From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl. The purpose of the present study was to determine (a) whether Cl
plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl
]i for
activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl
-
and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na+ and 0-194 mM Cl
. The artificial seawater bathing the BMF initially contained no Na+. After dialysis was halted, adding 50 mM Na+ to the artificial seawater caused an amiloride-sensitive pHi increase under both
normo- and hypertonic conditions. The computed Na-H exchange flux (JNa-H) increased with increasing [Cl
]i under both normo- and hypertonic conditions, with similar apparent Km values (~120 mM). However, the maximal
JNa-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pHi requires
Cl
under both normo- and hypertonic conditions, but at any given [Cl
]i, JNa-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl
]i is not the primary shrinkage signal, but may act as an
auxiliary shrinkage signal. To determine whether the Cl
-dependent step is after the G-protein-dependent step,
we predialyzed BMFs to a Cl
-free state, and then attempted to stimulate Na-H exchange by activating a G protein.
We found that, even in the absence of Cl
, dialyzing with GTP
S or AlF3, or injecting cholera toxin, stimulates Na-H
exchange. Because Na-H exchange activity was absent in control Cl
-depleted fibers, the Cl
-dependent step is at
or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF3 indicates that the G
protein is a heterotrimeric G protein.
Cell swelling generally initiates a rapid sequence of
events that results in the efflux of ions and water, a volume-regulatory decrease (VRD) that returns cell volume toward normal (for reviews see Hoffmann and Simonsen, 1989; Hallows and Knauf, 1994; Lang et al.,
1995
). The ion efflux may be mediated by K/Cl cotransport (Dunham and Ellory, 1981
; Jennings and Shulz,
1990
; Jennings and Schulz, 1991
; Jennings and Al-Rohil, 1990
), KCl efflux through parallel K+ and Cl
channels (Knoblauch et al., 1989
; Welling and O'Neil,
1990
; Banderali and Roy, 1992
), or K-H exchange
(Cala, 1980
, 1983
, 1985
). Conversely, cell shrinkage often
initiates a volume-regulatory increase (VRI), a rapid sequence of events that results in the influx of ions and
water (Lang et al., 1995
; Hoffman and Simonsen, 1989
;
Hallows and Knauf, 1994
). The influx may be mediated
by Na/K/Cl cotransport (Geck et al., 1980
; Eveloff and
Calamia, 1986
) or Na-H exchange augmented by Cl-HCO3 exchange (Kregenow et al., 1985
; Grinstein et
al., 1983
; Jennings et al., 1986
). It is believed that, in some cells, the VRD and VRI responses are reciprocal,
with cell swelling stimulating VRD and inhibiting VRI,
and cell shrinkage having the opposite effects (Lang et
al., 1995
).
Over a longer time frame, hypertonicity may stimulate osmotic-response elements in some cells, increasing the transcription of enzymes that catalyze the production of intracellular osmolytes. With a delay of ~48 h,
mIMCD-3 (renal medullary collecting duct), PAP-HT25 (rabbit inner medulla), and MDCK cells respond
to hypertonicity by inducing aldose reductase (Spring
and Siebens, 1988; Garcia-Perez and Burg, 1991
; Burg,
1995
). This enzyme converts glucose to the relatively
impermeant sorbitol, causing osmotic swelling. Other osmolytes, such as inositol, betaine, taurine, and glycerophosphocholine may also be concentrated, and
thereby promote re-swelling (Burg, 1995
; Garcia-Perez
and Burg, 1991
).
A major unanswered question in cell physiology is
how cells sense cell-volume changes and transduce
them to the appropriate changes in ion transport. One
important clue may be the observation, made by Parker
(1986), that Cl
is necessary for the shrinkage-induced
activation of Na-H exchange in dog red blood cells. In
earlier work on muscle fibers from the giant barnacle,
we confirmed this observation and additionally showed
that the shrinkage-induced activation of the Na-H exchanger specifically requires Cl
in the intracellular
fluid (Davis et al., 1994
). The precise role of Cl
in this
process is unclear. However, there is precedent for involvement of Cl
in other biological processes. For example, Cl
increases the affinity of the
subunit of the
heterotrimeric G protein Go for GTP
S (Higashijima et
al., 1987
).
A second important clue into the shrinkage signal-transduction system is that, in barnacle muscle fibers
(BMFs),1 the shrinkage-induced activation of the Na-H
exchanger appears to be mediated by a G protein
(Davis et al., 1992a). Thus, the effect of shrinkage on
the exchanger is inhibited by dialyzing the fiber with
GDP
S, and mimicked either by dialyzing with GTP
S or by injecting the fibers with activated catalytic subunit
of cholera toxin (CTX).
The purpose of the present study was to explore the
role of Cl in the shrinkage-induced activation of Na-H
exchange in internally dialyzed barnacle muscle fibers.
We used microelectrodes to monitor intracellular pH
(pHi) and calculated the Na-H exchange rate ( JNa-H) from the rate of pHi increase and the intracellular buffering power. Because internal Cl
is required for the
shrinkage-induced activation of Na-H exchange in BMFs,
we hypothesized that the primary signal the cell senses during shrinkage may be an increase in [Cl
]i. To test
this hypothesis, we determined the [Cl
]i dependence
of Na-H exchange, both under normo- and hypertonic conditions. We found that, even under normotonic
conditions, the Na-H exchanger is inactive in the absence of Cl
. Increasing [Cl
]i causes a monotonic rise
in JNa-H but, at a given [Cl
]i, JNa-H is always greater under hypertonic conditions. Thus, an increase in [Cl
]i
is not the primary shrinkage signal. In a second series
of experiments, we asked whether the Cl
-dependent
step in the activation of the Na-H exchange is before or
after the G-protein step. We found that, even in BMFs
depleted of Cl
, we could activate Na-H exchange with
GTP
S, AlF3, or CTX. Thus, the Na-H exchanger does
not require Cl
per se. Moreover, the Cl
-dependent
step precedes or is concurrent with the G-protein step in the signal-transduction cascade.
General
Barnacles were obtained from Bio-marine Enterprises (Seattle,
WA) and kept in an aerated aquarium at 4°C. After dissection, barnacle muscle fibers were kept at 4°C for a period of up to 36 h
in our standard artificial seawater (ASW) (see Solutions, below).
Before experiments, fibers were incubated for at least 1 h in a
Ca++-free artificial seawater (see Solutions, below) to prevent contraction during the subsequent cannulation (see below). This
Ca++-free solution also contained 0.5 mM SITS (4-acetamido-4-isothiocyanostilbene-2,2
-disulfate) (United States Biochemical Corp., Cleveland, OH) to permanently block the activity of
the Na+-driven Cl-HCO3 exchanger (Boron, 1977
). The fibers
were cannulated in a Ca++-free (or a Ca++- and Cl
-free) ASW
also containing SITS.
Solutions
Artificial seawaters.
All ASWs were nominally HCO3 free. The
standard ASW, in which fibers were incubated before the experiments, consisted of (mM): 440 Na+, 10 K+, 11 Ca++, 45.5 Mg++,
558 Cl
, 5 EPPS
(the anionic form of N -(2-hydroxyethyl)piperazine-N
-3-propanesulfonic acid; Sigma Chemical Co., St. Louis,
MO), and 5 of the neutral form of EPPS (pKEPPS
8.0). The usual
Ca++-free ASW was made by replacing the Ca++ in the standard
ASW with Mg++. ASWs containing 0 Na+ were made by replacing
Na+, mole for mole, with N-methyl-D-glucammonium (NMDG+)
that was produced by using HCl to titrate the free base N-methyl- D-glucamine (Sigma Chemical Co.). The ASW containing 50 mM
Na+ was made by diluting the standard ASW with the 0-Na+ ASW.
Dialysis fluids.
All the dialysis fluids (DFs) were Na+ free. The
standard pH-7.2 DF contained 34 mM Cl, and consisted of
(mM): 243 K+, 7 Mg++, 175 glutamate, 34 Cl
, 2 EGTA, 44 of the
anionic form of HEPES (United States Biochemical Corp.), 56 of
the neutral form of HEPES, 0.5 phenol red, and 4.0 Tris/ATP.
The standard pH-6.5 DF also contained 34 mM Cl
, and consisted of (mM): 255.5 K+, 7 Mg++, 160 glutamate, 34 Cl
, 2 EGTA, 71.5 of the anionic form of MES (2-(N-morpholino)-
ethanesulfonic acid; Sigma Chemical Co.), 28.5 of the neutral
form of MES, 0.5 phenol red, and 4.0 Tris/ATP. Cl
-free DFs
were made by replacing all of the Cl
with L-glutamate. DFs with
[Cl
] values above 34 mM were made by replacing glutamate,
mole for mole, with Cl
. Dialysis fluid pH was adjusted upward
with KOH and downward with either HCl (for Cl
-containing
DFs) or L-glutamic acid (for Cl
-free DFs). In the DFs containing
GTP
S, 1 mM ATP was substituted with 1mM GTP
S, keeping
the total nucleotide concentration at 4 mM. Aluminum fluoride
was added to the DFs as 10 mM KF + 100 µM AlCl3.
Measurement of pHi and Membrane Potential
The technique for measuring intracellular pH (pHi) in internally
dialyzed muscle fibers has been published elsewhere (Russell et
al., 1983). Single, isolated BMFs were horizontally cannulated in
a Ca++-free solution. A length of cellulose-acetate dialysis tubing with a molecular weight cutoff of ~6 kD was threaded into one cannula, through the muscle fiber, and out the opposite cannula. The pH electrodes were fabricated according to the design of
Hinke (1967)
. Glass microelectrodes used for measuring membrane voltage (Vm) were filled with 3 M KCl when the dialysis
fluid contained Cl
, but with 1 M K-glutamate when the DF was
Cl
free.
Experimental Protocols
General.
After cannulating the fiber and inserting the dialysis
tubing, we began dialysis with a pH-7.2 DF at a rate of 5 µl min1.
All DFs were Na+ free. The pH and Vm electrodes were inserted
through opposite cannulas so that their tips were within ~500
µm of each other. The central region of the fiber was isolated
from the cut ends with grease seals. We then began superfusing
the fiber with a Na+-free ASW at a rate of 1 ml min
1.
Studies on the [Cl]i dependence of Na-H exchange.
In these experiments, after an initial ~30-min period of dialysis with a DF having
a pH of 7.2 and a [Cl
] between 34 and 194 mM, dialysis continued for an additional ~50-60 min with a DF that was otherwise
identical, except for having a pH of 6.5. Thus, the total time for
dialysis was ~80-90 min. Previous work has shown that 60-90
min is sufficient for either 22Na (Russell et al., 1983
) or 36Cl (Boron et al., 1978
; Russell and Brodwick, 1988
) in the DF to achieve
isotopic equilibrium. After dialysis was halted, pHi was allowed to
stabilize for ~15 min in a Na+-free ASW.
Studies with GTPS or AlF3 in Cl
-depleted cells.
The protocol was
similar to that above, except that the initial period of dialysis with
the pH-7.2/Cl
-free DF continued for ~140 min to deplete the
cell of Cl
. During this time, the fiber was superfused with a Na+-
and Cl
-free ASW. This pH-7.2/Cl
-free DF was then switched to
an identical solution that also contained either 1 mM GTP
S or
10 mM AlF3. After ~40 min, we switched to an identical solution
in which the pH of the DF was lowered to 6.5 to acid load the fiber. We continued dialyzing with this solution for ~60 min.
Thus, the total dialysis time was ~240 min.
Studies on Cl-depleted cells injected with CTX.
This protocol was
similar to the one in the GTP
S and AlF3 experiments. The major difference was that, ~160 min after initiating dialysis, we microinjected the BMFs with CTX, and only then inserted the microelectrodes. The injection fluid was the pH-7.2/0-Cl
DF containing the dithiothreitol-activated CTX to a final intracellular concentration of ~3 × 10
6 M. During the microinjection and
electrode insertion, the fiber was briefly exposed to an ASW that
lacked Ca++ (to prevent contraction). Also, during the microinjection and electrode insertion, the fiber was dialyzed continuously with the pH-7.2/0-Cl
DF. After an additional ~110 min dialysis with this DF, and an additional ~60 min with a pH-6.5/
0-Cl
DF, we halted dialysis and allowed the fiber to stabilize for 120 min before assaying as described above.
Statistics
Values are reported as means ± SEM. Groups of data were compared using a two-sample t test assuming unequal variance.
Effect of Increasing Internal Cl under
Normotonic Conditions
Fig. 1 A illustrates an experiment in which a muscle fiber was acid loaded by internally dialyzing it with a Na+-free DF containing 34 mM Cl at pH 6.5. As noted in
METHODS, all fibers were pretreated with SITS to eliminate Na+-driven Cl-HCO3 exchange, and all were superfused with a Na+-free ASW for ~90 min. The terminal portion of this dialysis period is shown in Fig. 1 A.
After we halted dialysis (Fig. 1 A, a), pHi continued to
drift downward (Fig. 1 A, ab), probably because the tip
of the pH electrode in this experiment was rather distant from the dialysis tube. The mean pHi at b was 6.72 ± 0.01 (n = 20). Inasmuch as the fiber had been dialyzed
with a Na+-free DF, and superfused with a Na+-free
ASW, [Na+]i should have been extremely low. Exposing the fiber to an ASW containing 50 mM Na+ produced a slow increase in pHi (Fig. 1 A, bc), due mainly
to the basal activity of the Na-H exchanger (Davis et al.,
1994
). We chose to use a modest level of Na+, 50 mM,
because this concentration is high enough to support Na-H exchange, but not so high as to compete with
amiloride for binding sites on the transporter. We computed the total acid-extrusion rate (JTotal) as the product of the pHi recovery rate of segment bc and the previously measured intrinsic buffering power (Davis et al.,
1994
). The pHi increase of Fig. 1 A, bc was inhibited by
adding 1 mM amiloride to the 50-Na+ ASW (Fig. 1 A,
cd). The broken lines in the figure emphasize the slopes
in the absence and presence of amiloride. The delay between the application and action of amiloride presumably reflects the time for the drug to reach the interstices
of the BMF. For the purposes of this study, we will
present values for Na-H exchange rate (JNa-H) as the
amiloride-sensitive component of Jtotal; that is, the difference in flux values between bc and cd in Fig. 1 A. For 20 fibers, the mean JNa-H was 23 ± 4 µM min
1 (Table I).
Table I.
JNa-H at Different [Cl |
To address the question of whether increasing
[Cl]i stimulates Na-H exchange under normotonic conditions, we performed the experiment shown in Fig. 1
B, in which we dialyzed the fiber with a Na+-free DF
containing 194 mM Cl. After we halted dialysis, pHi
drifted upward very slowly (Fig. 1 B, ab). The mean pHi
at point b was 6.71 ± 0.02 (n = 12). Exposing the cell to
50 mM Na+ produced a rapid intracellular alkalinization (Fig. 1 B, bc) that was largely blocked by amiloride
(Fig. 1 B, cd). The mean JNa-H for these 12 experiments
was 138 ± 26 µM min
1 (see Table I). Therefore, under normotonic conditions (975 mosM/kg), increasing
[Cl
] in the DF from 34 to 194 mM dramatically stimulates the Na-H exchanger, increasing JNa-H from 23 to
138 µM min
1.
Effect of Increasing Internal Cl under Hypertonic Conditions
Fig. 2 A illustrates 1 of 13 experiments in which we examined the effect of hypertonicity on Na-H exchange in fibers dialyzed with 34 mM Cl. The first part of the protocol was
identical to that of Fig. 1 A: the fiber was acid loaded by
dialyzing with a Na+-free DF containing 34 mM Cl
, dialysis was halted, and pHi was allowed to stabilize (Fig. 2
A, ab). The mean pHi at point b was 6.70 ± 0.02 (n = 13). As in Fig. 1 A, exposing the cell to an ASW containing 50 mM Na+ elicited, at most, a very slow alkalinization (Fig. 2 A, bc).2 At point c, we switched to a Na+-free
ASW made hypertonic (1,600 mosM/kg) by the addition of mannitol. The self-limited increase in pHi (Fig.
2 A, cd) was presumably due to the concentration of intracellular buffers, as previously described. Indeed, in a
previous study, we found that exposing a muscle fiber
to the same hypertonic solution caused intracellular buffering power, measured at a pHi of ~6.8, to double
(Davis et al., 1994
). After pHi stabilized, exposing the
cell to a hypertonic ASW containing 50 mM Na+ produced a slow pHi increase (Fig. 2 A, de). Applying
amiloride not only blocked this pHi increase, it unmasked a slow acidification (Fig. 2 A, ef). The difference in the slopes of the pHi recoveries in segments de
and ef indicates that there was a modest rate of Na-H
exchange when cells dialyzed with 34 mM Cl
were
shrunken. In a total of 13 similar experiments, the
mean JNa-H was 86 ± 17 µM min
1 (see Table I), a figure
that takes into consideration the increased buffering
power in hypertonic solutions. This mean JNa-H, obtained under hypertonic conditions, is ~3.7-fold higher
than the mean JNa-H (see Fig. 1 A) obtained under normotonic conditions in cells dialyzed with 34 mM Cl
.
Effect of 194 mM Cl
To determine whether increasing [Cl]i also stimulates Na-H exchange under hypertonic conditions, we performed
the experiment shown in Fig. 2 B. This experiment is
identical to that shown in Fig. 2 A, except that the DF
contained 194 rather than 34 mM Cl. The mean pHi
at point b was 6.73 ± 0.01 (n = 8). Exposing the cell to
a normotonic ASW containing 50 mM Na+ produced a
rapid alkalinization (Fig. 2 B, bc), as observed above for
fibers dialyzed with 194 mM Cl
(Fig. 1 B). After we
switched to a Na+-free hypertonic solution, and pHi stabilized (Fig. 2 B, cd), increasing [Na+]o to 50 Na+ produced an even more marked alkalinization (Fig. 2 B,
de) that was largely blocked by amiloride (Fig. 2 B, ef).
The mean JNa-H for these eight experiments was 345 ± 43 µM min
1 (see Table I). Therefore, under hypertonic conditions, increasing the [Cl
] in the DF from
34 to 194 mM increased JNa-H fourfold, from 86 to 345 µM min
1.
[Cl]i Dependence of Na-H Exchange under Normo- and
Hypertonic Conditions
We have already seen that, under normotonic conditions, increasing the [Cl] of the DF from 34 to 194 mM caused an increase in JNa-H (Fig. 1, A vs. B). To determine the [Cl
]i dependence of Na-H exchange under normotonic conditions, we performed additional
experiments identical to those in Fig. 1 except that the
[Cl
] of the DFs was 74, 114, or 154 mM. The data are
summarized by the open circles in Fig. 3. In plotting
the data, we have assumed that [Cl
]i is the same as the
[Cl
] of the DF.
We also performed additional experiments to determine the [Cl]i dependence of Na-H exchange under
hypertonic conditions. These experiments were identical to those in Fig. 2, except that the [Cl
] of the DFs
was 74, 114, or 154 mM. We have assumed that the cells
behaved as perfect osmometers, so that increasing the
osmolality from 975 to 1,600 mosM/kg increased the
[Cl
]i by a factor of 1,600/975, or 1.64. Thus, [Cl
]i values for the hypertonic data, plotted as closed circles in Fig. 3, have values 1.64-fold higher than the corresponding [Cl
]i values for the normotonic data, plotted as open circles. Fig. 3 shows that, under both
normo- and hypertonic conditions, increasing nominal
[Cl
]i causes an increase in Na-H exchange activity. For
normotonic conditions (Fig. 3,
), a nonlinear least-squares curve fit (Hill coefficient = 2) produced an apparent K m for internal Cl
of 127 mM, and an apparent
Vmax of 201 µM min
1. For hypertonic conditions (Fig.
3,
), the apparent K m was ~112 mM, and the Vmax was
375 µM min
1. Therefore, we conclude that increasing
the nominal [Cl
]i stimulates Na-H exchange activity
under both normo- and hypertonic conditions. Furthermore, although hypertonicity had little effect on
the apparent Km for internal Cl
, it increased the Vmax
for Na-H exchange by nearly 90%.
Effect of Using Cl Substitutes other than Glutamate
From the above data, it would appear that increasing
[Cl]i stimulates Na-H exchange, both under normo-
and hypertonic conditions. However, to create DFs of
increasing [Cl
], we simultaneously lowered [glutamate].
Thus, it is possible that the increased JNa-H we observed
in high Cl DFs was due to decreasing [glutamate],
rather than increasing [Cl
]. Therefore, we examined
the effect of using two other Cl
substitutes, gluconate
and sulfamate. Our standard DF contained 34 mM Cl
and 160 mM glutamate. In the present series of experiments, done on matched fibers, we dialyzed BMFs with
DFs containing 34 mM Cl
plus 160 mM of either
glutamate, gluconate, or sulfamate. We assayed the fibers for Na-H exchange as in Fig. 1 A, under normotonic conditions. If glutamate were inhibiting Na-H
exchange at a [Cl]i of 34 mM, then replacing the
glutamate with either gluconate or sulfamate should
substantially increase JNa-H, that is, produce approximately the same JNa-H that we saw above at 194 mM Cl
(i.e., ~138 µM min
1), when we substituted 160 mM
glutamate for 160 mM Cl
. However, we found that although JNa-H was 16 ± 5 µM min
1 (n = 4) in fibers dialyzed with 160 mM glutamate, it was no higher in fibers
dialyzed with either 160 mM gluconate (JNa-H = 0 ± 7, n = 4) or 160 mM sulfamate (JNa-H = 14 ± 4, n = 5).
Thus, because glutamate is not inhibitory, Cl
must be
stimulatory.
Where Does Cl Play a Role in Activation of Na-H Exchange?
Because we can use GTPS, AlF3, or CTX to activate the
heterotrimeric G protein that ultimately activates the
Na-H exchanger, we are in a position to ask whether
the Cl
-dependent step in the shrinkage signal-transduction cascade is after the G protein. Our approach
was, first, to verify that complete Cl
removal (i.e., removing Cl
from both DF and ASW) does indeed block
Na-H exchange, and then to determine whether the
aforementioned G-protein activators are capable of
stimulating Na-H exchange in the absence of Cl
.
We Cl depleted fibers by exposing them to a Cl
-free ASW and dialyzing them with a
Cl
-free DF for a minimum of 160 min, and an average
of ~180 min. In the experiment shown in Fig. 4, we
pretreated the fiber with SITS, and then dialyzed for
120 min with a pH-7.2 DF that was free of both Na+ and
Cl
. During this time, the ASW was also free of Na+ and
Cl
. We then switched the pH of the DF to 6.5 for an
additional 60 min to acidify the cell. Fig. 4 picks up the
experiment during this latter period of dialysis. After
we halted dialysis, pHi stabilized (Fig. 4, ab). Exposing
the cell to an ASW containing 50 mM Na+ did not significantly alter the trajectory of pHi (Fig. 4, bc). Neither
was the pHi trajectory affected by applying 1 mM
amiloride (Fig. 4, cd). In a total of six similar experiments, the mean JNa-H was
3 ± 7 µM min
1 (see Table
I), which is not significantly different from zero. Thus,
Cl
depletion completely blocks Na-H exchange under
normotonic conditions.
Effect of GTP
We had previously shown that, in the presence of Cl
and under normotonic conditions, GTP
S activates Na-H
exchange in barnacle muscle fibers (Davis et al., 1992a
).
To determine whether GTP
S also activates the exchanger in Cl
-depleted fibers, we performed a series
of experiments similar to the one shown in Fig. 5 A.
Our protocol was the same as for Fig. 4, except that the
DF contained 1 mM GTP
S for the final ~95 min of dialysis. In these experiments, the duration of dialysis with the Cl
-free DF, before the introduction of GTP
S,
was as long as 145 min, and averaged 135 min. Fig. 5 A
picks up the experiment during the latter part of dialysis with the pH-6.5 DF containing GTP
S. When dialysis
was halted, the pHi continued to drift downward (Fig. 5
A, ab) in this particular experiment. Exposing the cell to
a Cl
-free ASW containing 50 mM Na+ produced a substantial alkalinization (Fig. 5 A, bc) that was blocked by
amiloride (Fig. 5 A, cd). For the eight fibers in this study,
the mean JNa-H was 164 ± 23 µM min
1 (see Table I),
which is significantly greater than the above value for
Cl
-depleted cells in the absence of GTP
S,
3 ± 7 µM
min
1 (P < 0.0001). Thus, even in Cl
-depleted fibers,
GTP
S markedly stimulates Na-H exchange.3
Effect of AlF3 on Na-H exchange in Cl
To obtain further evidence that G -protein activation will
stimulate Na-H exchange even in the absence of Cl,
we examined the effect of introducing AlF3 into Cl
-
depleted fibers. Our protocol was similar to that in Fig.
5 A, except that AlF3 (10 mM KF plus 100 µM AlCl3) replaced GTP
S for the final 100 min of dialysis. Before
the introduction of AlF3, muscle fibers in this group of
experiments were Cl
depleted for as long as 175 min,
and an average of 140 min. Fig. 5 B shows the terminal
portion of dialysis with the pH-6.5, AlF3-containing DF.
When we halted dialysis, pHi quickly stabilized (Fig. 5
B, ab). However, when we exposed the fiber to a Cl
-free ASW containing 50 mM Na+, pHi rose very rapidly,
as was the case for cells dialyzed with GTP
S (see Fig. 5
A). The rapid pHi increase in the AlF3-dialyzed cells was
greatly inhibited by amiloride (Fig. 5 B, cd). In a total of
six experiments, the mean JNa-H was 296 ± 28 µM min
1
(see Table I), significantly greater than the control flux
of
3 ± 7 µM min
1 in Cl
-depleted cells not dialyzed
with AlF3 (P < 0.0001). Thus, AlF3 markedly stimulates
Na-H exchange, even in Cl
-depleted fibers.
In previous experiments from this laboratory, we
had shown that injecting CTX into BMFs the day before the experiment stimulates Na-H exchange. These
previous studies were performed on cells dialyzed with
34 mM Cl and superfused with an ASW containing Cl
(Davis et al., 1992a
). To determine whether CTX can
activate Na-H exchange in the absence of Cl
, we
needed to modify the protocol from the previous study
so that the Cl
depletion, the CTX injection, and the
Na-H exchange assay could all be done on the same
day. Because the total time between CTX injection and
Na-H exchange assay in the present study would be substantially less than in the previous one, we increased
the final intracellular CTX concentration to 3 × 10
6
M. The protocol for the first portion of the experiment
was similar to those shown in Fig. 5. The differences are
detailed in METHODS; for example, the microelectrodes
were not inserted until after CTX injection. In the experiment shown in Fig. 6 A, we exposed a fiber to a pH-7.2 DF that was free of both Na+ and Cl
. The Cl
depletion time before injection with CTX was as long as
170 min, and averaged 160 min. After we injected the
CTX and inserted the microelectrodes, we continued
to dialyze with the pH-7.2, 0-Cl
DF for an additional
~110 min. We then switched to a DF with a pH of 6.5 to acidify the cell. After halting dialysis, we allowed the
fibers to incubate for an additional 120 min before assaying for Na-H exchange. Thus, we assayed the fibers
~290 min postinjection. Fig. 6 A picks up the experiment ~75 min after dialysis had been halted. Exposing
the cell to a Cl
-free ASW containing 50 mM Na+
caused an increase in pHi (Fig. 6 A, bc) that was reversed by amiloride (Fig. 6 A, cd). The mean JNa-H for
five similar experiments was 100 ± 35 µM min
1 (see
Table I), significantly greater than the control flux of
3 ± 7 µM min
1 in Cl
-depleted cells not injected
with CTX (P < 0.02).
In the above CTX experiments, the JNa-H of ~100 µM
min1 was substantially less than in comparable experiments with GTP
S (164 µM min
1) or AlF3 (296 µM
min
1). One reason JNa-H may have been relatively low
in the CTX experiments is that, even though we assayed ~290 min after injecting the CTX, we may not
have allowed enough time for the CTX to have its maximal effect. Therefore, we asked whether, under our assay conditions (Fig. 6 A), CTX would activate Na-H exchange similarly in the presence and absence of Cl
.
To answer this question, we performed a second series
of experiments, identical to that shown in Fig. 6 A, except that the DF contained 34 mM Cl
, and the ASW
contained 558 mM Cl
. Because increasing values of
[Cl
]i increase Na-H exchange, we chose a DF with a
relatively low [Cl
] to minimize "background" Na-H exchange activity; that is, Na-H exchange independent of
CTX. As with Cl
-depleted cells (see Fig. 6 A), cells dialyzed with 34 mM Cl
and injected with CTX exhibited
significant Na-H exchange (see Fig. 6 B). For five experiments, the mean JNa-H was 135 ± 38 µM min
1 (see
Table I). The background Na-H exchange rate for 34 mM Cl
(see discussion of Fig. 1) was ~23 µM min
1.
Subtracting 23 from 135 µM min
1 produces a CTX-
dependent JNa-H of 112 µM min
1 for Cl
-containing
cells. This figure is indistinguishable from the JNa-H in CTX fibers that were Cl
depleted (i.e., 100 µM
min
1). Thus, under the conditions of our experiments, CTX produces a similar activation of Na-H exchange in the presence and absence of Cl
.
Role of Intracellular Cl in the Activation of Na-H Exchange
The first indication that Cl may play a role in
the activation of the Na-H exchanger was Parker's observation, made in dog erythrocytes, that the Na-H
exchanger fails to respond to cell shrinkage when extra- and intracellular Cl
is replaced with either thiocyanate or nitrate (Parker, 1983
). However, Cl
is not
required for Na-H exchange per se. When Parker activated the Na-H exchanger by shrinking the cells in the
presence of Cl
, and then fixed the cells by briefly exposing them to glutaraldehyde, the Na-H exchanger remained activated even in the absence of Cl
(Parker,
1984
). Similarly, Motais et al. (1989)
found that Cl
is
required for the cAMP-dependent activation of Na-H
exchange by isoproterenol in trout erythrocytes. In
these cells, once Na-H exchange was activated by cAMP
in the presence of Cl
, the exchanger remained active
even after NO
3 replaced Cl
(Motais et al., 1989
).
Thus, two signal-transduction processes leading to activation of the Na-H exchanger require Cl
, even though
the exchanger itself does not require Cl
. More recently, a Cl
-dependent acid-base transporter, presumably an amiloride-resistant Na-H exchanger, has been
found in the apical membrane of colonic crypt cells
(Rajendran et al., 1995
). There is one example in which Cl
inhibits Na-H exchange. In salivary acinar
cells, carbachol stimulates Cl
channels, leading to Cl
efflux and cell shrinkage. The simultaneous presence
of carbachol, a decreased [Cl
]i and cell shrinkage results in activation of Na-H exchange (Foskett, 1990
;
Robertson and Foskett, 1994
).
Perhaps our most unexpected result was that, even
under normotonic conditions, the Na-H exchanger is
markedly stimulated by increasing [Cl]i. As summarized by the open symbols in Fig. 3, increasing [Cl
]i
from its "normal" level of 34 mM to 194 mM caused JNa-H
to increase six-fold. This result suggests that the exchanger may be the target of a Cl
-dependent signal-transduction system even under normotonic or "basal"
conditions. If this were the case, then one might predict that reducing [Cl
]i to zero would eliminate Na-H
exchange under normotonic conditions, a prediction
verified in Fig. 4. Thus, it appears that, at least in barnacle muscle, Cl
is required to elevate the Na-H exchanger to even its basal level of activity, and to maintain this basal activity.
Three aspects of the data obtained under hypertonic conditions are noteworthy. First, as was the case
under normotonic conditions, increasing the [Cl]i
produces a graded increase in JNa-H (Fig. 3,
). Second,
at any given [Cl
]i, the JNa-H is always greater under hyper- than under normotonic conditions. For example,
at a [Cl
]i of 114 mM, JNa-H was 89 µM min
1 under
normotonic conditions (Fig. 3, a). Increasing the osmolality, while holding [Cl
]i fixed at 114 mM, would
be expected to increase JNa-H approximately twofold to
~191 µM min
1 (interpolated point b). Thus, an increase in [Cl
]i cannot be the primary signal for triggering the shrinkage-induced increase in JNa-H. Third,
shrinkage not only increases JNa-H by shifting the exchanger from the normo- to the hypertonic curve in
Fig. 3, it also increases JNa-H because the loss of cell water increases [Cl
]i. Thus, in a cell dialyzed to a [Cl
]i
of 114 mM, increasing the osmolality from 975 to 1,600 mosM/kg, will increase [Cl
]i to 187 mM. As shown in
Fig. 3, this elevation in [Cl
]i would increase JNa-H by
nearly 45%, from ~191 µM min
1 (Fig. 3, b) to 275 µM
min
1 (Fig. 3, c). Thus, the increase in [Cl
]i that accompanies shrinkage is an auxiliary shrinkage signal.
As summarized in Fig. 3, the K m values for intracellular Cl are similar under normo- and hypertonic conditions (127 vs. 112 mM). This observation is
consistent with the notion that Cl
plays similar roles in
activating the Na-H exchanger at normal and low cell
volume. We propose the following model for barnacle muscle fibers: the "shrinkage signal," which leads to activation of the Na-H exchanger, is amplified in such a
way that the gain of the hypothetical amplifier increases with increasing [Cl
]i. In euvolemic cells, the
basal shrinkage signal is small, but greater than zero. At
a "normal" [Cl
]i of 34 mM, the amplification of this
small signal is weak. Thus, the combination of a euvolemic cell and a [Cl
]i of 34 mM produces a very low
JNa-H (i.e., 23 µM min
1).4 However, raising [Cl
]i to
194 mM in a euvolemic cell increases the amplification
of even this weak shrinkage signal, producing a modestly high JNa-H (i.e., 138 µM min
1). In shrunken cells,
the shrinkage signal is large. However, at a normal
[Cl
]i of 56 mM (produced by shrinking a cell with an
initial [Cl
]i of 34 mM), the amplification is weak, producing a rather modest JNa-H (i.e., 86 µM min
1). However, the combination of a shrunken cell and a [Cl
]i
increased to 318 mM produces a robust JNa-H (i.e., 345 µM min
1).
Activation of Na-H by GTPS, AlF3, and CTX in the Absence
of Cl
In previous work, we showed that, at a pHi of 6.8, Cl is
required for the shrinkage-induced activation of Na-H
exchange (Davis et al., 1994
). Studying barnacle muscle fibers at a pHi of ~7.2, we also showed that a G protein is involved in this process (Davis et al., 1992a
). In
particular, we showed that dialyzing with GDP
S blocks
the shrinkage-induced activation of the exchanger. The present study extends the previous G -protein work by
demonstrating that GTP
S and CTX both activate the
exchanger at pHi 6.8. In addition, we extend the earlier
work by demonstrating that AlF3 also activates the exchanger. Because AlF3 stimulates heterotrimeric G proteins, but not low-molecular-weight G proteins (Kaziro
et al., 1991
; Bigay et al., 1985
), our new observations
with AlF3 imply that activation of a heterotrimeric G
protein can activate the exchanger.5 In earlier work, we
showed that activating the PKA or PKC pathways fails to
stimulate the exchanger (Davis et al., 1992a
). Thus, the
most straightforward explanation for our data is that
the signal-transduction cascade triggered by cell shrinkage includes a heterotrimeric G protein.
One of the goals of the present study was to determine the order of the Cl-dependent and G -protein
steps in the shrinkage signal-transduction cascade. One
possibility is that Cl
acts at a step somewhere in the
G -protein cycle. Indeed, Cl
appears to increase the affinity of
o for GTP
S (Higashijima et al., 1987
). An increase in affinity of
for GTP would stabilize
in the
GTP-bound or active state. A Cl
requirement of the G
protein in the signal-transduction cascade could explain the Cl
-dependent activation of Na-H exchange
in dog and trout RBCs, and in BMFs. To determine the
order of the Cl
-dependent and G-protein steps in our
BMF experiments, we attempted to activate Na-H exchange with GTP
S, AlF3, and CTX in Cl
-depleted
cells. As noted above, we found that Cl
depletion
blocks Na-H exchange under normotonic conditions,
at pHi
6.8 (Fig. 4). Even in such Cl
-depleted cells,
introducing GTP
S (Fig. 5 A), AlF3 (Fig. 5 B), or CTX
(Fig. 6) activates the Na-H exchanger. Because each of these three agents acts at the level of the G protein,6
and is nevertheless able to bypass the blockade introduced by Cl
removal, we can conclude that the Cl
-
dependent step must precede or be concurrent with
the G -protein step.
Address correspondence to Walter F. Boron, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax: 203-785-7678; E-mail: walter.boron{at}yale.edu
Received for publication 27 February 1997 and accepted in revised form 12 September 1997.
Portions of this work have been published in preliminary form (Hogan, E.M., B.A. Davis, and W.F. Boron. 1995. Biophys. J. 9:A356).We thank Dr. Catherine Berlot for helpful discussions, Drs. Raphael Zahler and Gordon Cooper for assistance in performing the curve fitting, Mr. Duncan Wong for computer programming and assistance in preparing the figures, and Mr. Francisco Rodriguez for technical assistance. We also thank Dr. Mark Bevensee for help with the final revisions.
This work was supported by National Institutes of Health grant NS18400.
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