Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
Submitted 13 January 2003 ; accepted in final form 8 April 2003
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ABSTRACT |
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bicarbonate; carbon dioxide; intracellular pH; acid-base; volume reabsorption; out-of-equilibrium solutions
PCO2 also influences
reabsorption in the proximal
tubule, which is responsible for reabsorbing
80% of the filtered
. Cogan
(11) found that acute
respiratory alkalosis (lowering plasma PCO2 by 20 mmHg)
decreased
reabsorption
(JHCO3) by
25% in free-flow micropuncture
experiments in rats. Moreover, Sasaki et al.
(23) found that although acute
metabolic alkalosis (i.e., an increase in basolateral
[
] and pH with no change in
PCO2) inhibits
reabsorption, raising
PCO2 sufficiently to normalize basolateral pH (i.e.,
producing isohydric hypercapnia) reverses the inhibition.
The above-mentioned studies clearly demonstrate that an acute increase in
PCO2 and/or the accompanying decrease in basolateral pH
leads to an increase in JHCO3. Conversely, an
acute increase in basolateral []
and/or the accompanying increase in basolateral pH leads to a decrease in
proximal
reabsorption. However,
the equilibrium that interrelates CO2,
, and H+ had previously
made it impossible to determine the importance of basolateral CO2
per se and of
per se,
independently of basolateral pH, in controlling proximal
reabsorption. In 1995, our
laboratory introduced the concept of exploiting the relatively slow
equilibrium CO2 + H2O
H2CO3 to generate out-of-equilibrium (OOE)
solutions (29). The approach
is to use a rapid-mixing technique to combine two solutions with different
characteristics. With a judicious choice of the above parameters, it is
possible to generate OOE solutions with virtually any combination of
[CO2], [
], and pH, at
least within a range of several pH units that encompasses all pH values of
pathophysiological interest. In the present study, we exploit this OOE
approach to determine the extent to which
reabsorption by the S2 segment of
the rabbit proximal tubule is affected by the isolated removal of
from the basolateral or
"bath" solution (keeping bath PCO2 fixed at
5% and bath pH fixed at 7.4), or by the isolated removal of CO2
(keeping bath
fixed at 22 mM and
bath pH fixed at 7.4). Our results, which are the first to examine an isolated
change in CO2 or
on any
physiological parameter in a vertebrate cell, show that the isolated removal
of basolateral
stimulates
reabsorption, whereas the isolated
removal of basolateral CO2 inhibits
reabsorption.
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METHODS |
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The Yale Animal Care and Use Committee approved the following procedures.
Pathogen-free, female New Zealand White rabbits, obtained from Covance
(Denver, PA) and weighing 1.42.0 kg, were euthanized by a single
overdose of 3 ml (20 mg) of intravenous pentobarbital sodium. The kidneys
were then removed, transferred to a dissection dish, sliced into transverse 1-
to 1.50-mm-thick sections, and further dissected by hand to yield individual
midcortical1 S2
segments2
1.51.7 mm in length. We perfused these tubules in a manner similar to
that originally described by Burg and co-workers
(7), using several
modifications described by Baum et al.
(1). In particular, we doubly
cannulated the tubule (i.e., using holding, perfusion, and exchange pipettes)
at the perfusion end of the tubule and singly cannulated the tubule (i.e.,
using a holding pipette as well as a calibrated collection pipette with a
volume of
55 nl) at the collection end. The collection pipette was coated
with cured Sylgard (Sylgard 184 Silicone Elastomer Kit, Dow Corning, Midland,
MI) and filled with liquid Sylgard (the silicone elastomer base) at the distal
50 µm of the pipette. About 80% of the tubules (depending on how they
landed on being transferred to the chamber) were cannulated and perfused
orthograde. For these, typically 0.10.2 mm of the proximal part of the
proximal convoluted tubule was held in the perfusion pipette, and typically
0.20.3 mm of the proximal straight tubule was held in the collection
pipette. The mean length of perfused tubules in our
JV/JHCO3 experiments (where
JV is the fluid reabsorption rate), as measured with an
eyepiece micrometer, was 1.33 ± 0.06 mm (n = 16 tubules),
representing the distal end of the proximal convoluted tubule. The mean
luminal perfusion rate in our
JV/JHCO3 experiments was
13.0 ± 0.6 nl/min. We superfused the basolateral (i.e., bath) side of
the tubule with a solution at 37°C and flowing at 7 ml/min.
Solutions and Experimental Protocol
Dissection and luminal perfusion. The compositions of
solutions 14 (Table
1) were similar to those described by Baum et al.
(1), except for changes
required in the composition of solution 4 to make it compatible with
the OOE solutions (solutions 5 and 6). The tubules were
dissected in Hanks' solution (solution 1) at 4°C. Following
transfer of the tubule to the chamber filled with cold Hanks' solution, we
perfused the tubule lumen with a solution at 37°C buffered to pH 7.40 with
5% CO2/22 mM
(solution 2) and delivered by gravity. In experiments whereby we
measured JHCO3, this luminal solution also
contained dialyzed [3H]methoxyinulin (
30 µCi/ml).
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Warm-up period. After establishing luminal perfusion, we initiated
flow of a 37°C bath "warm-up" solution (solution 3)
that was buffered to pH 7.40 with 5% CO2/22 mM
. After a 20- to 30-min warm-up
period, we switched the bath solution from solution 3 to solution
4 (which was identical to our OOE solutions except that it contained both
5% CO2 and 22 mM
), or
to solution 5 or 6 (the OOE solutions). In any case,
coincident with the switch to the bath solution for the first data-collection
period, we used the calibrated collection pipette to remove and discard the
fluid that had accumulated in the holding pipette.
Data collection. We allowed the tubule to stabilize in the bath solution for the first data-collection period for 58 min. After this period, we again used the calibrated collection pipette to remove and discard the luminal fluid that had accumulated in the holding pipette and then began a series of three (or four) timed and calibrated collections. The first two (or three) were subsequently analyzed for [3H]methoxyinulin for use in the calculation of JV, and the third was analyzed for total CO2 for use in the calculation of JHCO3. We then usually switched to a different bath solution (solution 4, 5, or 6) for the second data-collection period. We repeated the entire procedure of allowing the tubule to stabilize for 58 min in the new solution and then performing three (or four) timed and calibrated collections.
OOE solutions: technical considerations. We generated OOE
solutions using the approach outlined in a previous paper
(29). In brief, we used a
dual-syringe pump (model 552222, Harvard Apparatus, South Holliston,
MA) to drive two 140-ml syringes (Monoject 140 ml, Sherwood Medical
Industries, Ballymoney, UK), one containing solution 5A (or
6A) and the other containing solution 5B (or 6B)
(Table 1; see
Fig. 1, A and
B). The A and B solutions each flowed at 3.5 ml/min, for
a total flow of 7 ml/min. Separate lengths of Tygon tubing (
-in. outer
diameter x
. inner
diameter, Norton Performance Plastics, Akron, OH) which has a relatively low
permeability to CO2, carried the outflow of each syringe to one of
two inlets of paired computer-actuated five-way valves (Eagle P/N
E41PP-00000, Clippard Instrument Laboratory, Cincinnati, OH). In
one position, these two valves, which have zero dead space, sent one pair of
solutions (e.g., solutions 5A and 5B) toward the chamber and
an alternate pair (e.g., solutions 6A and 6B) to a waste
recepticle; in the alternate position, the two valves reversed the
destinations of the two pairs of solutions. We actually used a series of four
such valve pairs, connected to one another in daisy-chain fashion, so that we
could switch among up to five bath solutions. All bath solutions, including
the ones that were not OOE, were delivered as described above by a pair of
syringes driven by a separate dual-syringe pump.
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The two outputs of the valve assembly, carrying solutions
5A+5B or 6A+6B to generate OOE solutions, or
carrying two identical streams of either solution 4 or solution
7, were carried by separate lengths of Tygon tubing to separate 22-cm
lengths of 15-gauge stainless steel tubing, which, in turn, were enclosed
within a jacket that was perfused with water by a circulating bath at
38°C. The downstream ends of the two lengths of stainless steel
tubing were connected to 5-cm lengths of Tygon tubing, which carried the
now-warmed pair of solutions to the two arms of a polypropylene T
(
x
x
. Tee P/N
636577, Cole-Parmer Instrument, Vernon Hills, IL). The base of the T
was connected to a length of Tygon tubing stuffed with nylon mesh (35-µm
nylon thread, Small Parts, Miami Lakes, FL) to generate turbulence and thereby
ensure adequate mixing of the two components of an OOE solution. The opposite
end of the tubing was situated at the entrance to the slot of the chamber (see
below); the total volume of the system from the point of mixing at the middle
of the T to the beginning of the chamber slot was 14 µl. From within the
valve assembly to the T, the two members of a pair of pieces of Tygon tubing
were of identical length. This precaution ensured that, following a switch in
the valve assembly, each member of the solution pair reached the T at the same
instant.
OOE solutions: theoretical considerations. Our OOE approach
exploits the slow, first equilibrium in the following reaction sequence
![]() | (1) |
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Figure 1C
summarizes the concentrations of CO2, H2CO3,
, and
as computed from Eqs.
13. Take the pure CO2 solution, for example. As
indicated by the top half of the left column on
Fig. 1C, solution
5A contained 2,210 µM CO2 (which corresponds to a
gas mixture of 10% CO2) at a pH of 5.40 (2 pH units below the
target pH of 7.40). Because the pK of the overall
equilibrium is
6.1, the [
] in
solution 5A was 441 µM. The top half of the
middle column of Fig.
1C shows, at the idealized instant of perfect mixing, the
result of combining solutions 5 and 6 in a 1:1 ratio. This
OOE pure CO2 solution contained 1,105 µM CO2 (which
corresponds to a gas mixture of 5% CO2) and
223 µM
; this
is
1% of the value in an
equilibrated 5% CO2/22 mM
solution. In principle, we could
have reduced the contaminating [
]
even further by lowering the pH of solution 5A below 5.40. The
bottom half of Fig.
1C provides a similar analysis for the pure
solution. Because solution
6B contained 44 mM total
at a
pH of 9.40 (2 pH units above the target pH of 7.40), the [CO2] in
solution 6B was 19.6 µM (which corresponds to a gas mixture of
0.09% CO2). Thus mixing this solution in a 1:1 ratio with
solution 6A initially yielded an OOE pure
solution containing nearly 22 mM
and 9.8 µM CO2
(which corresponds to a gas mixture of 0.04% CO2); this
[CO2] is <1% of the value in an equilibrated 5%
CO2/22 mM
solution.
Again, we could have reduced the contaminating [CO2] even further
by raising the pH of solution 6B above 9.40.
Our design criteria for the pure CO2 OOE solution (solution
5) were that solution 5B should be heavily buffered but have a
pH just high enough (i.e., 7.55) to drive the pH of the OOE solution to 7.40.
This combination of design features yields an OOE solution, as measured with a
PCO2 electrode (MI 720, Microelectrodes, Londonderry,
NH) at the end of the Tygon tubing that connects to the chamber, that is
indistinguishable from 5%. In our experience, attempting to generate a pure
CO2 solution by using a solution B with an extremely high
pH (e.g., pH 10) yielded an OOE solution with a [CO2] markedly
lower than the target value. We presume that rapid mixing of solutions
A and B creates short-lived microscopic pockets with a high
[CO2] (contributed by solution A) and a high
[OH] (contributed by solution B) that allows the
uncatalyzed reaction
to consume CO2 at a high rate.
Our design criteria for the pure
solution (solution 6)
were that solution 6A should be heavily buffered but have a pH just
low enough (i.e., 6.99) to drive the pH of the OOE solution to 7.40. This
approach yields an OOE solution that, when assayed as described in the
preceding paragraph, has a PCO2 indistinguishable from
zero. Attempts to generate a pure
solution by using a solution A with an extremely low pH (e.g., 5.00)
yielded an OOE solution with a [CO2] markedly higher than zero. We
presume that short-lived microscopic pockets with a high
[
] (from solution B) and a
low pH/high [H+] (from solution A) allowed the reaction
to raise [H2CO3] to such high levels that the
uncatalyzed reaction H2CO3
CO2 +
H2O produces CO2 at a high rate.
Because of the considerations discussed in the two previous paragraphs, we
suspect that it might be challenging to generate pure CO2 solutions
at an extremely alkaline target pH or pure
solutions at an extremely acidic
target pH.
Finally, it should be noted that we employed OOE solutions only in the bath. Although it is theoretically possible to generate OOE solutions for the lumen, the mixing of the A and B solutions would be extremely challenging, and the relatively slow transit of the fluid down the lumen would permit more extensive equilibration than we observe in the bath.
The chamber. The chamber (see
Fig. 1B), which was
machined out of polycarbonate, contained a channel 14-mm long x 2.5-mm
wide by 2.5-mm high. The bottom of the slot was formed by a coverslip. About
one-fourth of the way (measured from the solution inflow) along this slot was
the perfused tubule, with its long axis parallel to the axis of bath flow. The
chamber sat atop a heating plate (series 20 chamber and platform with integral
TS70B thermistor, Warner Instruments, Hamden, CT) that was clamped to a
temperature of 41°C. The combination of the prewarmed solution and the
heating plate yielded a bath temperature of 37.5°C (confirmed with a
miniature electronic temperature probe Digi-Sense with a type-K thermocouple,
Cole Parmer). A length of Tygon tubing from the T (see above) entered the
chamber through a port that was colinear with the long axis of the slot. As it
terminated at the entrance to the slot, the Tygon tubing had an inner diameter
of 1.6 mm, which was modestly less than the width of the chamber slot (2.5 mm)
to promote laminar flow along the slot and minimize the formation of dead
zones. Such dead zones would have permitted the equilibration of the bath
solution within these zones and, to some extent, thwarted our goal of
surrounding the tubule with an OOE solution. At the opposite end of the slot,
the bath solution was continuously sucked away. The height of the bath
solution within the slot was 1.2 mm.
We calculate that the time required for the newly mixed solution (7 ml/min)
to travel from the middle of the T to the tubule (total volume = 14 µl for
the tubing + 10 µl for 1/4 of the chamber slot = 24 µl) was 200 ms.
Given an extracellular CO2 concentration
([CO2]o) of 1,105
µM3 for
solution 5 at the instant of mixing (see middle column of
Fig. 1C, top
portion) and a rate constant of 0.08 s1, the
reaction CO2 + H2O
H2CO3
(Eq. 1) would consume CO2 and produce
H2CO3 at the rate of
88 µM/s. Thus after 200 ms
(see right column of Fig.
1C, top portion), the preceding reaction would
consume 17.7 µM CO2 or
1.6% of the original 1,105 µM
CO2. Simultaneously, the reaction
(Eq. 2) would produce 17.7 µM
, which represents 0.08% of the
present in an equilibrated 5%
CO2/22 mM
solution. As
a result, pH would fall by a trivial amount, [CO2] would fall to
1,087 µM CO2 (which corresponds to a gas mixture of 4.92%
CO2), and [
] would rise
by a trivial amount.
Conversely, given an extracellular
concentration
(
) of 22 mM
for solution 6 at the instant of mixing, the initial
[H2CO3] would be 2.765 µM (see middle column
of Fig. 1C, bottom
portion). Because the reaction H2CO3
CO2 + H2O (Eq. 1) has a rate constant of 32
s1, the preceding reaction would consume
H2CO3 and produce CO2 at the rate of
88
µM s1. Thus, after 200 ms (see right
column of Fig. 1C,
bottom portion), the preceding reaction would produce 17.7 µM
CO2, or
1.6% of that contained in an equilibrated 5%
CO2/22 mM
solution. As
a result, pH would rise by a trivial amount,
[
] would fall by a trivial amount,
and [CO2] would rise to 27.5 µM (which corresponds to a gas
mixture of 0.12% CO2). In a parallel series of experiments, we used
a pair of liquid-membrane pH-sensitive electrodes to behave as a
double-barreled PCO2 microelectrode
(28). We used this combination
to monitor PCO2 in the chamber slot at room temperature
under conditions mimicking those in the kidney-tubule experiments. While
generating a pure
solution, we
found that the [CO2] in the chamber slot was indistinguishable from
zero (not shown). However, this gratifying result was achieved only after
employment of the above considerations in chamber design.
Osmolality and pH of solutions. The osmolality of all solutions was measured using a vapor-pressure osmometer (model 5100C, Wescor, Logan, UT). The osmolality was adjusted, if necessary, to 300 ± 2 mosmol/kgH2O by adding either water or NaCl. The pH was adjusted to the value indicated in Table 1 using either HCl or NaOH. All solutions were made using water filtered through a Milli-Q biocel system (Millipore, Bedford, MA).
Measurement of JHCO3 and JV
We measured JHCO3 and JV
using an approach similar to that used by McKinney and Burg
(16). We assayed total
CO2 in aliquots of the perfusate and collected fluid using a WPI
NanoFlo device (World Precision Instruments, Sarasota, FL) to measure the
-dependent decrease in the
fluorescence at 340 nm of NADH as phosphoenolpyruvate carboxylase
(from "plant") and malate dehydrogenase (from porcine heart),
respectively, catalyzed the following two reactions
(13,
26)
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We computed JV from Vi, L, and the
concentration of [3H]methoxyinulin in the perfusate (Co)
and collected luminal fluid (Ci)
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Measurement of Intracellular pH
We calculated intracellular pH (pHi) from the fluorescence
excitation ratio of BCECF using the approach outlined previously for
osteoclasts (21). We loaded
perfused tubules with the dye by exposing them from the bath at room
temperature for 5 min to solution 7 containing 10 µM of
BCECF-AM (B-1170, Molecular Probes, Eugene, OR). The microscope was a Zeiss
IM-35 inverted microscope, equipped with a x40/numerical aperture 0.85
objective and apparatus for epiillumination. The light source in the
fluorescence experiments was a 100-W tungsten halogen lamp. Using a system of
dual filter wheels (Ludl Electronic Products, Hawthorne, NY), one wheel
carrying chromic filters and the other carrying neutral-density filters, we
alternately excited the entire field with light in wavelengths of 495 ±
5 and 440 ± 5 nm (Thermo Oriel, Stratford, CT), using the
neutral-density filters to equalize, as nearly as possible, the intensity of
incident light at the two wavelengths. The emitted fluorescent light, after
passing through a 510-nm long-pass dichroic mirror and a 530-nm long-pass
filter in the filter cube beneath the turret, was amplified by an image
intensifier (KS-1381 intensifier, Video Scope, Dulles, VA), before it was
captured with a charge-coupled-device camera (CCD 72, Dage M.T.I., Michigan
City, IN).
To limit both photobleaching of the dye and photodynamic damage of the
tubule cells, we limited illumination to 370 ms for the 490-nm light
(producing an I490 image), followed immediately by
370 ms for the 440-nm light (producing an I440
image). This pair of excitations was repeated at intervals varying from 6 to
20 s; between the 490- and 440-nm excitations, we kept the tubule in the dark
by closing the shutter on the filter wheel. Each cycle of excitations had the
following protocol.The filter wheel was rotated, under computer control, to
put the 490-nm filter in the light path. With the shutter of the filter wheel
still closed, we snapped an image to be used for background subtraction and
then opened the shutter. After a delay of 100 ms to allow for stabilization of
the shutter, we collected and averaged four successive video frames using an
image-processing board (DT3155, Data Translation, Marlboro, MA). The shutter
was then closed and, after storage of the averaged I490
image, the 440-nm filter was rotated into position and the
I440 image was obtained in the same way. Finally, we
subtracted the average pixel intensity of the matched background image (see
above) from the I490 and I440 images.
Software developed in our laboratory, using the Optimas (Media Cybernetics,
Silver Spring, MD) platform, controlled all aspects of data acquisition and
real-time storage to the hard disk of an Intel-based computer running
Microsoft Windows 98SE. This software also allowed us to monitor
I490/I440 fluorescence-excitation
ratios for selected areas of interest during the experiments. After the
experiment, we transferred the data to a CD-ROM for later analysis using
Optimas-based software. For the analysis, we outlined two areas of interest,
each of which typically represented 9% of the tubule length. In BCECF-loaded
tubules, the mean I490 signal was typically
3,000-fold greater than the I490 signal in tubules
not loaded with BCECF. The sum of the I490 values of the
pixels in the area of interest was divided by the sum of the corresponding
I440 values. The resulting ratio is strongly dependent on
pHi but relatively insensitive to other factors, such as dye
concentration.
We converted the I490/I440 ratios
to pHi values using the high-K+/nigericin technique of
Thomas et al. (27), as
modified by Boyarsky et al.
(4), to permit a one-point
calibration at pHi 7.00 for each experiment. At the end of each
experiment, we introduced into the bath a pH 7.00 high-K+/nigericin
solution (100 mM KCl, 32.2 mM HEPES-free acid, 1 mM CaCl2, 1.2 mM
MgSO4, titrated to pH 7.00 with 32.8 mM
N-methyl-D-glucamine) that drives pHi toward
7.00. Potential limitations of this approach are discussed elsewhere
(8). The
I490/I440 ratios of the entire
experiment were normalized by dividing them by the
I490/I440 ratio corresponding to
pHi 7.00, and we then calculated pHi using the following
equation (4)
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Because the nigericin used in the calibration can stick to the tubing and valves and later dissociate and enter nominally nigericin-free solutions in later experiments (2, 22), at the end of each pHi experiment we routinely flushed ethanol (70% in water) and then water (filtered through a Milli-Q biocel system) through the tubing and valves that carried the nigericin to the chamber, as well as the chamber itself.
Data Analysis
Statistical analyses were performed using the Analysis Toolpack of Microsoft Excel. Two sets of data were considered significantly different if the P value of the paired or unpaired t-test was <0.05. Results are given as means ± SE, with the number of tubules (n) from which it was calculated.
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RESULTS |
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In the JHCO3/JV
experiments described in the next two subsections, we constantly perfused the
lumen of the tubule with an equilibrated 5% CO2/22 mM
at pH 7.40
(Table 1, solution 2)
containing [3H]methoxyinulin. We usually collected luminal samples
for data analysis during two periods, each characterized by different bath
solutions that were delivered from different pairs of syringes, driven by
different syringe pumps. To verify that these two data-collection periods are
equivalent, we performed control experiments in which we delivered the
standard equilibrated
solution (solution 4) for each of the two data-collection periods,
first from one pair of syringes driven by one pump, and then from another pair
of syringes driven by another pump. As summarized in
Fig. 2, we observed no
significant difference between the two data-collection periods for either
JHCO3 or JV.
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|
A comparison of the open and hatched bars on
Fig. 3, A and
B, illustrates the effect of using the OOE approach to
remove basolateral while holding
bath [CO2] fixed at 5% and holding bath pH fixed at 7.40. Compared
with those exposed to the equilibrated 5% CO2/22 mM
solution at pH 7.40 (open bars),
tubules exposed to the corresponding pure CO2 solution (hatched
bars) had a mean JHCO3 that was >25% higher
(96.5 vs. 76.5
pmol·min1·mm1).
JV was
61% higher (1.03 vs. 0.64
nl·min1·mm1).
Viewed differently, the presence of basolateral
reduces the reabsorption of both
and fluid.
Considering only the data for the equilibrated 5% CO2/22 mM
solution at pH 7.40 (open bars in
Fig. 3, A and
B), our mean JHCO3 value
of 76.5
pmol·min1·mm1
is similar to the value of 52.3
pmol·min1·mm1
reported by Baum et al. (1) for
midcortical and juxtamedullary proximal convoluted tubules. Similarly, our
mean JV value of 0.64
nl·min1·mm1
is similar to the value of 0.72
nl·min1·mm1
reported by Baum et al. and is also similar to the value of
0.5
nl·min1·mm1
obtained by Quigley et al.
(20) on superficial S2
segments of rabbit proximal tubules.
Effect on JHCO3 and JV of Isolated Removal of Basolateral CO2
A comparison of the open and crosshatched bars in
Fig. 3, A and
B, illustrates the effect of using the OOE approach to
remove basolateral CO2 while holding bath
[] fixed at 22 mM and holding bath
pH fixed at 7.40. Compared with tubules exposed to the equilibrated 5%
CO2/22 mM
solution at
pH 7.40, those exposed to the corresponding pure
solution had a
JHCO3 that was nearly 40% lower (48.4 vs. 76.5
pmol·min1·mm1).
JV was nearly 25% lower (0.49 vs. 0.64
nl·min1·mm1).
Viewed differently, adding basolateral CO2 to a pure
solution enhances the reabsorption
of both
and fluid.
Effect on pHi of Isolated Removal of Either
Basolateral or
CO2
One would expect that tubules exposed to the different bath solutions
summarized in Fig. 3 would have
different values of pHi. Because such changes in pHi
could conceivably affect various solute transporters and thereby alter
JHCO3 and/or JV, we
examined the effect on pHi of switching the bath solution from 5%
CO2/22 mM at pH 7.40 to
the corresponding OOE solutions lacking either
or CO2.
Figure 4A shows an
experiment in which we used BCECF to monitor pHi in cells of an
isolated perfused S2 segment. Initially, the luminal and bath solutions were
buffered with HEPES (Table 1, solution 7). In a total of 38 tubules under these conditions, the
mean initial pHi was 7.19 ± 0.03. For two reasons, this
value is substantially higher than the values of 6.896.92 previously
reported by this laboratory for rabbit S3 segments
(9,
17). First, mean
pHi values steadily increase from the S3 to S2 to S1 segments
(14). Second, the luminal
solution in the present study contained lactate, which, presumably like
lactate in the salamander proximal tubule
(24) and acetate in the rabbit
S3 segment (18), produces a
intracellular alkalinization as it enters the cell with Na+ across
the apical membrane and exits with H+ across the basolateral
membrane.
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Switching the lumen to 5% CO2/22 mM
(solution 2) in
Fig. 4A caused an
abrupt and sustained decrease in pHi (segment ab),
reflecting the influx of CO2 across the apical membrane, the
intracellular reaction
,
and the continuous exit of
across
the basolateral membrane via the electrogenic Na-HCO3 cotransporter
(3). The mean pHi
under these conditions was 6.99 ± 0.02 (n = 38).
At point b we added equilibrated 5% CO2/22 mM
(solution 4) to the bath
in the continued presence of luminal
. On
one hand, we expect this maneuver to cause an abrupt pHi decrease
as the basolateral influx of CO2 leads to the intracellular
generation of H+ and
and to cause a sustained pHi decrease as the elevated intracellular
[
]
(
) promotes
the efflux of
through the
electrogenic Na-HCO3 cotransporter. On the other hand, based on
previous results from this laboratory in rabbit S3 segments
(9,
10,
17), we expect the addition of
basolateral
to
cause an increase in pHi for two reasons. First, the increase in
bath [
] would tend to slow net
efflux across the basolateral
membrane. Second, basolateral CO2 and/or
stimulates apical Na/H exchange
and H+ pumping. In our experiments, the alkalinizing tendencies
dominated (bc) as the mean pHi increased from 6.97
± 0.02 to 7.09 ± 0.04 (n = 18; P < 0.0002,
paired 2-tailed t-test). The latter value is also summarized by the
open bar in Fig. 4B.
The absence of a transient, CO2-induced acidification during
bc in Fig. 4A
probably reflects the limited time resolution of data acquisition.
Removing
from
the bath in Fig. 4A
caused a transient pHi increase due to the basolateral efflux of
CO2, followed by a relaxation of pHi (cd). At
point d, we switched the bath to the pure CO2 solution
(solution 5). The abrupt pHi decrease is the consequence
of the CO2 influx and formation of H+ and
. We expect the new steady-state
pHi to depend on the balance between enhanced
efflux (reflecting the increased
) via
electrogenic Na-HCO3 cotransport (which would tend to lower
pHi) and enhanced H+ extrusion if it is basolateral
CO2 that stimulates apical Na/H exchange and H+ pumping.
Under the conditions of our study, the acidifying influences dominated
(de) as the mean pHi fell from 7.02 ± 0.03 to 6.90
± 0.03 (n = 17; P < 0.001, paired 2-tailed
t-test). The latter condition, with one additional data point, is
also summarized by the hatched bar in Fig.
4B. Note that it is under this pure CO2
condition that
reabsorption was
maximal (see Fig.
3A).
Removing pure CO2 from the bath
(Fig. 4A) caused a
rebounding pHi increase (ef). At point f, we
introduced the pure solution
(solution 6) to the bath. We expect the increase in bath
[
] to inhibit
efflux via electrogenic
Na-HCO3 cotransport, or even to produce a net influx of
. Indeed, we observed a sustained
pHi increase (fg) as the mean pHi rose from
7.05 ± 0.06 to 7.33 ± 0.13 (n = 6; P <
0.02, paired 2-tailed t-test). The latter condition, with one
additional data point, is also summarized by the crosshatched bar in
Fig. 4B. Note that it
is under this pure
condition that
reabsorption was minimal (see
Fig. 3A).
Finally, removing the pure
solution from the bath causes pHi to drift toward its initial value
(gh).
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DISCUSSION |
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Removing Basolateral Stimulates
Reabsorption
As shown in Fig. 5, which is
a replot of the relationship between the JHCO3
data in Fig. 3A and
the pHi data in Fig.
4B, one way of interpreting the data from the present
study is that JHCO3 depends mainly on
pHi and that removing basolateral
enhances
reabsorption by lowering
pHi. Although the present data are consistent with this hypothesis,
the hypothesis suffers from three weaknesses.
|
First, the pHi-dependency model requires that decreasing
pHi from 7.1 to 6.9 would increase apical H+ extrusion
via the Na/H exchanger and H+ pump (see
Fig. 6) by 50%, whereas, at
least in the S3 segment of the rabbit proximal tubule, actual data show that
acid extrusion is remarkably insensitive to pHi changes in the
above pHi range, particularly in the absence of
(9,
10).
|
Second, the pHi-dependency model requires that decreasing
pHi from 7.1 to 6.9 would increase the basolateral efflux of
by 50% because changes in
basolateral
efflux must, in the
steady state, parallel changes in apical H+ extrusion. Although the
pHi dependence of the electrogenic Na-HCO3 cotransporter
(NBC) in the proximal tubule is unknown, one would expect that, if anything,
lowering pHi per se would inhibit NBC, just as lowering
pHi inhibits
exchange (5,
19,
25). For example, lowering
pHi from 7.1 to 6.9 at a fixed extracellular [CO2] of 5%
would lower the concentration of intracellular
, the ostensible substrate for NBC,
from
11 to
7 mM.
Finally, it is important to address the mechanism by which removing
basolateral lowers pHi.
Imagine that we start with equilibrated
in the
bath. Removing bath
should enhance
basolateral
efflux via NBC,
thereby lowering pHi. Note that enhancing the basolateral efflux of
is tantamount to enhancing
reabsorption. According to this
analysis, the fall in pHi is the result of enhanced
reabsorption rather than the cause
of it.
How then might the removal of basolateral
enhance
reabsorption? One possibility is
that the reduction in basolateral
[
] slows or even reverses the
backflux of
through the tight
junctions from the bath to the lumen. Another possibility, which is not
mutually exclusive, is that the reduction in basolateral
[
] enhances the net offloading of
at the extracellular face of
NBC.
Removing Basolateral CO2 Inhibits
Reabsorption
Given the relationship between JHCO3 and
pHi summarized in Fig.
5, the most tempting explanation for why removing basolateral
CO2 inhibits
reabsorption by 37% is that the removal of CO2 causes
pHi to increase, which in turn inhibits apical H+
extrusion. The present data do not rule out this hypothesis. However, if the
hypothesis is true, then we must explain how the pHi increase also
inhibits basolateral
efflux,
because as the cell approaches the new steady state, apical H+
extrusion and basolateral
efflux
must somehow fall in parallel. We expect that the rise in pHi per
se should, if anything, increase the basolateral efflux of
. Moreover, at a fixed
intracellular [CO2] ([CO2]i), a rise in
pHi ought to increase
, which
should further increase the basolateral efflux of
. On the other hand, the decrease
in bath [CO2] should lead to a fall in
[CO2]i, which would mitigate the rise in
and thereby
perhaps slow
efflux. Thus, for the
pHi hypothesis to be correct, removing bath CO2 must
indeed lower
, and the
inhibitory effect on NBC of lowering
per se must
overwhelm the stimulatory effect of raising pHi per se.
As shown in Fig.
4B, removing bath CO2 caused pHi to
rise from 7.09 to 7.33, and we calculate that
may have
simultaneously fallen from 10.8 to 9.4
mM.5 Even if the
Km for intracellular
were 10 mM, a decrease in
from 10.8
to 9.4 mM would decrease the transport rate by only a trivial amount. An
additional complication is that preliminary data indicate that when NBC
operates with a Na+:
stoichiometry of 1:2, it transports
rather than
(15). Thus it is likely that
NBC in the proximal tubule, which appears to function with Na+:
stoichiometry of 1:3, transports
one Na+, one
, and one
. We calculate that the removal of
bath CO2 actually causes
to rise
from 6.7 to 10.1
µM,6 and predict
that, if anything, this rise in
would
stimulate NBC. In other words, for the pHi hypothesis to be
correct, the anticipated inhibitory effect of decreasing
by 13%
would have to outweigh the anticipated stimulatory effects of raising
pHi by 0.24 and raising
by
50%
and produce a net 37% decrease in electrogenic Na-HCO3
cotransport.
As an alternative hypothesis, we suggest that basolateral CO2
per se may directly enhance the machinery of
reabsorption, stimulating in
parallel both apical H+ extrusion and basolateral
efflux. According to this
hypothesis, changes in steady-state pHi caused by removing or
adding back CO2 are the consequence, rather than the cause, of
altered rates of H+ and
transport. This CO2-triggering hypothesis is consistent with
earlier observations that the application of basolateral
, but
not of luminal
,
causes an increase in both steady-state pHi
(17) and H+
extrusion rates (9,
10). If this hypothesis is
correct, then it would have been the CO2, and not the
, present in the earlier
equilibrated
solutions that caused proximal tubule steady-state pHi and
H+ extrusion rates to increase. Further experiments will be
required to distinguish between the pHi hypothesis and the
alternative CO2-sensor hypothesis for producing the observed
changes in JHCO3.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
1 The junction between the proximal straight tubule and proximal convoluted
tubule was in the midcortex.
2 The dissected length of tubule included the distal-most 1.31.4 mm of
the proximal convoluted tubule plus the proximal-most 0.20.3 mm of
proximal straight tubule.
3 Assuming 1) a temperature of 37°C and thus a
PH2O of 47 mmHg, 2) a barometric
pressure of 760 mmHg, 3)5%CO2 in the gas cylinder, and
4) a CO2 solubility of 0.031 mM/mmHg. Thus
[CO2] = (760 47) x 5% x 0.031 = 1.105 mM =
1,105 µM.
4 The reabsorbate of the proximal tubule is very close to isosmotic. Even if
the reabsorbate were pure NaHCO3, its
[] could be no higher than one-half
the osmolality (assuming an osmotic coefficient of unity).
5 If we assume that [CO2]i is 5% when 5% CO2
is present in both lumen and bath, but only 2.5% when CO2 in
present in the lumen but not the bath, then removing bath CO2
causes to
fall from 10.8 to 9.4 mM.
6 We assume that the pK of the reaction
is 10.3 and thus that
.
When CO2 is present in the bath, pHi is 7.09 and the
computed
is
10.8 mM. We calculated that under these conditions,
is 6.7
µM. When CO2 is absent from the bath, pHi is 7.33 and
we assume that
is 9.4 mM
(see footnote 5). We calculate that under these conditions,
is 10.1 µM.
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REFERENCES |
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