 0.4 µA/cm2 and 17.7 6
1.5 mV) or vehicle control (None, no steroids) (4.1 6 0.3
µA/cm2 and 13.0 6 1.4 mV). The differences in Ieq were
statistically significant among all groups. There was no
statistical difference in Vte between the Aldo- and the
Dex-treated M-1 cells. The data summarized in Fig. 1
indicates that both Aldo and Dex stimulate Na1 transport in M-1 cells.
The next series of experiments was performed to
determine whether the stimulatory effect of Aldo is
additive to that of Dex. A nonadditive effect would
argue against a separate regulatory influence of Aldo.
As shown in Fig. 2, exposure of cells to Dex (100 nM)
and Aldo (1 µM) increased Ieq from 7.5 6 0.6 (Dex only)
to 10.2 6 0.5 µA/cm2 (n 5 9, P , 0.05). Vte in cells
treated with both hormones (36.4 6 3.4 mV) was also
higher than in cells treated with Dex only (30.5 6 4.5
mV); however, the difference was not statistically significant.
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HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
(n 5 12, P , 0.01). Vte also decreased from 22.6 6 1.7 to
16.8 6 2.3 mV (P , 0.05). These results indicate that at
least a component of the stimulatory effect of Dex is
probably mediated through activation of mineralocorticoid receptors.
In some studies, lower concentrations of mineralocorticoids (10–100 nM) have been used to stimulate Na1
transport in cultured cells of the collecting duct. In the
next series of experiments, we lowered the concentration of Aldo to 100 nM and examined whether stimulation of the Ieq was maintained. As can be seen in Fig. 5,
Aldo, at 100 nM, still caused a significant increase in Ieq
and Vte compared with cells which were not treated
with steroids. On the other hand, Aldo stimulation of Ieq
and Vte was less than that caused by Dex, similar to the
results of Fig. 1.
Response of M-1 cells to acute treatment with hormones. In contrast to adrenal steroids, whose effects
occur predominantly over hours to days, several other
hormones acutely influence Na1 transport in CCD.
Among these we investigated the effects of phorbol
Fig. 5. Stimulation of Ieq (left) and Vte (right) by lower concentration
of Aldo (100 nM) or Dex. Lower concentration of Aldo (100 nM) or Dex
(100 nM) significantly increased Ieq and Vte in comparison to nontreated cells (P , 0.05, n 5 8). Stimulation by Dex was highest among
all groups. * Significantly different from None group.
Fig. 6. Acute effect of phorbol myristate acetate (PMA) on Ieq (top)
and Vte (bottom). PMA (1028 M) did not significantly change Ieq or Vte
in M-1 cells treated with Dex (k) or Aldo (p); n, effect of adding
vehicle only.
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Fig. 4. Effect of mineralocorticoid antagonist (spironolactone) on
glucocorticoid stimulation of Ieq (left) and Vte (right). Spironolactone
(Spironolac, 10 µM) significantly inhibited both Ieq and Vte (* P , 0.05,
n 5 12) in Dex-treated cells.
esters as activators of protein kinase C (PKC), EGF,
elevations in [Ca21]i, and AVP.
PMA at 1029 M has been shown to acutely inhibit Na1
reabsorption and K1 secretion in the rabbit CCD (22)
and to inhibit Na1 channels in rat CCD (17), although
Rouch et al. (40) did not find an effect in the perfused
rat CCD. In our experiments, addition of PMA did not
affect either Vte or Ieq (Fig. 6) in M-1 cells treated with
either Dex (n 5 10) or Aldo (n 5 6). The results suggest
that activation of PKC as expected from treatment with
PMA does not significantly inhibit basal Na1 transport
in these cells.
EGF, administered acutely in the rabbit CCD, was
shown to drastically decrease Na1 reabsorption with a
corresponding fall in Vte (50). Further studies showed
that the effect of EGF is peritubular and depends on the
influx of Ca21 across the basolateral membrane (51).
This suggested that the effect of EGF is mediated
through an increase in [Ca21]i. In our studies, as shown
in Fig. 7, addition of EGF to the bath did not affect
either Vte or Ieq in M-1 cells treated with either Dex (n 5
8) or Aldo (n 5 5). Figure 7 also shows the inhibition of
transepithelial Vte and Ieq by amiloride. Since an EGF
effect in intact CCD is known to be mediated by a
change in activity of [Ca21]i, we checked whether EGF
did increase [Ca21]i in M-1 cells. Alternatively, we also
examined whether an induced increase in [Ca21]i would
have any influence on Ieq and Vte in these cells. As
shown in Fig. 8, top, using fura 2 to measure [Ca21]i,
addition of EGF elicited a small increase in [Ca21]i.
Subsequent addition of thapsigargin (1 µM) caused a
substantial increase in [Ca21]i that partially recovered.
Six similar experiments were conducted. In another set
of four parallel experiments, thapsigargin was added to
Dex-treated M-1 cells on Transwells; no change in Ieq or
Vte was seen (Fig. 8, bottom).
Unlike PMA or EGF, AVP was shown to activate Na1
reabsorption in the rabbit CCD in an early phase (5, 8,
12), which was soon followed by an inhibitory effect (12,
HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
23). In contrast to the rabbit CCD, addition of AVP (1
µM) to rat CCD produced a sustained increase in Na1
transport that was even greater when rats were treated
with deoxycorticosteroid hormones (12, 37, 48). In nine
experiments on M-1 cells, addition of AVP (1026 M)
caused a transient large increase in Ieq and Vte (Fig. 9)
and a much smaller sustained increase in Ieq. To
investigate whether the stimulation of Ieq was due to
activation of luminal Na1 conductance, we conducted a
series of seven experiments in which AVP was added
after inhibiting the luminal Na1 channels with amilo-
Fig. 8. Response of Ieq and Vte to changes in intracellular Ca21
([Ca21]i ). Top: an actual tracing showing that EGF moderately
increases [Ca21]i measured as a change in fluorescence ratio (R340/380 )
of fura 2 trapped intracellularly. Thapsigargin (Thapsig, 1 µM)
substantially but transiently increased [Ca21]i. Bottom: thapsigargin, which increases [Ca21]i did not significantly affect Ieq in Dextreated M-1 cells (p); k, effect of adding vehicle only.
Fig. 9. Response of M-1 cells to arginine vasopressin (AVP). Addition
of 1 µM AVP to bath elicited a transient and large increase in Ieq (top)
and Vte (bottom) of Dex-treated M-1 cells (p); k, no effect of adding
vehicles.
ride (10 µM). As shown in Fig. 10, addition of AVP in the
absence of amiloride (top tracing) caused the usual
transient increase in Ieq similar to Fig. 9 above. In
paired experiments, addition of 10 µM amiloride first
(Fig. 10, bottom tracing) caused rapid and almost
complete inhibition of Ieq as observed earlier (see Fig.
7). In the continued presence of amiloride, addition of
AVP caused a substantial transient increase in Ieq
similar to that observed in the absence of amiloride.
These results indicate that AVP stimulation of Ieq in
M-1 cells is not secondary to activation of amiloridesensitive Na1 channels.
Response to protease inhibition. Recently, Vallet et al.
(49) reported a novel autocrine mechanism for activa-
Fig. 10. Response of M-1 cells to AVP after pretreatment of cells with
amiloride. Addition of 1 µM AVP to control M-1 cells (j) caused
transient increase in Ieq as shown in Fig. 9 above. In paired
experiments, addition of 10 µM amiloride (l) inhibited Ieq almost
completely. In the continued presence of amiloride, addition of AVP (1
µM) still caused a transient and large increase in Ieq. Seven similar
experiments were conducted.
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Fig. 7. Acute effect of epidermal growth factor (EGF) on Ieq (top) and
Vte (bottom). EGF (10 ng/ml) did not significantly influence Ieq or Vte
in M-1 cells treated with Dex (k) or Aldo (n); p, effect of adding
vehicle only. Last segment of the tracing shows that amiloride (Amil,
10 µM) completely blocked Ieq and Vte in all groups.
F1003
F1004
HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
DISCUSSION
The present studies demonstrate that M-1 cells represent a useful model for studying the regulation of Na1
transport by CCD cells. The cells respond to either
glucocorticoids or mineralocorticoids; this response is
mediated at least in part by mineralocorticoid receptors
(spironolactone sensitive). Acute regulation in these
cells is distinctive in that AVP has an effect indepen-
Fig. 11. Trypsin stimulation of Ieq in M-1 cells pretreated with
aprotinin. In 12 experiments, addition of trypsin (200 µg/ml) to the
luminal solution significantly increased Ieq (P , 0.05) in M-1 cells
that were pretreated with aprotinin (p, top tracing). Control cells
showed no change (j, bottom tracing).
Fig. 12. Ethidium bromide-stained bands of PCR products as labeled
and as described in text. Band between a and b lanes is a ladder
showing 1636, 1018, and 506 nucleotide bands. For the a-, b-, and
g-subunits in absence (2) of Aldo, bands were clearly present in other
experiments.
dent of Na1 channels, but PKC activation and increases
in intracellular calcium are without effects, in contrast
to the rabbit CCD.
Most studies aimed at investigating the effects of
hormones on Na1 regulation in the CCD, especially
chronic exposure, have relied on preparations derived
from whole animals or amphibian cultured cells. The
main drawback of the whole animal studies is that
hormone treatments induce unavoidable secondary
changes that themselves affect transport. For example,
administration of steroids in vivo can affect renal
hemodynamics, distal fluid delivery, and electrolyte
and hormone status. Although the A6 amphibian culture model has been extraordinarily valuable in delineating the mechanisms and regulation of Na1 transport, possible differences with mammalian tissues need
to be examined. These considerations underlie the use
of a mammalian cell culture model in the present
studies to examine CCD Na1 transport.
Although M-1 cells are being increasingly utilized as
a mammalian CCD model with which to study Na1
transport, the regulation of transepithelial Na1 transport in these cells has not been fully characterized. This
characterization is crucial if these cells are to be used to
understand the behavior of the intact CCD. A principal
aim of our study was therefore to characterize the
response of M-1 cells to hormones known to alter Na1
transport in the intact mammalian CCD. Two aspects
of hormonal responses were addressed: chronic (hours/
days) regulation by Aldo and Dex, and acute regulation
(minutes) by EGF, AVP, PKC activation by PMA, and
intracellular calcium.
The Ieq measured in these studies is clearly mediated
predominantly by epithelial Na1 channels (ENaC).
Both Ieq and Vte were immediately and almost completely inhibited by low concentrations of amiloride. In
addition, the mRNA of all three subunits of ENaC were
detected by RT-PCR; this confirms the findings (Northern blots) of Letz et al. (28) on the presence of mRNA for
all three subunits of the epithelial Na1 channel in M-1
cells. Other investigators have characterized whole cell
currents and the single channel properties of the Na1
channels in these cells (10, 11, 25, 28). The conductances are amiloride sensitive and have selectivity of
Li1 . Na1 : K1 (10, 11, 28); single channels have
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tion of Na1 transport in A6 cells. They reported the
identification of a novel serine protease, CAP1, which
activated Na1 channels. To determine whether mammalian CCD cells express a similar mechanism of activation of Na1 transport, protocols similar to those reported in A6 cells were followed. Aprotinin (28 µg/ml), a
protease inhibitor, was added to the apical solution for
,12 h and Vte, Rte, and Ieq were measured. Ieq was
reduced by 49 6 9% (to a mean of 1.6 6 0.3 µA/cm2, n 5
12) in cells exposed to aprotinin. Trypsin (200 µg/ml),
was then added to the luminal solution of both aprotininexposed and control cells. As can be seen in Fig. 11, Ieq
increased significantly (by 103 6 27%, P , 0.05) over
5–10 min in cells previously treated with aprotinin.
Application of trypsin to cells that had not been exposed
to aprotinin had no effect on Vte or Rte. Amiloride
abolished Ieq in all cells. These data are consistent with
an endogenous protease, which normally activates Na1
transport in mammalian CCD cells, as it does in A6
cells. Inhibition of this protease activity with aprotinin
decreases this activation, and application of trypsin can
lead to reactivation.
RT-PCR. No PCR products were obtained in control
reactions in which the RT was omitted from the cDNA
synthesis. All three primer sets yielded the expected
products of ,913, 785, and 541 bp as shown in Fig. 12.
Data are from cells treated with vehicle only or Aldo.
Appropriately sized PCR products were consistently
obtained with Aldo-treated cells. Although Fig. 12
suggests that Aldo directly increases the mRNA levels
of all three subunits in M-1 cells, these results were not
quantitated for the present series of experiments.
HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
22, 50). The lack of response of the M-1 cells does,
however, parallel the lack of response of the rat CCD to
similar stimuli (40). Whether these differences represent species differences or differences based on culture
conditions cannot be clarified by the present data.
Others have examined the response of M-1 cells to some
agents; Stoos and colleagues (45, 46) have described
that M-1 cell transport is inhibited by 1) endotheliumderived relaxing factor (coculture with endothelial cells
and stimulation with bradykinin or acetylcholine) and
2) the combination of atrial natriuretic factor and
bradykinin. However, both inhibitory responses were
complex in that single agents or cGMP analogs alone
had no effect (45, 46).
Antidiuretic hormone (or arginine vasopressin, AVP)
transiently stimulated Ieq in the present studies (Fig.
9). The response of the intact CCD to AVP differs with
species. In the rabbit, AVP inhibits Na1 transport after
an initial brief stimulation (5, 23); in the rat CCD, AVP
stimulates Na1 transport, particularly in tubules from
deoxycorticosterone-treated animals (12, 37, 48). In
cultured CCD cells from rabbit, AVP stimulates Na1
transport (8). Other studies have reported that a component of AVP stimulation of Ieq in cultured cells is
mediated through a mechanism other than activation
of Na1 luminal channels. For example, Canessa and
Schafer (8) observed an amiloride-insensitive Ieq that
was inhibited by ouabain. Using primary cultures of
rabbit CCD, Nagy et al. (33) reported that AVP activated a DIDS-insensitive Cl2 conductance through a
cAMP pathway. In fact, Letz and Korbmacher (29)
reported recently that cAMP stimulated cystic fibrosis
transmembrane conductance regulator-like channels
in M-1 cells. In our study, AVP transiently stimulated
Ieq even after inhibiting luminal Na1 channels with
amiloride. The AVP-induced increase in Ieq in M-1 cells
is not secondary to activation of amiloride-sensitive
Na1 channels and may represent activation of Cl2
channels similar to other studies (29, 32, 33).
The inhibitory response to some hormones has been
linked to an increase in [Ca21]i (5). This increase in
[Ca21]i has been suggested to be secondary to an initial
increase of apical Na1 entry, which in turn leads to an
increase in [Ca21]i via basolateral Na1/Ca21 exchange
(6, 19). In our experiments increasing [Ca21]i by thapsigargin did not affect Ieq or Vte. Rouch et al. (40) reported
similar lack of inhibition of salt and water transport by
thapsigargin and ionomycin in rat CCD, although both
increased [Ca21]i. In this context, one possibility is that
stimulation by mineralocorticoids may decrease the
effectiveness of some inhibitory hormones, particularly
those linked to an increase in [Ca21]i. In agreement
with this hypothesis, Frindt et al. (18, 19) have postulated that mineralocorticoid-induced increases in apical Na1 conductance are sufficiently high that the
apical membrane is not rate limiting any more. In this
context, the second messenger pathway of the response
to hormones in these cells deserves further investigation.
An endogenous protease activity does appear to
activate transport in M-1 cells, as has been recently
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relatively low conductances (,5–8 pS). Using patch
clamp techniques, Chalfant et al. (10) have further
characterized the Na1 channel in M-1 cells as having
complex kinetics involving more than two open and two
closed states. In addition to these typical amiloridesensitive channels, M-1 cells also possess amilorideinsensitive, nonselective cation channels (1, 26); but
these are probably not involved in the amiloridesensitive transepithelial properties reported here.
M-1 cells in the present studies responded to both
Dex and Aldo. Although the degree of stimulation with
Aldo or Dex is relatively small, this stimulation was
consistent and significant. It should be noted that in
other in vitro studies, where higher absolute currents
were obtained (11, 25, 47), measurements were obtained in smaller wells (diameter 12 mm) and/or in
undefined media, such as PC-1 media, which contained
serum factors. In our studies, it was important to use
defined media to avoid nonspecific effects that may be
induced by serum or other undefined factors. Part of
this response was probably mediated via mineralocorticoid receptors, since the response was sensitive to
spironolactone and since Aldo augmented transport
even when Dex was present at maximally effective
concentrations. In other studies of cultured collecting
duct cells, both Aldo and Dex were also found to
stimulate electrogenic Na1 transport (24, 34). Response
to both glucocorticoids and mineralocorticoids contrasts with the usual concept that mineralocorticoids
and not glucocorticoids regulate distal nephron ion
transport. However, several previous studies using a
variety of models (including whole animals and primary cultures) have demonstrated effects of glucocorticoids, particularly synthetic agents (4, 13, 27, 34, 42,
43). Glucocorticoids are potent ligands for mineralocorticoid receptors, but mineralocorticoid target tissues
are usually ‘‘protected’’ by metabolism of endogenous
glucocorticoids by 11-b-hydroxysteroid dehydrogenase
(20). Glucocorticoid receptors may also modulate Na1
transport in collecting duct cells directly (27, 34).
However, endogenous glucocorticoids probably have
little regulatory role in CCD Na1 transport (via either
glucocorticoid or mineralocorticoid receptors) because
of the action of 11-b-hydroxysteroid dehydrogenase in
principal cells. Withdrawal of both hormones in M-1
cells resulted in loss of Vte and Ieq. This finding is
reasonably consistent with findings in other preparations. For example, Reif et al. (37) showed that Vte and
Na1 transport were minimal in CCD derived from rats
that have not been treated with excess mineralocorticoids. In sum, M-1 cells can be used to study steroid
stimulation of Na1 transport, but the relative response
is small compared with some models, rendering distinction between glucocorticoid and mineralocorticoid responses difficult.
In terms of acute regulation, M-1 cells did not
respond to either EGF, PMA to stimulate PKC, or
increases in intracellular calcium with thapsigargin.
This clearly differs from the response of the microperfused rabbit CCD, which responds to each of these
stimuli with an abrupt decrease in Na1 transport (19,
F1005
F1006
HORMONAL REGULATION OF TRANSPORT IN M-1 CELLS
Address for reprint requests: L. L. Hamm, Tulane Medical Center,
Section of Nephrology, SL45, 1430 Tulane Ave., New Orleans, LA
70112.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Received 17 October 1997; accepted in final form 27 August 1998.
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