From the Departments of Medicine and Physiology & Biophysics, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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Nerve growth factor (NGF) inhibits
transepithelial HCO3 Na+/H+ exchangers mediate the
electroneutral exchange of Na+ and H+ across
plasma membranes and play key roles in a variety of cell functions,
including cell volume regulation, pHi regulation, epithelial
sodium reabsorption, and cell growth and proliferation (1-3). At least
five mammalian isoforms of Na+/H+ exchange
(NHE1-5)1 have been
identified, which differ in their tissue distribution and responses to
extracellular stimuli (3-5). One of the most prominent features of
Na+/H+ exchange is its stimulation by growth
factors. This stimulation is rapid, is observed in many different cell
types, and occurs with virtually all mitogens (1-6). Furthermore,
growth factors stimulate all NHE isoforms examined to date (NHE1, NHE2,
and NHE3) (3, 4, 7, 8). The mechanisms involved in growth factor activation of Na+/H+ exchange have been studied
extensively because of the close association of increased exchanger
activity with cell proliferation and oncogenic transformation (1,
4-11).
The mammalian kidney expresses NHE1-4, and
Na+/H+ exchangers are present on both apical
and basolateral membranes of tubule epithelial cells. The ubiquitously
expressed isoform NHE1 is present on the basolateral membrane of most
nephron segments, where it is involved in housekeeping functions such
as cell volume and pHi regulation (12-17). NHE4 is also a
basolateral isoform localized to collecting ducts and distal tubules,
where it may play a specialized role in the control of cell volume (18,
19). NHE3 is localized to the apical membrane of proximal tubule and thick ascending limb cells (20-22), where it mediates the reabsorption of NaCl and NaHCO3 in the proximal tubule and the
reabsorption of NaHCO3 in the thick ascending limb
(23-27). NHE2 is an apical isoform in the kidney, but its functional
significance is unclear (5, 26-29). The kidney is also a site of
production of numerous growth factors, including epidermal growth
factor, insulin-like growth factor, platelet-derived growth factor,
nerve growth factor (NGF), and hepatocyte growth factor (30). Based on
their prominent stimulation of Na+/H+ exchange
activity in other systems, these factors could influence multiple
cellular processes in renal tubules, including urinary acidification,
through effects on Na+/H+ exchange activities.
At present, however, the effects of locally produced growth factors on
Na+/H+ exchangers and their related functions
in renal tubules are poorly understood.
The medullary thick ascending limb (MTAL) of the mammalian kidney
participates in the regulation of acid-base balance by reabsorbing a
sizeable fraction of the HCO3 In view of their potent stimulation of Na+/H+
exchange activity in other systems, we recently investigated the
effects of growth factors on HCO3 Tubule Perfusion--
MTALs from male Sprague-Dawley rats
(60-90 g; Taconic Farms Inc., Germantown, NY) were perfused in
vitro as described previously (32, 34, 38). In brief, the tubules
were dissected from the inner stripe of the outer medulla, transferred
to a bath chamber on the stage of an inverted microscope, and mounted
on micropipettes for perfusion at 37 °C. The composition of the
perfusion and bath solutions for specific protocols is given below.
Experiments were carried out using 7 S NGF (37). Solutions were
prepared as described (34, 37).
Measurement of Net
HCO3
The protocol for study of transepithelial
HCO3 Measurement of Intracellular pH--
pHi was
measured by use of the pH-sensitive dye
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and a
computer-controlled spectrofluorometer (CM-X, Spex Industries) coupled
to the perfusion apparatus as described previously (34, 36, 38). The
tubules were perfused in the same manner used for
HCO3 Measurement of Na+/H+ Exchange
Activities--
Na+/H+ exchange activities
were determined using two previously described methods (34, 36). In the
first method (34), Na+/H+ exchange rates
(JNa/H, pmol/min/mm) were determined by
measuring initial rates of pHi recovery after addition of
Na+ to the lumen or bath solutions. The tubules were
perfused and bathed initially in HEPES-buffered solution that contained
145 mM Na+, 4 mM K+,
147 mM Cl
Apical membrane Na+/H+ exchange activity
was also assessed using a second method (36). MTALs were perfused and
bathed in the control solution used for
HCO3 Statistical Analysis--
Results are presented as
means ± S.E. Differences between means were evaluated using
Student's t test for paired or unpaired data, as
appropriate. p < 0.05 was considered statistically significant.
NGF Inhibits HCO3 NGF Does Not Affect Apical Na+/H+ Exchange
Activity, but Inhibits Basolateral Na+/H+
Exchange Activity--
To identify the mechanism by which NGF inhibits
HCO3
The MTAL also contains a basolateral membrane
Na+/H+ exchanger that is active at the resting
pHi (36). The results in Fig. 2B show that NGF
decreased basolateral Na+/H+ exchange activity
by at least 50% at all pHi values studied. For data points
grouped over the pHi interval 6.9-7.1, NGF decreased the
basolateral Na+/H+ exchange rate from 14.8 ± 0.6 to 6.8 ± 0.7 pmol/min/mm (p < 0.001). NGF
also decreased the steady-state pHi reached after addition of
Na+ to the bath solution (7.33 ± 0.08 without NGF
(n = 4) versus 6.99 ± 0.03 with NGF
(n = 5); p < 0.005).2 These results
demonstrate that NGF inhibits basolateral membrane Na+/H+ exchange activity in the MTAL. Under
basal conditions (absence of NGF), basolateral membrane
Na+/H+ exchange rates were approximately
one-third of apical membrane Na+/H+ exchange
rates at comparable pHi (Fig. 2, A and
B).
To confirm that the Na+-dependent pHi
recovery inhibited by NGF represents basolateral membrane
Na+/H+ exchange, we measured initial rates of
pHi recovery after bath Na+ addition in the absence
and presence of bath EIPA. The results in Fig.
3 show that EIPA blocked pHi
recovery nearly completely both in the absence and presence of NGF.
With EIPA in the bath, the recovery rate was reduced to 0.7 ± 0.1 pmol/min/mm (n = 3) in the absence of NGF and to
0.6 ± 0.1 pmol/min/mm (n = 3) in the presence of
NGF (90% inhibition). These data are consistent with previous results
(36) and demonstrate that Na+/H+ exchange is
the only Na+-dependent acid extrusion mechanism
operating in the basolateral membrane under the conditions of our
experiments.
NGF Secondarily Inhibits Apical Membrane
Na+/H+ Exchange Activity--
Previously in
the MTAL, we demonstrated a functional interaction between the
basolateral and apical membrane Na+/H+
exchangers, in which primary inhibition of basolateral
Na+/H+ exchange activity resulted secondarily
in inhibition of apical Na+/H+ exchange
activity and thereby HCO3 NGF Inhibits HCO3
To examine further the role of basolateral membrane
Na+/H+ exchange in regulation by NGF, we tested
whether pretreatment with NGF would alter the inhibition of
HCO3 In the MTAL of the rat, both apical and basolateral membrane
Na+/H+ exchangers play a role in determining
the rate of transepithelial HCO3 NGF Inhibits Basolateral Membrane Na+/H+
Exchange Activity in the MTAL--
Stimulation of
Na+/H+ exchange by growth factors has been a
consistent, virtually universal finding (1, 3, 4). An increase in
Na+/H+ exchange activity is believed to be
important early in the mitogenic response to stabilize pHi
within a range permissive for cell growth (1, 6, 9, 40). In contrast,
we found that NGF inhibits basolateral membrane
Na+/H+ exchange activity in the MTAL. This
inhibition was rapid and was observed under conditions in which the
activity of the basolateral exchanger was studied independently of
effects of NGF on other transporters, which suggests that the NGF
signaling pathway is coupled directly to inhibition of the basolateral
exchanger. In addition, the physiological relevance of this inhibition
was established by the demonstration that it is necessary for
NGF-induced inhibition of HCO3
Although the mechanism by which NGF inhibits basolateral membrane
Na+/H+ exchange activity was not identified in
our experiments, some insights can be obtained from previous studies of
the signaling pathways involved in NGF regulation of
HCO3
Regulation of the epithelial exchanger isoforms NHE2 and NHE3 by growth
factors has been studied less extensively than regulation of NHE1.
However, the available evidence indicates that, like NHE1, NHE2 and
NHE3 are activated by growth factors (2, 4) and can support cell
proliferation when expressed in NHE-deficient cell lines (7, 8).
Furthermore, several growth factors have been shown to stimulate NHE3
activity in the brush-border membrane of intestinal epithelial cells
(45). NHE3 is the apical membrane isoform responsible for
H+ secretion and HCO3 Inhibition of Basolateral Membrane Na+/H+
Exchange Activity Is Necessary for NGF-induced Inhibition of
HCO3
Although basolateral membrane Na+/H+ exchange
is present in most segments of the nephron, its role in transepithelial
HCO3
The mechanism by which the NGF-induced inhibition of basolateral
membrane Na+/H+ exchange results in inhibition
of HCO3
NGF and its receptors are expressed in the kidney (30, 51-54), but
their roles in the regulation of renal function remain to be
determined. Our results demonstrating that NGF inhibits HCO3 absorption in
the rat medullary thick ascending limb (MTAL). To investigate the
mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane
Na+/H+ exchange activities were determined from
rates of pHi recovery after lumen or bath Na+
addition. NGF (0.7 nM in the bath) had no effect on apical
Na+/H+ exchange activity, but inhibited
basolateral Na+/H+ exchange activity by 50%.
Inhibition of basolateral Na+/H+ exchange
activity with ethylisopropyl amiloride (EIPA) secondarily reduces
apical Na+/H+ exchange activity and
HCO3
absorption in the MTAL (Good,
D. W., George, T., and Watts, B. A., III (1995) Proc.
Natl. Acad. Sci. U. S. A. 92, 12525-12529). To
determine whether a similar mechanism could explain inhibition of
HCO3
absorption by NGF, apical
Na+/H+ exchange activity was assessed in
physiological solutions (146 mM Na+) by
measurement of the initial rate of cell acidification after lumen EIPA
addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF
inhibited apical Na+/H+ exchange activity.
Inhibition of HCO3
absorption by NGF was
eliminated in the presence of bath EIPA or in the absence of bath
Na+. Also, NGF blocked inhibition of
HCO3
absorption by bath EIPA. We conclude
that NGF inhibits basolateral Na+/H+ exchange
activity in the MTAL, an effect opposite from the stimulation of
Na+/H+ exchange by growth factors in other
systems. NGF inhibits transepithelial HCO3
absorption through inhibition of
basolateral Na+/H+ exchange, most likely as the
result of functional coupling in which primary inhibition of
basolateral Na+/H+ exchange activity results
secondarily in inhibition of apical Na+/H+
exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal
tubule HCO3
absorption.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
filtered
at the glomerulus (31). The proton secretion required for this
HCO3
absorption is mediated virtually
completely by apical membrane Na+/H+ exchange
(25). Furthermore, the regulation of
HCO3
absorption is achieved largely
through regulation of this apical exchanger (24, 25, 27, 31-35). The
MTAL also contains a basolateral membrane
Na+/H+ exchanger that is active at the resting
pHi (33, 36). In general, it has been assumed for renal tubules
that basolateral membrane Na+/H+ exchange
opposes transcellular HCO3
absorption
because it diminishes net base efflux (2, 13, 14). Contrary to this
view, however, we recently demonstrated that inhibiting basolateral
Na+/H+ exchange activity with ethylisopropyl
amiloride (EIPA) decreased transepithelial
HCO3
absorption in the MTAL (36). This
decrease was the result of a functional interaction between the
basolateral and apical membrane Na+/H+
exchangers, in which primary inhibition of basolateral
Na+/H+ exchange activity secondarily inhibited
apical Na+/H+ exchange activity, thereby
decreasing HCO3
absorption (36). These
studies established that the HCO3
absorption rate in the MTAL is dependent not only on the activity of
the apical membrane Na+/H+ exchanger, but also
on the activity of the basolateral membrane Na+/H+ exchanger. However, physiological
factors that regulate HCO3
absorption
through effects on basolateral membrane Na+/H+
exchange activity have not been identified.
absorption by the MTAL. These studies demonstrated that NGF inhibits HCO3
absorption in the MTAL through a
tyrosine kinase-dependent signaling mechanism (37). This
inhibition was unexpected since it cannot readily be explained by the
classical action of growth factors to stimulate
Na+/H+ exchange activity. Instead, our results
suggested that NGF may inhibit Na+/H+ exchange
activity in the MTAL. The purpose of the present study was to examine
directly the effects of NGF on apical and basolateral membrane
Na+/H+ exchange activities to determine the
mechanism by which NGF inhibits HCO3
absorption. Our results demonstrate that, in contrast to the virtually
universal stimulation of Na+/H+ exchange by
growth factors in other cells, NGF inhibits basolateral membrane
Na+/H+ exchange activity in the MTAL. We also
demonstrate that the inhibition of basolateral
Na+/H+ exchange activity mediates NGF-induced
inhibition of transepithelial HCO3
absorption.
EXPERIMENTAL PROCEDURES
Absorption--
To
measure transepithelial HCO3
absorption rates, tubules were perfused and bathed in control solution
that contained 146 mM Na+, 4 mM
K+, 122 mM Cl
, 25 mM
HCO3
, 2.0 mM
Ca2+, 1.5 mM Mg2+, 2.0 mM phosphate, 1.2 mM
SO42
, 1.0 mM citrate, 2.0 mM lactate, and 5.5 mM glucose (equilibrated with 95% O2 and 5% CO2, pH 7.45, at
37 °C). Bath solutions also contained 0.2 g/100 ml fatty acid-free
bovine albumin. In one series of HCO3
transport experiments (see Fig. 5B), Na+ in the
bath solution was replaced completely with
N-methyl-D-glucammonium (NMDG+).
Experimental agents were added to the bath solutions as described under
"Results." The length of the perfused tubule segments ranged from
0.48 to 0.67 mm.
absorption was as described (32,
37). The tubules were equilibrated for 20-30 min at 37 °C in the
initial perfusion and bath solutions, and the luminal flow rate was
adjusted to 1.4-2.0 nl/min/mm. Two or three 10-min tubule fluid
samples were then collected for each period (initial, experimental, and
recovery). The tubules were allowed to re-equilibrate for 5-15 min
after an experimental agent was added to or removed from the bath
solution. The absolute rate of HCO3
absorption
(JHCO3
,
pmol/min/mm) was calculated from the luminal flow rate and the
difference between total CO2 concentrations in perfused and
collected fluids (32). When repeat measurements were made at the
beginning and end of an experiment (initial and recovery periods), the
values were averaged. Single tubule values are presented in the
figures. Means ± S.E. (n = number of tubules) are
presented below.
transport experiments, except
that the lumen and bath solutions were delivered via rapid flow systems
that permit complete exchange of the solutions in <2 s (36, 38).
Intracellular dye was excited alternately at 500- and 440-nm
wavelengths, and emission was monitored at 530 nm using a photon
counter. Intracellular dye was calibrated using high
K+/nigericin standards at the end of each experiment to
convert fluorescence excitation ratios
(F500/F440) to
pHi values, as described previously (34, 38).
, 2.0 mM
Ca2+, 1.5 mM Mg2+, 1.0 mM phosphate, 1.0 mM
SO42
, 1.0 mM citrate, 2.0 mM lactate, 5.5 mM glucose, and 5 mM HEPES (equilibrated with 100% O2, titrated
to pH 7.4). The perfusate also contained furosemide to block
Na+-K+-2Cl
cotransport-mediated
changes in cell Na+ concentration or volume. After a stable
pHi was reached, Na+ was replaced completely with
NMDG+ in the lumen and bath solutions. Bilateral
Na+ replacement unmasks a background acid loading process
that reduces the pHi to 6.5-6.7 (34). Apical
Na+/H+ exchange activity was then determined by
measurement of the rate of pHi increase after readdition of
Na+ to the tubule lumen. H+ flux rates were
calculated as (dpHi/dt) ×
i × V, where dpHi/dt is the initial slope of
the record of pHi versus time (pH units/min)
measured over the first 4 s following an experimental maneuver
(34),
i is the intrinsic intracellular buffering power
(mmol/liter·pH unit), and V is cell volume/mm of tubule
length (nl/mm).
i was measured in the presence and absence
of NGF as a function of pHi using previously described methods
(34). V, determined from inner and outer tubule diameters
(12, 24, 34), was 0.30 ± 0.01 nl/mm. NGF had no effect on
i or V. To determine the pHi dependence
of apical membrane Na+/H+ exchange, the
Na+-dependent pHi recovery was
interrupted at various points along the recovery curve by luminal
Na+ removal (NMDG+) plus EIPA, which unmasks
background acid loading (34, 39). At the point of interruption, the
Na+/H+ exchange rate is calculated as the
difference between the net recovery rate and the background acid
loading rate (34, 39). This approach permits the
Na+/H+ exchange rate to be determined over a
range of pHi values, with appropriate corrections for a
variable background acid loading rate. Basolateral membrane
Na+/H+ exchange rates were determined using
similar protocols, i.e. by measuring rates of pHi
recovery after readdition of Na+ to only the bath solution.
In experiments in which apical or basolateral
Na+/H+ exchange activity was measured, EIPA was
present on the opposite side of the tubule to eliminate any
contribution of the contralateral exchanger to the
Na+-induced changes in pHi.
transport experiments, and apical
Na+/H+ exchange activity was determined by
measuring the initial rate of cell acidification in response to rapid
addition of 50 µM EIPA to the tubule lumen. In this
analysis, JNa/H = (dpHi/dt) ×
T × V, where dpHi/dt is
the initial rate of pHi decrease after lumen EIPA addition,
T is the sum of
i and buffering power due to
HCO3
/CO2 (computed as 2.3 [HCO3]i) (36), and V is cell volume
measured in control solution (0.28 ± 0.02 nl/mm). The basis for
this approach is that, prior to EIPA addition, H+ extrusion
via the apical Na+/H+ exchanger balances
background acid loading to maintain pHi constant (36, 39). When
the apical exchanger is inhibited, the initial rate of cell
acidification estimates the steady-state rate of apical
Na+/H+ exchange that balances background acid
loading (36).
RESULTS
Absorption--
Previously, we demonstrated that NGF inhibits
HCO3
absorption in the MTAL (37). This
finding was confirmed in three experiments in the present study (Fig.
1). Addition of 0.7 nM NGF to
the bath decreased HCO3
absorption by
30%, from 13.5 ± 0.8 to 9.3 ± 0.6 pmol/min/mm
(p < 0.05). The inhibition was observed within 15 min
after addition of NGF to the bath and was reversible.
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Fig. 1.
NGF inhibits
HCO3
absorption in the MTAL. The absolute rate of
HCO3
absorption
(JHCO3
)
was measured under control conditions and after addition of 0.7 nM NGF to the bath. Data points are average values for
single tubules. Lines connect paired measurements made in the same
tubule. The p value is for the paired t test.
Mean values are given under "Results."
absorption, we examined directly
the effects of NGF on apical and basolateral membrane
Na+/H+ exchange activities.
Na+/H+ exchange rates were determined from
rates of pHi recovery measured after addition of
Na+ to the lumen or bath solutions (see "Experimental
Procedures"). The results in Fig.
2A show that NGF had no effect
on apical membrane Na+/H+ exchange activity
over the pHi range 6.7-7.7. The apical exchanger exhibited a
sigmoidal dependence on pHi, as described previously (34). A
Hill plot of the combined data gave a Vmax of 68 pmol/min/mm, an apparent pK of 7.26, and a Hill coefficient
of 1.8, values in close agreement with those obtained previously for
MTALs in isosmotic solutions (34). NGF also had no effect on the
steady-state pHi reached after lumen Na+ addition
(7.74 ± 0.05 without NGF (n = 6)
versus 7.76 ± 0.05 with NGF (n = 4);
p = not significant). Thus, we found no evidence for
direct coupling of the NGF pathway to regulation of apical membrane
Na+/H+ exchange activity.
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Fig. 2.
NGF has no effect on apical membrane
Na+/H+ exchange activity, but inhibits
basolateral membrane Na+/H+ exchange
activity. MTALs were studied in the absence (closed
circles) and presence (open circles) of 0.7 nM NGF in the bath solution. Na+/H+
exchange rates (JNa/H) were determined at
various intracellular pH values from rates of pHi recovery
measured after addition of Na+ to lumen or bath solutions
(see "Experimental Procedures"). A, apical membrane
Na+/H+ exchange activity is not affected by
NGF. Data points are from six tubules without NGF and five tubules with
NGF. The line is a least-squares fit to the Hill equation; kinetic
parameters are given under "Results." B, basolateral
membrane Na+/H+ exchange activity is decreased
by NGF. Data points are from five tubules without NGF and five tubules
with NGF. Note the different scales for JNa/H in
the apical and basolateral plots.
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Fig. 3.
EIPA inhibits basolateral membrane
Na+/H+ exchange activity. Tubules were
studied in the absence (A) and presence (B) of
0.7 nM NGF in the bath. Basolateral
Na+/H+ exchange rates
(JNa/H) were determined from rates of
pHi recovery after bath Na+ addition in the absence
and presence of 50 µM bath EIPA. Data points are values
for single tubules. Data points without EIPA are from Fig. 2. Mean
rates are given under "Results." The values below the panels are
mean pHi ± S.E. at which JNa/H was
measured.
absorption
(36). If a similar mechanism accounts for the inhibition of
HCO3
absorption by NGF, then
inhibition of apical Na+/H+ exchange activity
may not have been observed in the experiments in Fig. 2A
because basolateral Na+/H+ exchange activity
was blocked due to the absence of bath Na+, thereby
preventing NGF from inhibiting the basolateral exchanger and inducing
the interaction that secondarily decreases apical Na+/H+ exchange activity. We therefore examined
the effect of NGF on apical Na+/H+ exchange
using a second protocol in which the basolateral
Na+/H+ exchanger was active. MTALs were
perfused and bathed with the control solution used for transepithelial
HCO3
transport experiments (146 mM Na+ and 25 mM
HCO3
, pH 7.4), and apical membrane
Na+/H+ exchange activity was determined by
measurement of the initial rate of cell acidification after lumen EIPA
addition (see "Experimental Procedures"). A typical experiment is
shown in Fig. 4A. Addition of
50 µM EIPA to the tubule lumen caused a rapid decrease in
pHi due to inhibition of apical membrane
Na+/H+ exchange activity (25, 36). Removal of
EIPA caused pHi to recover to its initial value (~7.05). NGF
was then added to the bath, and the pHi response to lumen EIPA
was repeated. NGF reduced the initial rate of cell acidification (Fig.
4A, dashed lines), indicating a decrease in the
steady-state rate of apical membrane Na+/H+
exchange (36). Similar results were obtained when the order of the
experimental conditions was reversed. For a total of six experiments
(Fig. 4B), apical Na+/H+ exchange
activity was decreased by 20 ± 4% in the presence of NGF. Thus,
NGF inhibits apical membrane Na+/H+ exchange
activity in the MTAL, an effect that likely mediates its action to
inhibit HCO3
absorption. These
findings, together with the results in Fig. 2, are consistent with the
view that NGF inhibits apical membrane Na+/H+
exchange activity secondary to inhibition of basolateral membrane Na+/H+ exchange activity, that is, NGF inhibits
apical Na+/H+ exchange activity in the
experiments in Fig. 4, but not in the experiments in Fig.
2A, because, in the latter protocol, basolateral membrane
Na+/H+ exchange was blocked due to the absence
of bath Na+, thereby preventing the interaction between
basolateral and apical Na+/H+ exchangers that
secondarily decreases apical Na+/H+ exchange
activity (see "Discussion").
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Fig. 4.
NGF secondarily inhibits apical membrane
Na+/H+ exchange activity. MTALs were
perfused and bathed in control solution (containing 146 mM
Na+ and 25 mM
HCO3 , pH 7.4), and apical membrane
Na+/H+ exchange activity was assessed from the
initial rate of cell acidification after lumen addition of 50 µM EIPA (see "Experimental Procedures").
A, the tracing shows the response of pHi
to lumen EIPA addition, first in the absence and then in the presence
of 0.7 nM bath NGF. NGF decreased the initial rate of cell
acidification (dashed lines), indicating inhibition of the
steady-state rate of apical membrane Na+/H+
exchange. B, effect of NGF on apical membrane
Na+/H+ exchange activity
(JNa/H) in six experiments similar to that in
A. Data points are values for single tubules. Lines and the
p value are as described in the legend to Fig. 1.
Absorption through Inhibition of Basolateral Membrane
Na+/H+ Exchange--
Transepithelial transport
experiments were carried out to test further the hypothesis that the
inhibition of HCO3
absorption by NGF
was dependent on its action to inhibit basolateral membrane
Na+/H+ exchange. If NGF decreases
HCO3
absorption through inhibition of
basolateral Na+/H+ exchange, then NGF action
should be diminished under conditions in which the basolateral
exchanger is inhibited. To test this, we examined the effects of NGF on
HCO3
absorption in the presence of
bath EIPA and in the absence of bath Na+, two conditions
under which basolateral Na+/H+ exchange
activity is inhibited (36). The results in Fig.
5A show that, in tubules
bathed with 1 µM EIPA, addition of NGF to the bath
decreased HCO3
absorption only by 9%,
from 10.5 ± 0.4 to 9.5 ± 0.4 pmol/min/mm (n = 3; p < 0.025). When compared with the inhibition by
NGF in control experiments (Fig. 1), 1 µM bath EIPA
inhibited NGF action by >70%. The effect of NGF on
HCO3
absorption in tubules studied in
a Na+-free bath is shown in Fig. 5B. In these
experiments, the tubule lumen was perfused with control solution
(containing 146 mM Na+ and 25 mM
HCO3
); the bath solution was identical
except that Na+ was replaced completely with
NMDG+. Under these conditions, addition of NGF to the bath
had no effect on HCO3
absorption
(11.5 ± 1.0 pmol/min/mm in Na+-free bath
versus 11.4 ± 1.0 pmol/min/mm in Na+-free
bath + NGF, n = 4; p = not
significant). Thus, the effect of NGF to inhibit
HCO3
absorption was virtually
eliminated under two different conditions in which basolateral membrane
Na+/H+ exchange activity was
inhibited.3 These findings
indicate that inhibition of basolateral membrane Na+/H+ exchange is essential for NGF-induced
inhibition of HCO3
absorption.
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Fig. 5.
Inhibition of
HCO3
absorption by NGF is eliminated by conditions that inhibit basolateral
membrane Na+/H+ exchange. A,
effect of NGF (0.7 nM added to the bath) on
HCO3
absorption in the presence of
bath EIPA. EIPA (1 µM) was present in the bath throughout
the experiments. B, effect of NGF on
HCO3
absorption in tubules studied in
a Na+-free bath. Na+ in the bath was replaced
completely with NMDG+ throughout the experiments; the lumen
was perfused with control solution containing 146 mM
Na+.
JHCO3
,
data points, lines, and p values are as described in the
legend to Fig. 1. Mean values are given under "Results."
NS, not significant.
absorption by bath EIPA. Under
control conditions, addition of 50 µM EIPA to the bath
decreased HCO3
absorption by 40%,
from 13.2 ± 1.1 to 8.1 ± 1.1 pmol/min/mm (n = 5; p < 0.001) (Fig.
6A). In contrast, in tubules
bathed with NGF, addition of 50 µM EIPA to the bath
decreased HCO3
absorption only by
16%, from 10.5 ± 0.3 to 8.8 ± 0.3 pmol/min/mm (n = 4; p < 0.005) (Fig.
6B). Thus, pretreatment with NGF markedly attenuates the
inhibition of HCO3
absorption by bath EIPA. These
results suggest that NGF and bath EIPA inhibit
HCO3
absorption via a common
mechanism, namely inhibition of basolateral membrane
Na+/H+ exchange activity. When taken together,
our results support the conclusion that the inhibition of
HCO3
absorption by NGF is mediated
through inhibition of basolateral membrane
Na+/H+ exchange.
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Fig. 6.
NGF blocks inhibition of
HCO3
absorption by bath EIPA. The effect of adding 50 µM
EIPA to the bath on HCO3
absorption
was examined in the absence (A) and presence (B)
of NGF. In B, 0.7 nM NGF was present in the bath
throughout the experiments.
JHCO3
,
data points, lines, and p values are as described in the
legend to Fig. 1. Mean values are given under "Results."
DISCUSSION
absorption (25, 31, 36). Recently, we demonstrated that NGF inhibits
HCO3
absorption in the MTAL (37). The
objective of the present study was to investigate the role of
Na+/H+ exchange in this inhibitory response. We
found that NGF inhibits basolateral membrane
Na+/H+ exchange activity in the MTAL and that
this inhibition mediates the NGF-induced inhibition of transepithelial
HCO3
absorption. In contrast, apical
membrane Na+/H+ exchange activity is not
affected directly by NGF, but is inhibited secondary to the inhibition
of basolateral membrane Na+/H+ exchange
activity. To our knowledge, inhibition of
Na+/H+ exchange activity by peptide growth
factors has not previously been reported. Thus, our studies identify a
novel mechanism by which growth factors may regulate
HCO3
absorption in renal tubules,
namely inhibition of basolateral membrane
Na+/H+ exchange activity.
absorption. Immunocytochemical studies using isoform-specific antibodies (16, 17), studies of NHE transcripts (15, 41), and
functional studies of amiloride sensitivity (36) have indicated that
basolateral membrane Na+/H+ exchange activity
in the MTAL is mediated by NHE1. NHE1 is expressed in virtually all
cell types and tissues and is the exchanger isoform whose activation is
closely associated with the growth response (1, 4, 6). Our data suggest
that NHE1 is inhibited by NGF in the MTAL; however, we cannot rule out
the possibility that NGF may inhibit an as yet undetermined exchanger
isoform that may co-localize with NHE1 in the basolateral membrane. The
inhibition is not the result of a general cellular action of NGF
because NGF has been shown to stimulate Na+/H+
exchange activity in other systems (42, 43). At this point, we do not
know whether the inhibition of basolateral membrane Na+/H+ exchange activity that we observed
represents a general response of the MTAL cells to growth factor
stimulation or a specific response to stimulation by NGF.
absorption. The inhibition of
HCO3
absorption by NGF was additive to
inhibition by cAMP and was unaffected by inhibitors of protein kinase C
(37). Thus, the inhibition of basolateral membrane
Na+/H+ exchange activity by NGF is unlikely to
involve cAMP- or protein kinase C-dependent signaling
pathways. In contrast, the inhibition of
HCO3
absorption by NGF was eliminated
by pretreatment with the tyrosine kinase inhibitor genistein (37). This
suggests that tyrosine kinase pathways are involved in the NGF
inhibition of Na+/H+ exchange activity. The
signal transduction mechanisms by which growth factors activate
Na+/H+ exchange (NHE1) in other systems are
complex and may include calcium/calmodulin, mitogen-activated protein
kinases, phosphatidylinositol 3-kinase, small molecular weight GTPases,
and associated regulatory proteins (4, 5, 44-48). It remains to be
determined whether the inhibition of Na+/H+
exchange activity by NGF in the MTAL is mediated through these same
pathways or through signaling mechanisms distinct from those that
mediate exchanger activation.
absorption in the rat MTAL (21, 22, 24, 27, 35, 36). However, at least
two observations in the present study indicate that NHE3 activity is
not regulated directly by NGF: 1) NGF had no effect on lumen
Na+-dependent pHi recovery (Fig.
2A); and 2) NGF had no effect on
HCO3
absorption in the absence of bath
Na+ or in the presence bath EIPA (Fig. 5). Because apical
membrane Na+/H+ exchange mediates
HCO3
absorption in the latter
conditions (36), any direct coupling of the NGF pathway to stimulation
(or inhibition) of the apical exchanger should have been evident as a
change in the HCO3
absorption rate.
Thus, the stimulation of NHE3 by growth factors in other systems was
not observed with NGF in the MTAL, suggesting that growth factor
regulation of NHE3 activity also may be cell type-specific. As
discussed below, we did find that apical membrane Na+/H+ exchange activity was inhibited by NGF;
however, this inhibition occurs secondary to inhibition of basolateral
membrane Na+/H+ exchange activity.
Absorption--
Several
observations support the conclusion that NGF inhibits MTAL
HCO3
absorption through inhibition of
basolateral membrane Na+/H+ exchange. 1) NGF
inhibits basolateral Na+/H+ exchange activity
at physiological pHi values. 2) The inhibition of
HCO3
absorption by NGF is nearly
abolished in tubules bathed with EIPA to decrease basolateral
Na+/H+ exchange activity. 3) The inhibition of
HCO3
absorption requires
Na+ in the bath solution. 4) Pretreatment with NGF blocks
inhibition of HCO3
absorption by bath
EIPA, an effect mediated through inhibition of basolateral
Na+/H+ exchange activity (36). Taken together,
these findings indicate that inhibition of basolateral membrane
Na+/H+ exchange activity is an essential
component in NGF-induced inhibition of
HCO3
absorption. Furthermore, they
establish that the physiological function of basolateral
Na+/H+ exchange in renal tubules is not limited
to housekeeping functions such as regulation of pHi and cell
volume, but also includes a more specialized role in the regulation of
acid secretion and transepithelial NaHCO3 absorption.
absorption is not understood. In
renal tubules, HCO3
absorption is
achieved by secretion of protons across the apical membrane (which
titrate lumen HCO3
) and transport of
HCO3
across the basolateral membrane
(49). It is widely assumed that basolateral membrane
Na+/H+ exchange decreases the efficiency of
HCO3
absorption by decreasing net
basolateral base efflux and/or by increasing cell sodium concentration
or pHi and secondarily inhibiting apical proton secretion (2,
13, 14, 50). In contrast to this view, our results demonstrate that
inhibiting basolateral membrane Na+/H+ exchange
activity with either NGF (present study) or EIPA (36) markedly
decreases HCO3
absorption in the MTAL.
These studies provide the first identified role for basolateral
membrane Na+/H+ exchange in the physiological
regulation of renal tubule HCO3
absorption. Furthermore, they demonstrate that, contrary to the generally accepted view that it opposes
HCO3
absorption (2, 13, 14), the
presence of basolateral membrane Na+/H+
exchange enhances HCO3
absorption in
the MTAL.
absorption likely involves
interaction between the basolateral and apical membrane
Na+/H+ exchangers. In the MTAL, changes in the
rate of HCO3
absorption are mediated
through changes in apical membrane Na+/H+
exchange activity (24, 25, 31, 34, 35). In previous studies in which
EIPA was used to inhibit basolateral Na+/H+
exchange activity, we demonstrated that the basolateral and apical membrane Na+/H+ exchangers are functionally
coupled, wherein primary inhibition of basolateral
Na+/H+ exchange activity results secondarily in
inhibition of apical Na+/H+ exchange activity
(36). The evidence presented in this report that 1) NGF primarily
inhibits basolateral membrane Na+/H+ exchange
activity, 2) inhibition of basolateral Na+/H+
exchange activity is required for inhibition of
HCO3
absorption, and 3) apical
membrane Na+/H+ exchange activity is inhibited
by NGF only under conditions in which basolateral
Na+/H+ exchange activity is inhibited supports
the view that NGF inhibits HCO3
absorption via this coupling mechanism. Thus, our data are consistent with NGF inhibiting HCO3
absorption in
the MTAL through the following events. 1) Interaction of NGF with its
membrane receptor activates signaling mechanisms coupled to the
inhibition of basolateral membrane Na+/H+
exchange. 2) Inhibition of basolateral Na+/H+
exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity due to coupling
between the exchangers. 3) The decrease in apical membrane
Na+/H+ exchange activity diminishes luminal
acid secretion, thereby decreasing net
HCO3
absorption (25, 31, 36). This
mechanism can account for our observations that apical membrane
Na+/H+ exchange activity is inhibited by NGF
when studied by lumen EIPA addition in physiological (146 mM Na+) solutions (Fig. 4), but not when
studied by bilateral Na+ removal and lumen Na+
readdition (Fig. 2A): in the latter protocol, basolateral
membrane Na+/H+ exchange is blocked due to the
absence of bath Na+, which prevents NGF from inhibiting the
basolateral exchanger and inducing the basolateral
exchanger-dependent interaction that secondarily decreases
apical Na+/H+ exchange activity. Important
goals for future studies will be to identify the signaling pathway that
couples NGF to the inhibition of basolateral
Na+/H+ exchange activity and the mechanism of
interaction between the basolateral and apical membrane
Na+/H+ exchangers. Our results indicate,
however, that communication between the exchangers is activated in the
MTAL by growth factors and thus represents a physiologically relevant
pathway of transport regulation.
absorption in the MTAL through
inhibition of basolateral membrane Na+/H+
exchange activity establish directly that NGF can influence the function of renal tubules. The change in
HCO3
absorption induced by NGF
(30-40%) is similar to that observed with other regulatory factors,
such as chronic metabolic acidosis and alkalosis, dietary sodium
intake, and angiotensin II (31, 55). These findings suggest that NGF
may be involved in the control of urinary acidification, as discussed
previously (37). In addition, other stimuli that regulate
HCO3
absorption in the MTAL, including
angiotensin II, vasopressin, changes in osmolality, and aldosterone
(24, 31, 32, 55), have been shown to alter basolateral membrane
Na+/H+ exchange activity in renal cells (12,
33, 56-58). Thus, an important goal for future investigations will be
to determine whether basolateral membrane
Na+/H+ exchange may be an effector that
mediates the regulation of HCO3
absorption by a wide variety of physiological stimuli.
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ACKNOWLEDGEMENT |
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We thank L. Reuss for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by NIDDK Grant DK-38217 from the National Institutes of Health and a research fellowship from the National Kidney Foundation (to B. A. W.), with funds contributed in part by the National Kidney Foundation of Southeast Texas.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.
To whom correspondence should be addressed: 4.200 John Sealy Annex
0562, University of Texas Medical Branch, 301 University Blvd.,
Galveston, TX 77555-0562. Tel.: 409-772-2472; Fax: 409-772-5451.
2 In our analysis, the pHi range over which Na+/H+ exchange activities are measured is bracketed on the low end by the pHi reached after bilateral Na+ replacement and on the high end by the pHi achieved after lumen or bath Na+ readdition (34). Because the basolateral Na+/H+ exchange rate is less than the apical Na+/H+ exchange rate, the steady-state pHi reached after bath Na+ addition is less than that reached after lumen Na+ addition. This limits the pHi range over which the basolateral exchange rate is determined, which precludes kinetic analysis of the mechanism by which NGF inhibits exchange activity. Our data show clearly, however, that NGF inhibits basolateral membrane Na+/H+ exchange activity at the resting pHi normally measured in MTAL segments (7.0-7.1) (25, 36).
3
The small residual inhibition of
HCO3 absorption by NGF in the presence
of bath EIPA likely is due to incomplete inhibition of the basolateral
Na+/H+ exchanger by 1 µM EIPA at
physiological Na+ concentrations (36) rather than to an
exchanger-independent regulatory mechanism. 1 µM EIPA was
used in this experiment because this concentration has a negligible
effect on pHi (36); thus, the lack of effect of NGF on
HCO3
absorption could not be
attributed to an EIPA-induced cell acidification.
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ABBREVIATIONS |
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The abbreviations used are: NHE, Na+/H+ exchanger; NGF, nerve growth factor; MTAL, medullary thick ascending limb; EIPA, ethylisopropyl amiloride; NMDG+, N-methyl-D-glucammonium.
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REFERENCES |
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