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3)n
cotransport
,2, and1 Departments of Medicine and Physiology, Tulane University, School of Medicine, New Orleans, Louisiana 70112; and 2 East Carolina University, Greenville, North Carolina 27858
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We examined the effect of norepinephrine (NE) on intracellular
pH (pHi) and activity of
Na+
(aNai) in the
isolated perfused kidney proximal tubule of
Ambystoma, using single-barreled
voltage and ion-selective microelectrodes. In control
HCO
3 Ringer, addition of
106 M NE to the
bath reversibly depolarized the basolateral membrane potential (V1),
the luminal membrane potential
(V2), and the
transepithelial potential difference
(V3) and
increased pHi by 0.14 ± 0.02. These effects were mimicked by isoproterenol but were abolished after pretreatment with SITS or in the absence of
CO2/HCO3. Removal of bath Na+ depolarized
V1 and
V2,
hyperpolarized
V3, and decreased
pHi. These effects are largely
mediated by the electrogenic
Na+-(HCO3)n
cotransporter. In the presence of NE, the effects of
Na+ removal on membrane potential
differences and the rate of change of
pHi were significantly smaller.
Reducing bath HCO3 concentration from
10 to 2 mM at constant CO2 (pH
6.8) depolarized V1 and
V2, decreased
pHi, and lowered
aNai. These changes are also
due to
Na+-(HCO3)n.
In the presence of NE, reducing bath
[HCO3] caused a
smaller depolarizations of
V1 and
V2, and the rate
of pHi decrease was significantly
reduced. Our results indicate: 1) NE
causes an increase in pHi;
2) the NE-induced alkalinization is
mediated by a SITS-sensitive and HCO3-dependent transporter on the
basolateral membrane; and 3) in the
presence of NE, the reduced effects caused by basolateral
HCO3 changes or
Na+ removal are indicative of an
inhibitory effect of NE on
Na+-(HCO3)n
cotransport.
adrenergic agonists; electrogenic sodium-bicarbonate cotransport; intracellular pH; sodium/proton exchange
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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CATECHOLAMINES HAVE BEEN reported to affect
sodium, water, and HCO3 reabsorption
in the kidney proximal tubule (for a review, see Ref. 17). At least
a component of this effect seems to be independent of
hemodynamic or humoral factors and occurs through a direct action on
the tubular epithelium. For example, studies on the rabbit isolated
perfused tubule suggest that norepinephrine (NE) enhances fluid and
sodium reabsorption (6, 14), and microperfusion studies indicate that
adrenergic stimulation stimulates fluid uptake and affects
HCO3 reabsorption in the intact
proximal tubule (12, 13). At the cellular level, Beach and co-workers
(5) suggested that NE stimulates Na-K-ATPase, whereas Podevin and
Parini (23) found no effect on Na-K-ATPase by NE. Still, other studies
reported that the effect of NE in the proximal tubule is probably
mediated through Na/H exchange (16, 22). The characterization of the specific ion transport mechanisms and the type of receptor(s) involved
in mediating the action of NE on the proximal cell continue to be
actively studied.
We have reported earlier that NE decreased intracellular Na activity
(aNai) and activated
Na-K-ATPase in the Ambystoma proximal
tubule (2). Because transport of Na+ and
H+ is closely linked in the
proximal tubule cell, it is likely that NE would affect intracellular
pH (pHi) as well. This is
further enforced by the reported effects of NE on
HCO3 reabsorption. In the kidney
proximal tubule,
Na+-(HCO3)n
cotransport is an important mechanism that contributes to
Na+ and
HCO3 transport and
pHi regulation.
The
Na+-(HCO3)n
cotransporter was first demonstrated in the
Ambystoma kidney proximal tubule (9) and was then described in the mammalian proximal tubule as well as a
wide variety of cells. More recently, the
Na+-(HCO3)n
cotransporter from Ambystoma kidney
was cloned (25).
Na+-(HCO3)n
cotransport plays a major role in the reabsorption of
HCO3 in the proximal tubule, as it
could account for up to 90% of HCO3 flux across the basolateral membrane in that segment (24) and as such
is extremely important in regulation of
pHi (8). Only few studies have
addressed the regulation of this transporter, which could play a major
role in acid base homeostasis as well as in the regulation of
Na+ transport (16, 29). The
response of this transporter to humoral factors like catecholamines has
not been addressed before.
In this study, we demonstrate a direct effect of NE on
Na+-(HCO3)n
cotransport in the Ambystoma proximal
tubule. We used voltage (Ling-Gerard) and ion-selective microelectrodes
to monitor cell membrane potential differences (PDs),
pHi, and
aNai in the isolated perfused preparation. Our results indicate that NE causes an alkalinization of
pHi. The effect of NE is mimicked
by isoproterenol and abolished after treatment with SITS
or in the absence of HCO3 (HEPES
buffer). Whereas Na/H exchange does not seem to play a significant
role, our data indicate that the action of NE on
pHi is mediated, at least in part,
by its action on
Na+-(HCO3)n
cotransport.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Isolated Perfused Tubule Preparation
Tiger salamanders (Ambystoma tigrinum) in the neotenic phase were obtained from Charles Sullivan (Nashville, TN) and kept in an aquarium at 4°C. The tubules were isolated and perfused, as described by Sackin and Boulpaep (27), and the method will only be briefly summarized here. The animals were anesthetized by immersion in 0.2% tricaine methanesulfonate solution. The kidneys were removed, placed in chilled preoxygenated amphibian solution containing 1.5% polyvinylpyrrolidone, and cut transversely into several pieces. Single tubules (100 µm diameter), with glomeruli intact, were isolated from the ventral surface of the kidney. The early part of the tubule (700-1,000 µm in length) was dissected, and the ends were opened with very fine forceps. The tubule segments were transferred in a small amount of medium to the chamber and connected to a perfusion apparatus similar to that described by Burg et al. (11). This apparatus consists of two sets of three concentric pipettes. The tubule was mounted between the outer (holding) and the middle (perfusion) pipette by applying a slight suction through the holding pipette. A proper mechanical and electrical seal was thus formed at the constriction around the tubule. The perfusion fluid was delivered via the innermost (exchange) pipette of the right-hand side assembly at a rate of ~1 ml/min. Only a small amount of this fluid perfuses the tubule (~100 nl/min). The rest leaves the pipette through a drain where it contacts a 3 M KCl free-flowing salt bridge. At the collection side, the perfusate leaving the tubule enters the left-hand side middle pipette, where continuous suction was applied to prevent any accumulation of this fluid. The solution bathing the tubule was continuously exchanged at 3 ml/min. Experiments were conducted at room temperature (21-25°C). The tubules were visualized with an inverted microscope (Leitz, Wetzlar, Germany), and microelectrode impalements were made at a magnification ×320.Compositions of Solutions
The compositions of solutions are given in Table 1. Solutions were delivered by gravity to either bath or lumen through CO2-impermeable Saran tubing (Clarkson Equipment and Controls, Detroit, MI). The osmolalities of all solutions were measured prior to the experiment and verified to be ~200 mosmol/kgH2O. In Na-free solutions, Na+ was replaced by N-methyl-D-glucamine (NMDG+). Solution 3 (minimal substrate/HCO3), in which the amino
acids were deleted, was used when SITS was added to the solution.
All the salts used to prepare the solutions were obtained from Sigma
Chemical (St. Louis, MO). SITS (5 × 104 M), norepinephrine
(106 M), and isoproterenol
(105 M) were purchased from Sigma
and were added directly to the solutions immediately before use.
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Electrophysiological Measurements in the Isolated Tubule
The pH-and Na-sensitive microelectrodes were of the liquid ion exchanger type. Single-barreled microelectrodes were manufactured as described earlier by Sackin and Boulpaep (27). Briefly, aluminosilicate glass tubings (1.2 mm OD × 0.86 mm ID; Frederick Haer, Brunswick, ME) were pulled on a horizontal Flaming Brown puller (model P 80/PC; Sutter Instruments, St. Raphael, CA) and dried in an oven at 200°C for 2 h. With the electrodes in a closed vessel (300 ml), 10 µl of tri-n-butyl-chlorosilane was then introduced for 2 min, after which the silane fumes were vented, and the electrodes were left in the oven for an additional 30 min. The exchanger for Na+ or H+ (Fluka, Buchs, Switzerland), was then introduced into the tip of the electrodes by means of a very fine glass capillary. Na+ electrodes were backfilled with 150 mM NaCl. pH electrodes were backfilled with a buffer solution containing 0.04 M KH2PO4, 0.023 M NaOH, and 0.015 M NaCl, pH 7.0 (3). The electrodes were fitted with a holder with an Ag-AgCl pellet. Na+ electrodes were calibrated in 10 mM NaCl, 100 mM NaCl, and 100 mM KCl. The slope of each electrode was determined from the relation
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pH electrodes were calibrated in HEPES buffer-Ringer (Table 1; adjusted
to pH values 6, 7, and 8). The average slope of the pH electrodes was
59.5 ± 1.0 mV/pH. Ling-Gerard microelectrodes were pulled from 1-mm
OD borosilicate fiber capillaries obtained from Frederick Haer and
filled with 3 M KCl. Their resistances ranged from 25 to 50 M
, and
their tip potentials were <5 mV. After the tubule was properly
mounted and perfused, two adjacent cells were impaled through the
basolateral membrane, the first with a Ling-Gerard microelectrode and
the second with the pH or Na+-sensitive microelectrode. The
basolateral membrane potential (V1) was
obtained by measuring the voltage difference between a Ling-Gerard
microelectrode and a free-flowing 3 M KCl, Ag-AgCl (electrode tip ~5
µm) in the bath. The transepitheial membrane potential
(V3) was
obtained by measuring the voltage difference between a free-flowing 3 M
KCl electrode in the drain of the perfusion side and the bath
free-flowing electrode. The luminal membrane potential
(V2) was
calculated as the difference between the transepithelial PD
(V3) and the
basolateral membrane PD
(V1). The total
potential of the ion-sensitive electrode was obtained by measuring the
voltage difference between the Na+
or the pH electrode and the reference electrode in the bath. The pure
ionic potential was obtained by subtracting electronically V1 from the total
potential of Na+ or the pH
electrode. The bath was grounded through a platinum wire. All these
parameters were continuously recorded on a four-channel strip-chart
recorder (RS 3400; Gould, Cleveland, OH), and the data were
simultaneously stored on a hard disk.
Curve Fitting, Statistics, and Data Analysis
Initial rates of change of pHi (dpHi/dt) and aNai(daNai/dt) were determined by using a computer to fit pHi (or aNai) vs. time data to a linear regression line. In all the experiments, values are reported as means ± SE. Unless otherwise noted, statistical significance was judged from paired Student's t-tests. Measurements were determined under control and test conditions in the same tubule, and each tubule served as its own control (paired data); n is the number of observations.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effect of NE on pHi and Cell Membrane PD
Steady-state effects of NE. In the first set of experiments, we examined the effects caused by addition of 106 M NE to the bath on
V1,
V2,
V3, and
pHi when the tubules were bathed
and perfused with control solution (Table 1, solution 1). Figure
1 is an actual tracing of
such an experiment. Addition of NE to the bath usually caused small
depolarizations of
V1 and V3 and a
significant and sustained increase of
pHi. These changes were readily
reversed on removal of basolateral NE. In several experiments, like
that shown in Fig. 1, the removal of bath NE often caused a significant
transient hyperpolarization of
V1 before returning to steady-state value, whereas the recovery of
pHi was always monotonic. In
tubules perfused and bathed in
CO2/HCO3 control Ringer,
V1,
V2, and
V3 averaged
62 ± 4.1, 57 ± 3.8, and 5.5 ± 0.6 mV,
respectively. pHi was 7.29 ± 0.04 (10). The addition of
106 M NE reversibly
depolarized V1 by
5.7 ± 1.6, V2
by 5.4 ± 1.5, and
V3 by 0.5 ± 0.2 mV, and pHi increased by 0.14 ± 0.02 pH units (P < 0.001). The
action of NE was completely reversible.
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Effects of isoproterenol. The second
part of Fig. 1 shows the effect of addition and removal of
isoproterenol, a 
-adrenergic agonist on
V1,
V3, and
pHi. Addition of isoproterenol
(105 M) to the bath
mimicked the effect of NE causing a depolarization of
V1 and
V3 and an
increase in pHi. The recovery of
pHi on removal of isoproterenol
from the bath, compared with that in the case of NE, was often slow. In
four experiments, addition of isoproterenol to the bath depolarized
V1,
V2, and
V3 by 9.8 ± 2.9, 8.0 ± 1.8, and 1.7 ± 0.5 mV, respectively.
pHi increased from 7.38 ± 0.16 to 7.55 ± 0.11 pH units (P < 0.04). The effects of isoproterenol imply that activation of
-receptors is probably involved in mediating the action NE.
Lack of Effect of NE on Luminal Na/H Exchange or Na-Substrate Cotransport
General. Two luminal transport mechanisms, whereby NE could induce a change in pHi are Na/H exchange and Na-substrate cotransport. Luminal Na/H exchange (8) is generally accepted as one of the main mechanisms responsible for Na+ and HCO3 uptake across the proximal
tubule. Some studies (16, 22) suggest that adrenergic agonists enhance Na+ reabsorption in the proximal
tubule by stimulating brush-border membrane Na/H exchange.
Na-substrate cotransport includes sodium glucose, sodium amino acids
(30), and sodium-monocarboxylate (in this case, lactate) cotransport.
In the proximal tubule of Ambystoma,
Na-lactate cotransport has been shown to act as a potent acid-extruding
mechanism (28). Based on measurements of apical to basolateral membrane
resistance ratio, Morgunov (20) suggested that -receptor stimulation
may activate Na-substrate cotransport in the
Ambystoma proximal tubule.
Changes caused by the removal of luminal
substrates. To determine whether NE acts on either
luminal Na-substrate cotransport or Na/H exchange, we performed the
following experiment with a typical tracing shown in Fig.
2. The experiments were performed in the
absence of
CO2/HCO3
(HEPES buffered; solution 7) to
minimize the contribution of
HCO3-dependent transport mechanisms to
the pHi changes. As shown in Fig.
2A, removal of luminal substrates
(solution 8) caused a
hyperpolarization of
V1 and
V2 of 11.0 ± 2.4 and 15.3 ± 2.0 mV, respectively, and a depolarization of
V3 of 3.08 ± 0.93 mV (n = 4, P < 0.05). These changes are typical
of the proximal tubule response to the removal of luminal substrates
and are due to the electrogenic Na-glucose and Na-amino acid
cotransport (15, 19). The removal of luminal substrates also caused a
substantial decrease in pHi of
0.32 ± 0.04 pH units (segment ab,
Fig. 2A). The fall in
pHi is due to the inhibition of
luminal Na-lactate cotransport (28). The same maneuver of removing
luminal substrates was repeated in the presence of basolateral NE
(segment a'b', Fig.
2B). In four paired experiments, the
changes in V1,
V2, and
V3 and the
decrease in pHi induced by removal
of luminal substrates were not statistically different in the presence
or absence of NE. In addition, the rate of decrease in
pHi in the absence of NE was not
statistically different from that in its presence.
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Changes caused by removal and readdition of luminal Na+. The second step in the experiment of Fig. 2 was aimed at checking whether the activity of luminal Na/H exchange was affected by NE. In the continued absence of substrates, removal of luminal Na+ (solution 9) caused small transient changes in V1 and V3 (1), and pHi decreased further by 0.07 ± 0.02 pH units (P < 0.05; segment bc, Fig. 2A). Readdition of luminal Na+ reversed these effects on the membrane PDs, and pHi recovered (segment cd, Fig. 2A). These changes in pHi are most likely caused by luminal Na/H exchange. Similar effects were observed when luminal Na+ was removed in the presence of NE (segments b'c' and c'd', Fig. 2B). Both the decrease in pHi and the rate of change of pHi (during acidification and recovery) were not different in the presence or absence of NE. These findings suggest that luminal Na/H exchange activity is not affected by NE.
Changes caused by readdition of luminal substrates. The final step in this experiment involved readding substrates to the lumen, first in the absence of NE (Fig. 2A) and then in its presence (Fig. 2B). The readdition of substrates caused a depolarization of V1 and V2 of 11 ± 2.6 and 15.3 ± 3.3 mV, respectively, and a hyperpolarization of V3 of 3.1 ± 1.4 mV. pHi increased by 0.42 ± 0.09 pH units to its initial control value (segment de, Fig. 2A). The increase in pHi is due to reactivation of Na-lactate cotransport. These changes in membrane PDs and pHi were not statistically different from the changes in the presence of NE (segment d'e', Fig. 2B). Also the rate of increase of pHi in the presence of NE was not different from that in its absence. These results show that NE did not affect the changes in V1, V2, V3, or pHi caused by removal or readdition of substrates to the lumen and indicate that NE has no effect on Na-substrate cotransport.
NE Effect is Dependent on Presence of
HCO3
3.
Our initial experiments demonstrated a substantial effect of NE on
V1,
V2,
V3, and
pHi when the tubule was bathed and
perfused with a
CO2/HCO3
"control" solution (solution
1). Addition of
106 M NE to the bath
usually caused a small but significant depolarization of
V1,
V2, and
V3 and an
increase in pHi (see Fig. 1). To
determine whether the effect of NE was dependent on the
presence of
CO2/HCO3, we added NE to the bath when the tubules were bathed and perfused with
a
CO2/HCO3-free
solution (HEPES buffered, pH = 7.5; solution
7). As can be seen in the initial part of the experiement of Fig. 2B,
addition of NE in the absence of
CO2/HCO3 caused no sustained changes in the membrane PDs and no increase in pHi. Six other experiments were
performed, and similar results were obtained.
We also checked the effect of NE
(106 M) on
aNai. As can be seen in Fig.
3, addition of NE to the bath in the
presence of
CO2/HCO3
(solution 1) caused the usual
depolarizations of
V1 and
V3 and decreased
aNai. These changes were
reversed on removal of NE. Similar results were reported in an earlier
study (2). Switching from control CO2/HCO3
solution (solution 1) to HEPES
(solution 7) in the lumen did not
cause any changes in
V3,
V1, or
aNai. Switching from
CO2/HCO3
to HEPES in the bath caused a small depolarization of
V3 and a large
depolarization of
V1. The
aNai transiently and quickly
decreased before it recovered to a value slightly less than that in
control. These changes are caused by the activity of the electrogenic
Na+-(HCO3)n
cotransporter at the basolateral membrane (9). In the absence of
CO2/HCO3, addition and removal of NE to the bath caused no changes in
V3, V1, or
aNai. In Fig. 3, NE was added
and removed twice. The results of these experiments showing the effects
of NE on pHi and
aNai in the presence and
absence of
CO2/HCO3
are summarized in Table 2.
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The absence of an effect of NE on membrane PDs,
aNai, and pHi
in the absence of
CO2/HCO3 suggests that HCO3 transport is
involved in mediating the action of NE.
Inhibition of the effects of NE by
SITS. The stilbene derivative, SITS, inhibits most
HCO3-transporting mechanisms, notably
Na+-(HCO3)n
cotransport and Cl/HCO3 exchange. To
further investigate whether the action of NE is mediated by a
HCO3 transporter that can be blocked
by SITS, we monitored the effect of NE after pretreating the tubule with SITS. As shown in the experiment of Fig.
4A, NE was
added first, which caused the usual depolarizations of
V3 and
V1 and the
increase in pHi. Removal of NE
reversed these changes, and V3,
V1, and
pHi recovered. Addition of SITS (5 × 104 M) to the bath
caused a small hyperpolarization of
V1 and an increase in pHi. These effects
were similar to the ones observed by Boron and Boulpaep (9) on
application of SITS to the tubule and are due to inhibition by SITS of
the electrogenic Na+ and
HCO3 exit. The tubule was treated with SITS for a prolonged period of time (25 min) to ensure irreversible effects of SITS, and it was subsequently removed from the bath to
prevent its interaction with NE. When NE was then added to the bath, no
effects on V3,
V1, or
pHi were observed.
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To check whether SITS also inhibits the effect of NE on
aNai, we repeated the same
experiment of Fig. 4A using an
Na+-sensitive microelectrode. As
shown in Fig. 4B, exposing the tubule to SITS caused a hyperpolarization of
V3 and
V1, as observed
in Fig. 4A. The
aNai increased also, which is consistent with inhibition of
Na+-(HCO3)n
cotransport by SITS. After pretreatment with SITS, addition or removal
of NE to the bath did not cause any changes in
V3,
V1, or
aNai (compare with Fig. 3). In
the same experiment, addition of isoproterenol (105 M) to the bath, which
mimics the effect of NE, causing depolarizations of membrane PDs, an
increase in pHi (see Fig. 1), and
a decrease in aNai (2), did
not cause any effect on V3,
V1, and
aNai after pretreatment with
SITS. The results of the experiments depicted in Fig. 4 and the lack of
response to NE in the absence of
CO2/HCO3 (Table 2) suggest that HCO3 transport
is involved in mediating the effect of NE on the proximal tubule cell.
Role of
Na+-(HCO3)n
Cotransport
3)n
cotransporter has been identified as one of the dominant pathways of
HCO3 transport across the basolateral membrane of the renal proximal tubule of Ambystoma
tigrinum (8). This mechanism extrudes
Na+,
HCO3, and a net negative charge from
cell to the peritubular space; is independent of
Cl; and is inhibited by the
disulfonic stilbene derivative SITS. Na+-(HCO3)n
cotransport is expected to affect
pHi and
V1 in the
following ways. 1) Removal of
basolateral Na+ should lead to a
decrease in pHi, a decrease in
aNai, and a depolarization of
V1 resulting from
HCO3 exit from the cell along with
Na+.
2) Reducing the concentration of
HCO3 in the bath should also lead to a
decrease in pHi, a decrease in
aNai, and a depolarization of
V1 driven by an
enhanced gradient for HCO3 exit from
the cell together with Na+.
3) The effects of both maneuvers
described in 1 and
2 are expected to be more evident in
the presence rather than in the absence of external
HCO3.
4) Finally, SITS should reduce the
effects on pHi,
aNai, and
V1 caused by removal of bath Na+ or lowering
bath HCO3. These predicted changes in
pHi or
aNai and membrane PDs caused
by the above maneuvers were measured in the presence and absence of NE
to examine the effect of NE on the activity of
Na+-(HCO3)n
cotransport in the proximal tubule.
Effects of basolateral
Na+ removal on
membrane PDs, pHi,
and/or aNai in the presence and absence of NE. IN THE PRESENCE OF
LUMINAL SODIUM AND SUBSTRATES. In the first set of
experiments, we examined the effect of NE on
Na+-(HCO3)n
cotransport by removing basolateral Na+ in the absence and presence of
NE while monitoring
V3,
V1, and pHi. As can be seen in Fig.
5, removal of bath
Na+ (solution
5) caused hyperpolarization of
V3, a
depolarization of
V1, and a rapid
and sustained decrease in pHi
(Fig. 5, segment abc). These changes
are similar to those observed by Boron and Boulpaep (9) and are
primarily caused by
Na+-(HCO3)n
cotransport as described above. In five tubules,
V3 hyperpolarized
from 5.4 ± 0.5 to 9.7 ± 2.5 mV
(P < 0.01),
V1 depolarized
from 66.4 ± 8.5 to 40.8 ± 9.0 mV
(P < 0.005), and
pHi decreased from 7.3 ± 0.05 to 7.1 ± 0.1 pH units (P < 0.03). The rate of pHi decrease
(dpHi/dt)
was 1.20 ± 0.29 pH units/min. When
Na+ was returned to the bath,
V3,
V1, and
pHi recovered fully
(segment def). The
pHi recovery was typically
preceded by a transient acidification (segment
de), and the initial rate of
pHi recovery was 1.25 ± 0.2 pH
units/min. The same protocol was then repeated in the presence of NE.
As can be seen in Fig. 5,
right, removal of bath
Na+ in the presence of NE caused
V3 to
hyperpolarize and
V1 to depolarize. pHi transiently alkalinized before
it decreased to a stable, more acidic value than control
(segment a'b'c').
These changes were completely reversed on restoring bath
Na+ to normal with
pHi transiently acidifying before
it recovered (segment
d'e'f'). In the presence of NE, the
initial rate of pHi increase on
readdition of bath Na+ was
1.07 ± 0.13 pH units/min. In paired experiments on the
same tubules, similar to that of Fig. 5, the peak depolarization of V1 and the rate
of pHi decrease on removal of bath
Na+ were significantly smaller in
the presence of NE. Also, the rate of
pHi increase on readdition of bath
Na+ was significantly less in the
presence of NE. These results are summarized in Table
3 and indicate that NE probably inhibits Na+-(HCO3)n
cotransport.
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3)n
cotransporter. The aNai is
expected to decrease also for the same reason. However, this decrease
in pHi and
aNai may in turn accelerate Na+ uptake from the lumen, driven
by an enhanced Na+ gradient across
the luminal membrane. To prevent the contribution of luminal
Na+ transport from affecting the
changes in aNai, pHi, or membrane PDs caused by
basolateral
Na+-(HCO3)n
cotransport, we removed bath Na+
in the absence of luminal Na+ and
substrates. Under these conditions (no luminal
Na+ or substrate), removal of bath
Na+ depolarized
V1 by 17.0 ± 2.5 mV (n = 7) and
V2 by 19.6 ± 2.6 mV (n = 5), hyperpolarized
V3 by 3.9 ± 1.2 mV (n = 8), and decreased aNai by 5.1 ± 0.8 mM
(n = 5).
pHi also decreased by 0.21 ± 0.02 pH units (n = 3), at an initial
rate of acidification of 0.53 ± 0.05 pH units/min. All the changes
were completely reversed on readdition of
Na+ to the bath. In the presence
of NE in the bath, removal of basolateral Na+, in the continued absence of
luminal Na+ and substrates, caused
a smaller depolarization of
V1 (11.0 ± 3.2 mV, n = 7;
P < 0.01) and a smaller
depolarization of
V2 (15.2 ± 3.5 mV, n = 7;
P < 0.03). The hyperpolarization of
V3 (4.0 ± 1.3 mV; n = 8) was not significantly
different from that in the absence of NE. The decrease in
aNai (4.0 ± 1.3 mM,
n = 8) and
pHi (0.14 ± 0.01 pH units,
n = 3), as well as the rate of
decrease of pHi (0.39 ± 0.04 pH units/min), were also significantly smaller in the
presence of NE. The results of these experiments confirm the same
observations as in the presence of luminal sodium and substrates and
further demonstrate that the activity of
Na+-(HCO3)n
cotransport is diminshed by NE.
IN THE PRESENCE OF SITS AND THE ABSENCE OF LUMINAL SODIUM AND
SUBSTRATES.
The effect of NE on
Na+(HCO3)n
cotransport has thus far been demonstrated by smaller changes, caused by removal of basolateral Na+,
primarily on V1,
and the rate of change of pHi or
aNai. If these changes were
indeed caused by an effect of NE on
Na+-(HCO3)n
cotransport, then blocking this cotransporter by SITS should
1) greatly reduce the changes in
membrane PDs, aNai,
and/or pHi, as well as the
rate of change of pHi or
aNai; and 2) abolish any significant
difference in the presence or absence of NE caused by removal of
Na+ from the bath. To check this
hypothesis, we removed basolateral Na+ in the presence and absence of
NE after pretreatment of the tubule with SITS. In experiments on four
tubules and after pretreatment with SITS, removal of bath
Na+ in the absence of NE caused a
depolarization of
V1 of 8.3 ± 4.3 mV, a depolarization of
V2 of 14 ± 4.0 mV, and a hyperpolarization of
V3 of 4.7 ± 2.6 mV. pHi decreased by 0.11 ± 0.007 pH units at a rate of 0.26 ± 0.02 pH units/min. The
changes in V1,
V2, V3, and
pHi, caused by removal of bath
Na+ after pretreatment with SITS,
are smaller than the respective changes in membrane PDs or
pHi in the absence of SITS
(compare in the presence of luminal sodium and
substrates), as is expected from the inhibition of
Na+-(HCO3)n.
However, these changes were not significantly different
(P > 0.05) from those caused by
removal of bath Na+ in the
presence of NE (also after treatment with SITS) where V1 depolarized by
10.0 ± 2.7 mV,
V2 depolarized by
16 ± 1.1 mV, and
V3 hyperpolarized
by 5.2 ± 2.6 mV. pHi decreased
by 0.08 ± 0.01 pH units, and the rate of
pHi decrease was 0.29 ± 0.04 pH units/min. The results of these experiments demonstrate that
inhibition of
Na+-(HCO3)n
cotransport (with SITS in this case) abolishes any significant effect
of NE on membrane PDs, pHi, or
aNai caused by removal of bath Na+. This observation is
consistent with an NE effect mediated by Na+-(HCO3)n
cotransport.
IN THE ABSENCE OF CARBON DIOXIDE/BICARBONATE.
The contribution of
Na+-(HCO3)n
cotransport (and other HCO3-dependent
mechanisms) to transport in the proximal tubule can also be abolished
or greatly reduced by removal of
CO2/HCO3
from the external solutions. In the absence of
CO2/HCO3,
removal of bath Na+ is expected to
cause similar changes to those during inhibition of
Na+-(HCO3)n
cotransport by SITS, namely, 1)
smaller changes in membrane PDs,
pHi, and/or
aNai, compared with control in
the presence of
CO2/HCO3; 2) no significant differences
between the presence or absence of NE caused by removal of
Na+ from the bath, if the NE
effect is mediated through
Na+-(HCO3)n
cotransport. To check this, tubules were exposed bilaterally to a
CO2/HCO3-free solution (HEPES buffered, solution
7). In the absence of
CO2/HCO3, removal of bath Na+ did not cause
significant changes in
V1 and
V2 (as opposed to the large depolarization observed in the presence of
HCO3) and hyperpolarization of
V3 of 3.4 ± 0.5 mV (P < 0.05, n = 4). The
aNai decreased by 8.7 ± 1.9 mM (n = 3), whereas
pHi remained unchanged. These
changes are significantly smaller than the respective changes caused by
removal of bath Na+ in the
presence of
CO2/HCO3
(compare in the presence of luminal sodium and substrates and Table 3).
In the presence of NE and still in the absence of
CO2/HCO3, removal of bath Na+ did not
significantly change
V1 and
V2, and
V3 hyperpolarized by 4.1 ± 0.4 mV. The
aNai decreased by 8.1 ± 1.2 mM, and pHi decreased by 0.05 ± 0.03 pH units. These changes were not statistically different in
the presence or absence of NE. These findings are also consistent with
an effect of NE on Na+-(HCO3)n
cotransport.
Effects of lowering bath
[HCO3] from 10 to 2 mM on membrane PDs,
pHi, and/or
aNai in the presence and
absence of NE. To further examine a possible effect
of NE on
Na+-(HCO3)n
cotransport, we lowered bath [HCO3] from 10 to 2 mM at
constant PCO2 in the presence and
absence of NE while monitoring
V3,
V1,
pHi, or
aNai. Lowering bath
[HCO3] will also lower
bath pH from 7.5 to 6.8. This maneuver is expected to drive
Na+-(HCO3)n
cotransport and cause changes in membrane PDs,
pHi, and
aNai qualitatively similar to
those caused by removal of basolateral
Na+. The predicted changes
attributed to
Na+-(HCO3)n
cotransport in this case are a depolarization of
V1 and a decrease
in pHi and
aNai.
EFFECT ON INTRACELLULAR PH.
As can be seen in Fig. 6, lowering bath pH
from 7.5 to 6.8 (solution 6) caused
a small depolarization in
V3, a large
depolarization of
V1, and a
substantial decreased in pHi
(segment abc). Returning bath pH to
normal (solution 1) resulted in
complete recovery of V3,
V1, and
pHi (segment
cde). Exposing the tubule to NE in the bath caused
the usual small depolarization of
V1 and a small
increase in pHi
(segment ea'). In the presence
of NE, lowering bath pH again from 7.5 to 6.8 caused a small
depolarization of
V3, a
depolarization of
V1, and a
decrease in pHi
(segment a'b'c'). In the
presence of NE, the initial depolarization of
V1 was typically
followed by a partial repolarization. Although the decrease of
pHi in the presence of NE was not
statistically different from that in the absence of NE, both the peak
depolarization of
V1 and the rate of decrease of pHi were
significantly smaller in the presence of NE. Restoring bath pH to 7.5 caused complete recovery of
V3, V1, and
pHi (segment
c'd'e'). During recovery in the presence of NE, the hyperpolarization of
V1 and the rate
of increase of pHi
(dpHi/dt)
were also significantly smaller than the respective changes in
V1 and
dpHi/dt
in the absence of NE. The results of this and similar experiments are
summarized in Table 4.
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|
3] on
aNai and membrane PDs in the
presence and absence of NE. As discussed in
Predictions, lowering bath
[HCO3], and consequently bath pH, is expected to decrease
aNai and depolarize
V1 resulting from
the efflux of Na+ and net negative
charge via the electrogenic
Na+-(HCO3)n
cotransporter. In our experiments, lowering bath pH caused a small
transient depolarization of
V3, a large
depolarization of
V1, and a fast
and sustained decrease in aNai
of 3.1 ± 0.3 mM (n = 8). In the
presence of NE, lowering bath pH caused a small transient decrease in
aNai. The maximal decrease in
aNai in the presence of NE
averaged 1.2 ± 0.6 mM (n = 8). Both the initial depolarization of
V1 and the
decrease in aNai on decreasing bath pH are attributed to the activity of
Na+-(HCO3)n
cotransport (9) and are significantly smaller in the presence of NE
(P < 0.02).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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General
In the proximal tubule, a major reabsorptive site in the nephron, the same pathways that regulate pHi contribute to net acid-base transport and HCO3 reabsorption across the tubule. This latter process is believed to involve two discrete steps: H+ efflux across the luminal
membrane and HCO3 efflux across the
basolateral membrane (for a review, see Ref. 18). Previous work on the
Ambystoma proximal tubule has
demonstrated that at least three main transport mechanisms are involved
in pHi regulation in this segment.
At the luminal membrane, Na/H exchange (8) and Na-substrate (in this
case, lactate) cotransport (28) are generally accepted as the two main
mechanisms responsible for acid extrusion. At the basolateral membrane,
Na+(HCO3)n
is most likely the predominant transporter that extrudes
HCO3 from the cell (9). In addition to
regulating pHi, these mechanisms
are responsible for a major portion of
Na+ and
HCO3 uptake across the tubular
epithelium in amphibian, as well as mammalian proximal tubules.
The data in the present study indicate that NE has a direct effect on
cellular H+ (or
HCO3) transport in the
Ambystoma proximal tubule. NE is
expected to have similar effects in the mammalian proximal tubule
considering that many transport mechanisms in this segment, including
Na+-(HCO3)n
cotransport, were initially identified in the
Ambystoma tubule. In the isolated
perfused proximal tubule, addition of NE to the bath caused an increase
in pHi, which was readily reversed
on removal of NE. This increase in pHi is a result of a direct action
of NE on the proximal cell, because the isolated tubule preparation
eliminates any changes caused by hemodynamic or other humoral factors.
In addition, this preparation preserves the integrity and polarity of
the epithelium. To our knowledge, the effect of NE on
pHi was never before monitored in
the intact kidney proximal tubule epithelium.
Whereas the steady-state effect of NE on
V1 was a small
and an often transient depolarization, its effect on
pHi was a sustained and
substantial increase. A similar small depolarization of
V1 and
V3 and an
increase in pHi were also caused
by isoproterenol, suggesting that the effect of NE may be mediated
through -receptor activation. An 
-adrenergic effect, however,
cannot be ruled out, since NE was not applied in the presence of an
-receptor antagonist.
These consistent effects of NE on membrane PDs and
pHi are undoubtedly caused by a
change in cellular transport (primary or secondary) pertaining to
H+. However, it is not possible to
conclude, based only on steady-state measurements, the effect on a
specific transport mechanism. For example, the sustained increase in
pHi could be due to activation of
H+-extruding mechanism(s) (such as
Na/H exchange) or an inhibition of acid-loading mechanism(s)
[such as
Na+-(HCO3)n
cotransport]. It could also result from differential effects on
both acid-loading and acid-extrusion mechanisms. For these reasons, we
designed this study to examine which specific transporters were
affected by NE.
Evidence Against Involvement of Na/H Exchange or Luminal Na-Substrate (Lactate) Cotransport
Several studies have reported that catecholamines interact with Na/H exchange in the proximal (tubule) cell. Nord and co-workers (22), using binding studies on the isolated proximal cells of the rabbit, suggested that the NE effect is elicited via2-adrenoreceptors and is
mediated through an effect on luminal Na/H exchange. Gesek and
Schoolworth (16) showed that -agonists stimulate EIPA-sensitive Na+ uptake in tubule suspensions.
Other studies attribute the effects of adrenergic catecholamines on
Na+ transport to stimulation of
Na-K-ATPase (4, 5). Our data do not support an effect of NE on luminal
Na/H exchange. We assayed for luminal Na/H exchange by removal of
luminal Na+ and substrate, which
resulted in an acute intracellular acid load as would be expected from
inhibition (or reversal) of luminal Na+-dependent acid extruding
mechanisms including Na/H exchange. Selective activation of Na/H
exchange by addition of luminal
Na+ resulted in an increase in
pHi, which was not different, and neither was the rate of increase of
pHi in the absence or presence of
NE (see Fig. 2). In a previous study (2), we used
Na+-sensitive microelectrodes to
assess the effect of NE on Na/H exchange. With the same protocol for
removal and readdition of luminal
Na+, as described above for the
pHi measurements, NE did not cause any change in the amount or rate of intracellular
Na+ recovery that can be
attributed to luminal Na/H exchange.
The Ambystoma proximal tubule is also
reported to have a basolateral Na/H exchanger (8). In our study, two
lines of evidence indicate that NE apparently does not affect this
mechanism. First, in the presence of SITS, the changes in
pHi and
aNai caused by removal and
readdition of basolateral Na+ were
not significantly different in the presence or absence of NE. Second,
the NE effects on pHi and
aNai caused by removal and
readdition of basolateral Na+ were
also abolished in the absence of
CO2/HCO3. Under both conditions, SITS and absence of
CO2/HCO3, the decreases in pHi and
aNai induced by basolateral Na+ removal are mediated at least
in part by basolateral Na/H exchange and were not affected by NE.
Another potent acid-extruding mechanism in the
Ambystoma proximal tubule is
Na-monocarboxylate cotransport at the luminal membrane (21, 28). In the
Ambystoma proximal tubule, luminal Na-lactate cotransport coupled to basolateral
H+-lactate cotransport results in
net uptake of Na+ and extrusion of
H+ much like a lactate-dependent
Na/H exchange. Whereas some studies indicated that -adrenergic
activation could possibly lead to stimulation of Na-substrate
cotransport (20), including Na-lactate, our studies do not show an
effect of NE on this transporter.
pHi changes caused by removal and
readdition of luminal substrates were not different in the presence or
absence of NE. Similarly, we have also shown that changes in the
activities of intracellular Na+,
caused by removal and readdition of luminal substrates, were not
different in the presence or absence of NE (2). We can therefore
conclude that NE does not significantly affect Na/H exchange or
Na-substrate cotransport in this preparation.
Evidence for Involvement of
Na+-(HCO3)n
Cotransport
3 transport is involved in
mediating the cellular effects of NE. In our experiments, steady-state
changes in aNai, pHi, and membrane PDs induced by
basolateral NE were completely abolished in the absence of
CO2/HCO3.
Moreover, SITS, a well-known inhibitor of most
HCO3-transporting mechanisms also
abolished the NE effects on pHi,
aNai, and membrane PDs. In the
Ambystoma proximal tubule, the major HCO3 transport mechanism is an
electrogenic Na+-(HCO3)n
cotransporter located at the basolateral membrane (9). This transporter
was recently cloned (25) from RNA isolated from the
Ambystoma proximal tubule. The model
for this transporter predicts that alterations in bath
[HCO3] or
[Na+] should produce
specific changes in
V1,
pHi, and
aNai, as discussed in
RESULTS. These predictions were tested
in this study and were used to confirm that NE reduces the activity of this transporter. Our evidence for an effect of NE on
Na+-(HCO3)n
cotransport is that removal of bath
Na+ in the presence of NE caused
1) a decrease in
pHi which was significantly slower
than in its absence, 2) a smaller
decrease in aNai on removal of
bath Na+ and
3) a depolarization of
V1 in the
presence of NE which was less than in its absence. A major part of the
depolarization of V1 on removal of
basolateral Na+ is due to the
electrogenic transport of Na+ and
HCO3. In fact, removal of bath
Na+ to induce a depolarization was
the assay used in cloning the Na+(HCO3)n
cotransporter from Ambystoma proximal
tubule (25). It is important to note that, although addition of NE to
the bath did not cause a smaller
pHi decrease on removal of bath
Na+, the rate of
pHi decrease was significantly
reduced (see Table 3). Similarly, the rate of
pHi increase on readdition of bath Na+ was reduced in the presence of
NE.
To further confirm that NE was inhibiting
Na+-(HCO3)n
cotransport as assayed by removal of bath Na+, we repeated this maneuver in
the presence of SITS or in the absence of
CO2/HCO3.
Blocking the transporter in both cases (SITS or removal of
CO2/HCO3) greatly reduced the changes in
V1,
pHi, and
aNai induced by removal of
basolateral Na+ and abolished any
differences in these parameters due to the presence of NE. These
findings are consistent with an effect of NE on
pHi that is primarily mediated
through
Na+-(HCO3)n
cotransport.
The second line of evidence to confirm that NE affects
Na+-(HCO3)n
cotransport is that lowering bath
[HCO3] from 10 to 2 mM at
constant PCO2 in the presence of NE
caused 1) a smaller depolarization
of V1 compared
with that in the absence of NE, 2) a
smaller decrease in aNai, and
3) a slower decrease in
pHi. As in the case of removal of
basolateral Na+, although the
magnitude of pHi decrease was not
different in the presence or absence of NE, the rate of
pHi decrease was greatly reduced
in the presence of NE. Similarly, the rate of
pHi recovery on restoring bath
HCO3 to normal was less in the
presence of NE. In both cases, a significantly smaller
H+ flux is mediated through
Na+-(HCO3)n
cotransport in the presence of NE.
It is unlikely that NE affected other
HCO3 -dependent,
pHi-regulating mechanisms, such as
the
Cl/HCO3
exchanger present in the mammalian proximal tubule. Inhibition of
Cl/HCO3
exchange by NE would cause an increase in
pHi, as observed in this study.
However, unlike the mammalian proximal tubule, the
Ambystoma proximal tubule seems to
have no or very little activity of
Cl/HCO3
exchange (9). Moreover, the assay employed in this study, namely,
removal and readdition of basolateral
Na+ would induce changes in
V1 (and in
aNai) that are caused
primarily by
Na+(HCO3)n
cotransport. The inhibition by NE of these changes in
V1 (and in
aNai), as shown in Fig. 5,
indicate a specific effect on
Na+-(HCO3)n
cotransport, since the
Cl/HCO3
exchanger is electroneutral.
In a previous study, we showed that NE stimulated Na-K-ATPase in the
proximal (tubule) cell (2). In the same study, we demonstrated that the
increased activity of the pump in the presence of NE was not due to a
change in aNai. Activation of
Na-K-ATPase by NE could conceivably lead to inhibition of
Na+-(HCO3)n
cotransport, due to decreased availability of intracellular
Na+. We have not examined whether
the NE-induced decrease in
aNai, as reported earlier (2),
is enough to cause inhibition of
Na+-(HCO3)n
cotransport, and the
Km
for Na+ is not known.
Alternatively, it is possible that the increase in
pHi, resulting from inhibition of
Na+(HCO3)n
cotransport by NE, leads to activation of Na-K-ATPase, which is a
pHi-dependent transporter (10).
This issue was not addressed in this study.
Few studies have investigated the regulation of
Na+-(HCO3)n
cotransport. Among these, one study (29) reported that this transporter
is sensitive to pHi through an
internal modifier site with optimal activity between
pHi 7.0 and 7.4. Others (26) have
reported that
Na+(HCO3)n
is inhibited by cAMP and calmodulin but is stimulated by protein kinase
C. The inhibition by NE may represent one of the humoral factors that
contribute to regulation of this transporter. It is interesting to note
that NE is also reported to increase cAMP, which makes cAMP a likely
candidate for a second messenger. The question of the second messenger, however, needs to be addressed further.
In conclusion, the results of this study demonstrate that NE directly
affects pHi in the
Ambystoma proximal tubule, leading to
an increase in pHi. Neither the
activities of luminal Na/H exchange nor Na-substrate (lactate)
cotransport is affected by NE. On the other hand, NE inhibits
Na+-(HCO3)n
cotransport at the basolateral membrane.
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ACKNOWLEDGEMENTS |
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This work was partly supported by American Heart Association (Louisiana Affiliate) Grant-in-Aid LA-96-GS-18.
