(Received for publication, September 12, 1995)
From the
Na/H
exchanger (NHE) activity
is regulated by several types of receptors directly coupled to distinct
classes (i.e. G
, G
, G
, and
G
) of heterotrimeric (
) GTP-binding
proteins (G proteins), which, upon activation, modulate production of
various second messengers (e.g. cAMP, cGMP, diacylglycerol,
inositol trisphosphate, and Ca
). Recently, four
isoforms of the rat Na
/H
exchanger
were identified by molecular cloning. To examine their intrinsic
responsiveness to G protein and second messenger stimulation, three of
these isoforms, NHE-1, -2, and -3, were stably expressed in mutant
Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1
cells). Incubation of cells with either
AlF
, a general agonist of G proteins, or
cholera toxin, a selective activator of G
that
stimulates adenylate cyclase, accelerated the rates of
amiloride-inhibitable
Na
influx mediated
by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short
term treatment with phorbol 12-myristate 13-acetate, which mimics
diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and
isobutylmethylxanthine) that lead to activation of cAMP-dependent
protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but
depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate
were blocked by depleting cells of PKC or by inhibiting PKC using
chelerythrine chloride, confirming a role for PKC in modulating NHE
isoform activities. Likewise, the PKA antagonist, H-89, attenuated the
effects of elevated cAMP
on NHE-1, -2, and -3,
further demonstrating the regulation by PKA. Unlike
cAMP
, elevation of cGMP
by
treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE
isoform activities, thereby excluding the possibility of a role for
cGMP-dependent protein kinase in these cells. These data support the
concept that the NHE isoforms are differentially responsive to agonists
of the PKA and PKC pathways.
Na/H
exchanger (NHE) (
)activity is present in the plasma membrane of all
mammalian cells and, depending on the cell type and membrane
localization, fulfills several distinct physiological functions,
including control of intracellular pH (pH
),
maintenance of cellular volume, facilitation of cell proliferation in
response to growth factor stimulation, and transepithelial
Na
reabsorption (reviewed in (1) ). This
functional diversity is accomplished by the actions of distinct
isoforms of the Na
/H
exchanger.
To
date, four members (NHE-1 to NHE-4) of this multigene family have been
identified and characterized by cDNA cloning (2, 3, 4, 5) and functional
expression studies(6, 7, 8, 9) .
More recently, the existence of a putative fifth (10) and
possibly sixth (11) isoform have been revealed by chromosomal
mapping in humans. Overall, they share 40-60% amino acid
identity (molecular mass ranging from
81 to 93 kDa) and exhibit
similar plasma membrane topologies, with 10-12 predicted
N-terminal transmembrane-spanning regions and a large C-terminal
cytoplasmic region. This latter region exhibits the greatest divergence
in amino acid sequence among the isoforms and contains one or more
potential sites for phosphorylation by different serine/threonine
protein kinases.
Previous studies have revealed a wide variety of
molecular signals, including neurotransmitters, growth factors, peptide
hormones, chemotactic factors, lectins, and osmotic shrinkage, that
rapidly modulate Na/H
exchanger
activity (for reviews, see (1) and (12) ). Many of
these stimuli transmit their signals via interactions with plasma
membrane receptors that are coupled to a diverse family of
heterotrimeric (
) GTP-binding proteins (G proteins) (for
reviews, see (13) and (14) ). Receptor-mediated
activation of G proteins leads to dissociation of
GTP from
the
subunits (which remain tightly associated) and their
release from the receptor. These subunits (
or
), in
turn, can directly bind and regulate a variety of effector molecules,
such as Ca
and K
channels, adenylate
cyclase, cGMP phosphodiesterase, and phospholipase C
, thereby
modulating intracellular ion levels and signaling pathways (i.e. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and
Ca
).
The response of the
Na/H
exchanger following activation
of different serine/threonine kinases is complex and dependent on cell
type (reviewed in (5) and (12) ). In most
nonepithelial cells, growth factors and phorbol esters that mediate
their effects through PKC generally accelerate exchanger
activity(15) . However, in some renal and intestinal epithelial
cells, the apical exchanger is inhibited (16, 17) under conditions where the basolateral
exchanger remains unaffected(18) . Moreover, agents that
elevate intracellular cAMP (cAMP
), which in turn
activates PKA, inhibit the apical exchanger of renal epithelial
cells(19, 20, 21) but stimulate exchanger
activity in hepatocytes (22) and macrophages (23) .
Raising cGMP
levels has been reported to increase (24) or decrease (25) exchanger activity, depending on
the cell type. Increasing intracellular Ca
has also
provided contradictory results, with exchanger activity being
stimulated (26, 27, 28, 29) or
depressed (29, 30) in a pattern that cannot always be
accounted for by the level of PKC activity. This has led to suggestions
of a possible regulatory role for
Ca
/calmodulin-dependent protein kinase II as a
mediator of some of these
effects(30, 31, 32, 33) . In
addition, Ca
/calmodulin itself appears to directly
bind and activate the NHE-1 isoform(34, 35) . These
molecular mechanisms are not fully resolved but clearly differ from
osmotic regulation of the exchangers, which is ATP-dependent (36) but does not appear to involve direct phosphorylation of
the exchanger, at least in the case of NHE-1(37) . This process
may involve other ancillary factors such as G proteins that are
independent of the PKA and PKC pathways (37, 38, 39) . At present, little information
is available concerning the stimuli that selectively modulate the
individual NHE isoforms and their mechanisms of action(8) .
In order to delineate Na/H
exchanger regulation by serine/threonine kinases in greater
detail, we have stably transfected individual NHE isoforms (NHE-1,
NHE-2, and NHE-3) into Chinese hamster ovary cells that are devoid of
endogenous exchanger activity (AP-1 cells). We reasoned that a common
cellular background should provide a useful model system in which to
compare distinct exchangers. The aim of the present study was to test
the hypothesis that the NHE isoforms have intrinsic capabilities to
respond to PKA and PKC, since previous studies suggested that they are
two of the major signaling pathways modulating
Na
/H
exchanger activity in various
cell types. The present results support the notion that these isoforms
are differentially responsive to these signaling pathways.
For experimentation, the cells were subcultured
at 5 10
cells/well in 24-well plates and grown to
confluence. The cells were arrested at the G
/G
stage by washing the monolayers with phosphate-buffered saline
and incubating in serum-free
-MEM medium for 17-20 h.
In experiments where exchanger activity was to be
determined from the rate of Na
influx at
constant H
concentration, pH
was clamped by incubating the cells in solutions of high
K
concentration containing the
K
/H
exchange ionophore
nigericin(41) . Because at equilibrium
[K
]
/[K
]
=
[H
]
/[H
]
,
the desired pH
was calculated from the imposed
[K
] gradient and the extracellular pH
(pH
= 7.5), assuming an intracellular
[K
] of 140 mM. Briefly, the
monolayers were washed twice with Na
solution and then
preincubated for 15 min in K
solution (70 mM KCl, 60 mM choline chloride, 1 mM NaCl, 2 mM CaCl
, 1 mM MgCl
, 5 mM glucose, 4 µM nigericin, 100 µM bumetanide, 20 mM HEPES-Tris, pH 7.5) at 37 °C. This
solution was then replaced with fresh K
solution
supplemented with 1 µCi/ml
NaCl, 1 mM ouabain, and in the absence or presence of 1 mM amiloride.
To extract the radiolabel, 0.25 ml of 0.5 N NaOH was added to each well, and the wells were washed with 0.25 ml of 0.5 N HCl. Both the solubilized cell extract and wash solutions were suspended in 5-ml scintillation fluid, and the radioactivity was assayed by liquid scintillation spectroscopy. Protein content was determined using the Bio-Rad DC Protein Assay procedure.
BCECF-loaded cells were mounted in the bottom of a laminar
flow-through temperature-controlled chamber (volume, 350 µl).
Silicone rubber was used to complete a water-tight seal. The chamber
was mounted on the stage of a Nikon inverted microscope equipped for
epifluorescence (Diaphot, Nikon, Tokyo, Japan). The light source was a
75-watt mercury-xenon arc lamp powered by a DC power supply. Excitation
light was passed through one of two differential interference filters
(440 or 490 nm; ±5 nm) mounted in a turret which could be
rotated by a computer-controlled stepping motor. The light was then
passed through a 510-nm dichroic mirror and a 40
Nikon UV-fluor
oil immersion lens with a numerical aperture of 1.3. All fluorescent
light passed back through the dichroic mirror and 515-nm bandpass
filter to reduce background fluorescence. The emitted fluorescence was
deflected to the eyepieces or to an intensified charge-coupled device
video camera (model 2468, Hamamatsu Photonics K.K., Hamamatsu City,
Japan). Emitted light at wavelengths between 510 and 530 nm was
captured during illumination at each excitation wavelength at the rate
of 32 frames/sec. All analyses were performed using a computer-based
image analysis system (Fluor-1; Universal Imaging, West Chester, PA).
Twelve frames were averaged to produce a gray scale image, which was
corrected on a pixel-by-pixel basis using background images that had
been acquired from cell-free areas of the coverslip. Autofluorescence
was undetectable, as determined by measuring non-BCECF-loaded cells
(<1.5% and <3% of the base-line fluorescence of BCECF-loaded
cells during excitation with 440 and 490 nm, respectively). For each
pair of images, the ratio of the fluorescence intensity at 520 nm
during excitation at 490 nm versus the intensity of
fluorescence at 520 nm during excitation at 440 nm was calculated,
again on a pixel-by-pixel basis. Individual cells were identified using
the image of fluorescence during excitation at 490 nm, and one
cytosolic area was defined and marked per cell. The ratios for each
defined area were stored on computer disk. The epifluorescence light
path was blocked with a shutter between fluorescence measurements to
minimize photo-bleaching of the BCECF and cell UV damage. Under this
protocol, one ratio image was acquired every 1.4 s. A pseudo-color
ratio image as well as a graph showing the ratio for each of the areas
of interest was displayed on a color monitor (ECM1311U, Electrohome,
Kitchener, Ontario). Stored ratios were imported into a spreadsheet
(Lotus 123) where pH calculations were performed.
Calibration of
intracellular pH was performed by perfusing the cells with high
potassium saline (120 mM potassium gluconate, 10 mM NaCl, 1 mM MgCl, 0.1 mM
CaCl
, 10 mM glucose, and 10 mM HEPES
containing 50 µM nigericin(41) . The pH of the
extracellular saline was varied between pH 6.2 and 8.7. Fluorescence
ratios were obtained at each calibration pH (after equilibrium was
reached), and a standard curve was generated using MicroCal Origin
(MicroCal Software Inc., Northampton, MA) running under Windows 3.1
(Microsoft Corp.) and exported to Lotus 123. Intracellular pH values
were then calculated from the experimental fluorescence ratios.
To detect the general participation of G proteins in
regulating NHE isoform activities in intact cells independently of
receptors, the effects of AlF were
examined in stably transfected AP-1 cells individually expressing
NHE-1, -2, or -3. AlF
interacts with G
proteins by forming a
G
GDP-Al
-F
complex that mimics GTP in a manner that closely resembles that
of nonhydrolyzable guanine nucleotide analogues such as GTP
S and,
therefore, is a convenient and general means of stimulating G
protein-mediated pathways(48) . As illustrated in Fig. 1, pretreatment of cells with AlF
for 60 min to activate G proteins stimulated
amiloride-inhibitable
Na
uptake by cells
expressing NHE-1 or -2 and inhibited uptake into cells expressing
NHE-3. No amiloride-inhibitable
Na
influx
was observed in the parental AP-1 cells (data not shown). Of course,
this regulatory pattern represents a composite effect that depends not
only on the transfected NHE isoforms but also on the exact cellular
complement of G proteins and effectors present in AP-1 cells.
Figure 1:
Influence
of AlF on activities of rat
Na
/H
exchanger isoforms stably
expressed in Chinese hamster ovary AP-1 cells. AP-1 cell transfectants
expressing rat NHE-1 (solid bars), NHE-2 (dotted
bars), or NHE-3 (striped bars) were grown to confluence
in regular
-MEM medium in 24-well plates. Cells were then made
quiescent by incubating in serum-free
-MEM overnight before
assaying for NHE activity. NHE activity was assessed by measuring the
initial rates of amiloride-inhibitable
Na
influx. Prior to
Na
influx measurements,
the cells were preincubated in AlF
(10
mM NaF, 20 µM AlCl
) for 20 or 60 min
in isotonic NaCl solution. The cells were then rapidly washed with
isotonic choline chloride solution.
Na
influx was initiated by the addition of choline chloride solution
containing 1 µCi/ml
NaCl, 1 mM ouabain,
AlF
and in the absence or presence of 1
mM amiloride.
Na
influx was
measured over a 12-min period at 22 °C (see ``Experimental
Procedures'' for further details). Low levels of background
Na
influx that were not inhibitable by 1
mM amiloride were subtracted from the total influx. NHE
activity is presented as a percentage of the amiloride-inhibitable
Na
influx determined under control
conditions. Each value is the mean ± S.D. (n =
8-12) from two or three experiments. C,
control.
To
isolate the actions of a specific class of G proteins, CTX was used
since it selectively activates G by ADP-ribosylating the
subunit near the GTP-binding site and inhibiting GTP
hydrolysis(48) . The liberated
independently
stimulates adenylate cyclase activity, and this response can be
enhanced or antagonized by the presence of the
subunits,
depending on the subtype of adenylate
cyclase(13, 49) . Thus, a cellular response to CTX
most likely indicates involvement of the cAMP-PKA pathway. As shown in Fig. 2A, CTX stimulated NHE-1 and -2 and inhibited NHE-3 in
a concentration-dependent manner, a pattern similar to that observed
for AlF
. The effects of CTX were not
affected in cells depleted of PKC by overnight incubation (18-24
h) with 200 nM PMA (50) (Fig. 2B) or
by 1 µM chelerythrine chloride(51) , a highly
specific and potent inhibitor of the catalytic domain of PKC (data not
shown). However, the effects were abrogated by 100 µM H-89, a highly selective PKA antagonist (52) (Fig. 2B). Thus, these data are consistent
with the notion that G proteins linked to the adenylate
cyclase-cAMP-PKA pathway are involved in differentially regulating
isoforms of the Na
/H
exchanger.
Unfortunately, specific involvement of G
in regulating the
NHE isoforms through the phospholipase C
-diacylglycerol-PKC
pathway could not be readily assessed due to the absence of a selective
agonist for this G protein. Therefore, to further define the signaling
pathways that function downstream of G
and G
,
specific activators of PKC and PKA were examined.
Figure 2:
Influence of cholera toxin on activities
of rat Na/H
exchanger isoforms stably
expressed in AP-1 cells. Confluent AP-1 cell transfectants expressing
rat NHE-1 (solid bars), NHE-2 (dotted bars), or NHE-3 (striped bars) were incubated in serum-free medium overnight
before assaying for NHE activity. A, prior to
Na
influx measurements, the cells were
preincubated in isotonic NaCl solution containing increasing
concentrations of cholera toxin (CTX; 1-1000 ng/ml) for
1 h. The cells were rapidly washed with Na
-free,
isotonic choline chloride solution and then incubated in choline
chloride solutions containing 1 µCi/ml
NaCl
(carrier-free), 1 mM ouabain, the varying concentrations of
CTX, and either in the absence or presence of 100 µM EIPA.
Na
influx was terminated after a 12-min
incubation period. Low levels of background
Na
influx that were not inhibitable by 100 µM EIPA were
subtracted from the total influx. NHE activity was defined as
EIPA-inhibitable
Na
influx and presented
as a percentage of control values. Each value is the mean ± S.D. (n = 8-14) from two to four experiments. B, to assess the involvement of serine/threonine protein
kinases in mediating the effects of CTX, cells were either depleted of
PKC activity by overnight incubation (18-24 h) with PMA (200
nM) (50) or exposed to the PKA antagonist H-89 (100
µM) (52) for 1 h in serum-free
-MEM medium
prior to CTX treatment. Cells were subsequently preincubated in
isotonic NaCl solution containing CTX (1 µg/ml) for 1 h and then
assayed for NHE isoform activities as described above. Values represent
the mean ± S.D. (n = 8-16) from two to
four experiments. Significant difference from control values was
determined by a two-tailed Student's t test and is
indicated by an asterisk (p < 0.05). C,
control.
Figure 3:
Influence of phorbol ester and cAMP
agonists on activities of rat Na/H
exchanger isoforms stably expressed in AP-1 cells. Confluent AP-1
cell transfectants expressing rat NHE-1 (solid bars), NHE-2 (dotted bars), or NHE-3 (striped bars) were incubated
in serum-free medium overnight before assaying NHE activity. Prior to
Na
influx measurements, the cells were
preincubated for 15 min in isotonic NaCl solution containing either 1
µM PMA, 1 µM 4
-PMA, 10 µM
forskolin (F), and 10 µM 1,9-dideoxyforskolin (1,9-ddF) (A) or 10 µM forskolin, 0.5
mM cpt-cAMP, 1 mM isobutylmethylxanthine (IBMX), and 1 mM dibutyryl-cGMP (db-cGMP) (B). The cells were rapidly washed with
Na
-free, isotonic choline chloride solution and then
assayed for EIPA-inhibitable
Na
influx in
the continuing presence of the various agents. NHE activity was
presented as a percentage of the EIPA-inhibitable
Na
influx determined under control
conditions. Each value is the mean ± S.D. (n =
12-16) from three or four experiments. Significant difference
from control values was determined by a two-tailed Student's t test and is indicated by an asterisk (p < 0.05). C, control.
In order to confirm that
the effect of forskolin was mediated by elevation of cAMP,
two additional agents known to increase cAMP
levels were
tested; cpt-cAMP (0.5 mM), a cell-permeable cAMP analog that
is relatively resistant to hydrolysis by phosphodiesterases, and
3-isobutyl-1-methylxanthine (1 mM), a nonspecific inhibitor of
phosphodiesterases. Similar to forskolin, both these agents increased
the transport activities of NHE-1 and -2 by approximately 50-100%
and depressed the activity of NHE-3 by 50-80% (Fig. 3B). The cell-permeant cGMP analogues
dibutyryl-cGMP (1 mM) (Fig. 3B) and
8-bromo-cGMP (1 mM) (data not shown) had no effect on
transport by any of the three isoforms, suggesting that cGMP-dependent
protein kinase does not regulate these NHE isoforms, at least when
expressed in this cell type. Virtually identical results were obtained
with these agents in multiple AP-1 cell lines expressing individual
isoforms (data not shown). Therefore, the results were not due to
random clonal isolation of AP-1 cell transfectants exhibiting aberrant
signaling.
Figure 4:
Influence of phorbol ester (PMA)
and forskolin (F) on resting pH in
untransfected AP-1 cells and on activities of rat NHE-1 and NHE-2 in
AP-1 cells under pH
-clamped conditions. A, untransfected AP-1 cells cultured to subconfluence
(
70-80%) on individual glass coverslips were deprived of
serum >5 h and then loaded with the cell-permeant pH fluorescent
dye, BCECF/acetoxymethyl ester. The coverslips were rinsed twice with
isotonic NaCl solution and then placed in the bottom of a laminar
flow-through temperature-controlled chamber. The chamber was sealed and
mounted on the stage of a Nikon inverted microscope equipped for
epifluorescence and then perfused with isotonic NaCl solution preheated
to 37 °C. Individual cells within the field of view were selected
(n
25 cells), and the fluorescence ratio was continuously
monitored as described under ``Experimental Procedures''.
After a 10-15-min equilibration period, the isotonic NaCl
perfusate solution was supplemented with either diluent
(Me
SO) (
), 1 µM PMA (
), or 10
µM forskolin (
) (arrow labeled stimulus).
At the conclusion of each experiment, the pH
of
cells for each coverslip was calibrated using the
K
-nigericin method(41) . The resting
pH
of untreated AP-1 cells was
7.0-7.1,
and the data for forskolin and PMA-treated cells were intentionally
offset to avoid overlap. Results are the mean ± S.E. and are
representative of at least three experiments. B, confluent
AP-1 transfectants expressing NHE-1 (filled bars) or NHE-2 (dotted bars) were preincubated for 15 min in a
K
-nigericin solution to set pH
at
7.2 (see ``Experimental Procedures'' for details) and also
contained diluent (Me
SO), 1 µM PMA, or 10
µM forskolin. At the end of this incubation period, the
solution was aspirated and replaced with the same solution supplemented
with 1 µCi/ml
NaCl, 1 mM ouabain in the
absence or presence of 1 mM amiloride. Isotope uptake was
terminated after 12 min, and the samples were processed as described
under ``Experimental Procedures.'' Data are presented as a
percentage of the amiloride-inhibitable
Na
influx determined under control conditions. Each value is the
mean ± S.D. (n = 12) from three experiments.
Significant difference from control values was determined by a
two-tailed Student's t test and is indicated by an asterisk (p < 0.05). C,
control.
To examine this possibility, pH was
clamped at 7.2 using K
-nigericin (see
``Experimental Procedures'') and amiloride-inhibitable
Na
influx was measured into cells exposed
to forskolin or PMA. As illustrated in Fig. 4B,
forskolin treatment increased NHE-1 and -2 activities by 47 and 36%,
respectively. Similarly, PMA treatment stimulated NHE-1 and -2 by 47
and 44%, respectively. The percentage stimulation under
pH
-clamped conditions was somewhat lower than with
unclamped cells (see Fig. 3). However, this is only an apparent
decrease, as the absolute rates of amiloride-inhibitable
Na
influx were higher in control,
forskolin-, and PMA-treated cells under pH
-clamped
conditions, presumably due to the different buffers used or to
differences in pH
between clamped versus nonclamped cells. Regardless, the results were qualitatively
similar using both assays of NHE function and lead to the same
conclusion. Both forskolin and PMA enhanced NHE-1 and -2 activities
even when the intracellular H
(substrate)
concentration and transmembrane pH gradient were held constant. Their
predominant effects were likely mediated by changes in the intrinsic
turnover number of the exchangers.
The conclusions drawn from Na
influx assays were confirmed and
extended by measuring NHE activity as the Na
-dependent
recovery of pH
in cells that had been acid-loaded by an
NH
prepulse(54) . One advantage of
this approach over isotope uptakes is the fact that physiological
concentrations (130 mM) of Na
can be used
extracellularly when studying the effects of potential stimuli. As
illustrated in Fig. 5, for untreated (diluent only) cells
expressing NHE-1, -2, and -3, cell acidification was followed by a
rapid return to resting cell pH
levels, and the
alkalinization was entirely dependent on the presence of
Na
, consistent with the involvement of NHE
activity. Furthermore, this response was inhibited by amiloride, as
previously reported using these cells(36) . Treatment of cells
expressing NHE-1 and -2 with PMA and forskolin accelerated the rate of
Na
-dependent pH
recovery following cell
acidification. In contrast, treatment of NHE-3 cells with PMA and
forskolin attenuated the rate of Na
-dependent recovery
of pH
following cell acidification, with the greatest
effect being observed with forskolin. These results are very similar to
those obtained by
Na
influx measurements (Fig. 3) and indicate that PMA and forskolin have converse
effects on NHE-1 and -2 compared with NHE-3. There was no
Na
-dependent recovery of pH
in parental
AP-1 cells following an acute intracellular acid load under any
condition tested (i.e. diluent (Me
SO), PMA, or
forskolin), confirming the absence of NHE activity in these cells.
Figure 5:
Influence of phorbol ester and forskolin
on rates of acid-induced pH recovery in AP-1 cell
transfectants expressing rat NHE-1, NHE-2, and NHE-3. Subconfluent
(
70-80%) AP-1 cell transfectants expressing NHE-1, NHE-2, or
NHE-3 were deprived of serum for >5 h and then loaded with
BCECF/acetoxymethyl ester. After mounting the coverslips on a Nikon
inverted microscope equipped for epifluorescence, the cells were
perfused with isotonic NaCl solution preheated to 37 °C. After a
10-15-min equilibration period, the isotonic NaCl perfusate
solution was supplemented with control diluent (Me
SO)
(
), 1 µM PMA (
), or 10 µM
forskolin (
) for 5 min. These agents were also present in all
subsequent solutions throughout the experiment. Cells were then
acidified by using the NH
prepulse
technique(54) . Briefly, cells were acid-loaded for 5 min in
NH
Cl solution (25 mM NH
Cl, 105 mM choline chloride, 1 mM MgCl
, 2 mM CaCl
, 5 mM glucose, 20 mM
HEPES-Tris, pH 7.4) followed by perfusion in Na
-free
choline chloride solution (130 mM choline chloride, 1
mM MgCl
, 2 mM CaCl
, 5 mM glucose, 20 mM HEPES-Tris, pH 7.4) for 5 min. This
treatment typically reduced pH
to
6.6 for all
cell types. A pH
recovery from the imposed acid
load was triggered by perfusion with isotonic NaCl solution. The data
are the mean ± S.E. and are representative of at least three
experiments of each kind.
Figure 6:
Effect of PKC inhibition on phorbol ester (PMA) and forskolin (F) regulation of rat NHE-1 (filled bars) and NHE-3 (striped bars) in AP-1 cells.
AP-1 cell transfectants expressing either rat NHE-1 or NHE-3 cells were
grown to confluence in 24-well plates. Prior to Na
influx measurements, the cells were
depleted of PKC activity by overnight preincubation with 200 nM PMA (A) or by exposure to diluent or the PKC antagonist
chelerythrine chloride (1 µM) for 1 h in serum-free
-MEM medium (B). In A, cells were subsequently
preincubated for 15 min in isotonic NaCl solution containing diluent, 1
µM PMA, or 10 µM forskolin, whereas in B, cells were treated with the different agents either alone
or in the combined presence of chelerythrine chloride. Cells were then
assayed for NHE isoform activities as described under
``Experimental Procedures.'' Values represent the mean
± S.D. (n = 12) from three experiments.
Significant difference from control values was determined by a
two-tailed Student's t test and is indicated by an asterisk (p < 0.05). C,
control.
If the antithetical effects of forskolin on NHE-1 and -3 were mediated through activation of PKA, both responses should be inhibited by antagonists of PKA, such as H-89. Indeed, the stimulation of NHE-1 and the inhibition of NHE-3 were both attenuated in the presence of H-89, strongly implicating PKA in this process (Fig. 7). It is interesting that NHE-1 activity in the presence of H-89 alone was significantly repressed by 75%, suggesting that a substantial portion of its basal activity was dependent on basal PKA activity. Opposite results, though quantitatively less dramatic, were obtained for NHE-3 activity, which showed a marginal 15% stimulation in the presence of H-89 alone.
Figure 7:
Effect of PKA inhibition on
forskolin-mediated regulation of rat NHE-1 (filled bars) and
NHE-3 (striped bars) in AP-1 cells. Confluent AP-1 cell
transfectants expressing either rat NHE-1 or NHE-3 were preincubated in
the absence or presence of the PKA antagonist H-89 (100
µM) for 1 h in serum-free -MEM medium. Cells were
then incubated for an additional 15 min in isotonic NaCl solution
containing either diluent or 10 µM forskolin (F)
in the absence or presence of H-89 prior to measurements of initial
rates of amiloride-inhibitable
Na
influx.
Each value is the mean ± S.D. (n = 12) from
three experiments. Significant difference from control values was
determined by a two-tailed Student's t test and is
indicated by an asterisk (p < 0.05). C,
control.
Studies defining the regulation of individual NHE isoforms by distinct intracellular signaling pathways have only recently been undertaken. The results from this study demonstrate that activation of the PKA or PKC pathway can lead to stimulation of NHE-1 and -2 as well as inhibition of NHE-3 when the exchangers are stably expressed in AP-1 cells.
Regulation of the NA/H
exchanger by numerous hormones and growth-promoting agents is well
documented (reviewed in (5) and (12) ). Since many of
these agents bind to cell surface receptors that ultimately activate
distinct serine/threonine protein kinases, it is likely that
heterotrimeric G proteins play an essential intermediary role in the
transmembrane signaling events that lead to altered
Na
/H
exchanger activity. Busch et
al. (47) have recently shown that microinjection of
GTP
S or purified G
subunits of transducin into Xenopus laevis oocytes stimulated native
Na
/H
exchanger activity by the PKA or
PKC pathways, respectively. However, it is unclear whether both
pathways activated the same or distinct isoforms of the exchanger in
oocytes. Using human embryonic kidney 293 cells as hosts, transient
expression of constitutively activated mutants of G
and G
enhanced Na
/H
exchanger activity, whereas G
and
G
were without effect(39) . Interestingly,
while G
appeared to exert its effects through the
phospholipase C
pathway, G
acted without
modifying intracellular levels of inositol phosphate and cAMP,
suggesting the involvement of a novel signaling pathway. In a
comparable study using COS-1 cells, transient expression of activated
G
, G
, and G
also
stimulated the Na
/H
exchanger, while,
on the contrary, G
inhibited its activity and
G
was without effect(45) . Depleting cells of
PKC activity abolished the enhancement caused by G
and
G
, but did not affect the stimulation mediated by
G
. Thus, G proteins such as G
appear to activate the Na
/H
exchanger by a distinct pathway that is independent of PKA and PKC.
Indeed, G
has recently been shown to activate the Jun
kinase/stress-activated protein kinase pathway(58) , suggesting
that this kinase may be linked to the regulation of the
Na
/H
exchanger. Again, however, it is
unclear which isoforms of the Na
/H
exchanger are being regulated in these cell types. The results
from our study partially clarify this issue by showing that specific
activation of G
(by cholera toxin) stimulated the
activities of NHE-1 and NHE-2 and inhibited that of NHE-3 through a
signaling pathway involving PKA. Further studies using this
heterologous expression system are currently ongoing to define the
involvement of other G proteins.
Previous studies have convincingly demonstrated that the phospholipase C-diacylglycerol-PKC pathway constitutes a major signaling route for activation of the ubiquitous NHE-1 isoform of the exchanger. Stable expression of human (59) and rabbit (8) NHE-1 in fibroblastic cells (PS120) has shown that this isoform is rapidly activated following acute cell stimulation by phorbol esters as well as by growth factors and other mitogens. Our results with rat NHE-1 expressed in AP-1 cells confirm these results; however, the precise molecular mechanism remains unclear.
Stimulation of NHE-1 by phorbol esters and other
growth-promoting agents was initially attributed to an increase in the
phosphorylation of a common set of tryptic peptide fragments in the
C-terminal region of the exchanger(15, 59) . These
data implied that the different agonists, which stimulated diverse
signaling pathways, ultimately transmitted their signals to a common
protein kinase that phosphorylated and activated the exchanger.
However, subsequent studies revealed that deletion of this region
(amino acids 635-815) only partially impaired (50%) activation,
whereas removal of another upstream region (amino acids 567-635),
which does not contain any of the phosphorylation sites, completely
abolished activation by several growth-promoting agents(60) .
The involvement of multiple regulatory regions to account for the
stimulation of NHE-1 by diverse agents has also been supported by
studies of Winkel et al.(61) , who demonstrated that
microinjection of a polyclonal antibody raised against amino acids
658-815 of NHE-1 ablated the stimulation mediated by endothelin-1
and -thrombin but was ineffective in preventing activation induced
by phorbol ester and hyperosmotic medium. These data suggested that
other mechanisms in addition to direct phosphorylation of NHE-1 may
play an important role in regulating its activity. One possible
mechanism that has been proposed is the participation of
exchanger-associated regulatory factors that themselves may also be
targets of protein kinases. In support of this argument, a 24-kDa
protein has recently been found to associate in situ with
NHE-1, although its functional significance has yet to be defined. (
)
In contrast to PKC-mediated activation of NHE-1,
evidence supporting a role for cAMP in the regulation of NHE-1 is
rather sparse and contradictory, and this has lead to the general view
that this isoform is not responsive to this second messenger. Previous
studies have shown that human (53) and rabbit (8) NHE-1
expressed in PS120 fibroblastic cells are unresponsive to cAMP
analogues. However, a subsequent study showed that when human NHE-1 was
stably transfected into opossum kidney (OK) cells, its activity was
inhibited by activation of PKA (induced by forskolin) or PKC (induced
by phorbol ester), suggesting possible cell-specific regulatory
effects(17) . In contrast, primary rat hepatocytes (22) and murine macrophages (23) showed significant
cAMP-induced stimulation of Na/H
exchanger activity. Subsequent investigations have revealed that
these tissues express only the NHE-1
isoform(2, 3, 62) . More recent studies have
found that the rat osteoblastic cell line, UMR-106, also expresses
NHE-1 exclusively and that it is cAMP-activable(57) .
Consistent with these studies, the data presented herein show that rat
NHE-1 stably expressed in AP-1 cells is also stimulated by agonists
that increase cAMP
accumulation, thereby suggesting that
this stimulatory response is an intrinsic property of this isoform.
In addition to rat NHE-1, the trout red cell also expresses a
Na/H
exchanger, called
NHE, that
is phorbol ester- and cAMP-activable in PS120 fibroblasts and has a
primary structure with highest identity to that of mammalian
NHE-1(53) . The trout
NHE contains two optimal consensus
sites for phosphorylation by PKA (R(R/K)X(S*/T*)) at
Ser
and Ser
, which, when simultaneously
mutated to Gly, partially reduced (by
72%) the ability of
cAMP
to activate the exchanger(63) . The residual
cAMP-activable activity was found to require amino acids 559-661
that may contain cryptic PKA sites that have yet to be identified or,
alternatively, may interact with cAMP/PKA-regulated accessory factors.
Interestingly, mutation of the two serine residues did not alter the
capacity of
NHE to be induced by phorbol ester, suggesting that
the actions of PKC are not convergent with those of PKA and are
mediated elsewhere in the exchanger. Furthermore, these results
suggested that the absence of cAMP regulation of human and rabbit NHE-1
in the same cell line (i.e. PS120) is likely not a consequence
of a dysfunctional PKA pathway but perhaps, as suggested above, due to
the absence of other cell-specific, cAMP/PKA-regulated factors that
interact with NHE-1. It is also worth noting that while rat NHE-1
contains several putative PKC consensus sequences
((R/K)
X
)(S*/T*)(X
(R/K
))
in its C-terminal region(2) , it does not contain a classical
consensus site for PKA. However, since there is overlap in consensus
sequence determinants among protein kinases(64) , one cannot
exclude the potential for PKA phosphorylation of NHE-1. In summary,
while it is difficult at the present time to reconcile the variable
regulation of NHE-1 by increasing cAMP
, several factors
operating independently or in combination may account for these
observations, such as cell-specific differences in the expression of
signaling components, putative exchanger-associated regulatory factors,
or perhaps species variation. Further studies are in progress to define
the molecular mechanism by which NHE-1 is regulated by PKA.
Unlike
NHE-1, much less is known about second messenger regulation of NHE-2.
In a SV-40-transformed rabbit S proximal tubule (RKPC-2)
cell line, native NHE-2, which appears to reside on the apical
membrane, was inhibited by 8-bromo-cAMP, whereas it was stimulated by
PMA(65) . However, heterologous expression studies have shown
that rabbit NHE-2 in PS120 fibroblastic cells was similarly activated
by phorbol esters as well as serum but was unresponsive to
cell-permeant cAMP analogues(8) . The results from the present
study partially corroborate these results by showing that rat NHE-2 in
AP-1 cells is also stimulated by phorbol ester but differ in that it is
enhanced by cAMP analogues as well. The variable responsiveness of
NHE-2 to cAMP probably depends on the cell type. Unlike NHE-1, the
C-terminal cytoplasmic domain of rat and rabbit NHE-2 contains several
classical consensus sequences for phosphorylation by PKA as well as
PKC. The question of whether kinase action is mediated through
phosphorylation of these sites or possibly via cell-specific,
exchanger-associated regulatory factors awaits future studies.
In
contrast to NHE-1 and NHE-2, the NHE-3 isoform of the
Na/H
exchanger in AP-1 cells is
unique in that it exhibits decreased rates of transport in response to
G protein and second messenger agonists of the PKA and PKC pathways. An
identical pattern of regulation is also observed in AP-1 cells when
this isoform is exposed to hyperosmotic medium(36) . This
distinctive regulation precisely mimics that observed for the
endogenous, apically targeted NHE-3 isoform in renal proximal tubule
opossum kidney cells where hyperosmolarity (66) and agonists of
the PKA and PKC signaling pathways (67, 68) inhibit
its activity. Thus, AP-1 cells provide a useful model for investigating
the mechanism by which these diverse stimuli converge to inhibit NHE-3.
Analogous results have partially been obtained using rabbit NHE-3
stably expressed in fibroblastic cells (PS120), which was inhibited by
acute exposure to PMA but unresponsive to elevated
cAMP(8) . On the other hand, the rabbit renal
Na
/H
exchanger in isolated brush
border membrane vesicles (presumably NHE-3) was inhibited by
PKA(19) . Thus, cAMP-mediated regulation of NHE-3 appears to be
cell-specific.
More recent structural analyses by us suggest that a
region between amino acids 579 and 684 of rat NHE-3 is essential for
the cAMP response. ()Interestingly, Levine and colleagues (69) have recently shown that the same region in rabbit NHE-3
also mediates inhibition by PKC. Examination of the cytoplasmic domain
of rat and rabbit NHE-3 reveals the presence of potential consensus
sequences for PKA as well as for PKC within or in close proximity to
this region. Thus, PKA and PKC may act at the same phosphorylation site
or may phosphorylate discrete sites within this region, which
nevertheless similarly influence exchanger activity. However, the
molecular signaling events that occur between activation of these
kinases and the responses of NHE-3 are unclear. For example, it is
unknown whether these protein kinases mediate their effects by direct
phosphorylation of NHE-3 or indirectly via phosphorylation-dependent
ancillary proteins. With regard to the latter, there is some in
vitro evidence that PKA-mediated inhibition of the rabbit renal
apical Na
/H
exchanger requires the
involvement of a regulatory protein that is separate from the kinase
and transporter(70) . Cell-specific expression of these factors
could account for the variable responsiveness of NHE-3 to individual
protein kinases. Further studies are ongoing to confirm this hypothesis
and identify the precise molecular mechanisms involved.