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restoration’: involvement of the Cl− channel and Na+-K+-Cl− cotransporter
Hearing Research 113 (1997) 99^109
Changes in the volume of marginal cells induced by isotonic `Cl3 depletion/restoration': involvement of the Cl3 channel and Na-K-Cl3 cotransporter Shunji Takeuchi *, Motonori Ando, Akihiko Irimajiri Department of Physiology, Kochi Medical School, Nankoku 783, Japan
Received 3 March 1997; revised 10 June 1997; accepted 18 July 1997
Abstract
Marginal cells constitute the endolymph-facing epithelium responsible for the secretion of endolymph by the stria vascularis in the inner ear. We have studied the possible involvement of Cl3 conductance and Na -K -Cl3 cotransport in the mechanism of changes in cell volume upon isotonic Cl3 depletion/restoration. Changes in cell volume were estimated from video-microscopic images with the aid of an image processor. Marginal cells shrank to 80% of their original volume in 30 s and to 65^70% in 90 s upon total replacement of [Cl]o ( 150 mM) by gluconate3 , and the original volume of the shrunken cells was restored within 2 min after restoration of Cl3 . The order of potency of anions to induce isotonic shrinkage was gluconate3 I3 F3 Br3 . The cell shrinkage caused by Cl3 depletion was partially inhibited by 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB, 0.2 mM), but not by either 4-acetamido-4P-isothiocyanato-stilbene-2,2P-disulfonic acid (SITS, 0.5 mM), bumetanide (10 WM) or ouabain (1 mM). The cell shrinkage caused by a reduction of [Cl]o from 150 mM to 7.5 mM was not affected by [K]o in the range of 3.6 mM to 72 mM. These results suggest that the main efflux pathway(s) responsible for the `Cl removal'-induced shrinkage depends on volumecorrelated Cl3 conductance (Takeuchi and Irimajiri, J. Membrane Biol. 150, 47^62, 1996) and that this pathway(s) is essentially independent of the Na -K -Cl3 cotransporter, the Na ,K -ATPase, and the K -Cl3 cotransporter. With regard to volume recovery after isotonic shrinkage, its critical dependence on the simultaneous presence of Na , K and Cl3 in the bath and its substantial inhibition by bumetanide (10 WM) both indicate a major role for Na -K -Cl3 cotransport. The strong influence on cell volume of solute fluxes working through the Cl3 channel and the Na -K -Cl3 cotransporter implies an essential role for these pathways in the ion transport mechanism(s) of the marginal cell.
V
V
s s s
V
Keywords :
Cell volume; Cl3 channel; Na -K -Cl3 cotransporter; Inner ear; Gerbil
1. Introduction
Several ion transport mechanisms in marginal cells have been reported (for review, see Wangemann, 1995) and similarities in ion transport mechanisms between strial marginal cells and vestibular dark cells have been pointed out (Wangemann, 1995). In addition to the apical IsK channel as a pathway for K secretion (Sunose et al., 1994; Marcus and Shen, 1994; Sunose et al., 1997), the importance of the basolateral Cl3 conductance in the normal function of marginal cells has * Corresponding author. Tel.: +81 (888) 80 2311; Fax: +81 (888) 80 2310.
been suggested by recent electrophysiological studies. These include: (i) marked depolarization of the transepithelial potential across the marginal cell layer upon depletion of basolateral Cl3 (Wangemann et al., 1995), (ii) relatively high density of 80 pS Cl3 channels in the basolateral membrane of marginal cells (Takeuchi et al., 1995), and (iii) a large whole-cell Cl3 conductance in dissociated marginal cells (Takeuchi and Irimajiri, 1996). The basolateral Cl3 conductance can provide a Cl3 e¥ux pathway (Wangemann, 1995). Besides conductive pathways and the Na ,K -ATPase, the Na -K -Cl3 cotransporter is essential in the normal function of stria vascularis since: (i) the dark cell in the vestibular labyrinth, an analogue to the mar-
0378-5955 / 97 / $17.00 ß 1997 Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 7 ) 0 0 1 3 4 - 2
HEARES 2889 28-11-97
100 Table 1 Composition of solutions (in mM) Solution control
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
Ca-free
Cl-free or gluconate3 ^ 150 ^ ^ ^ ^ 1 ^ 4 ^ 1.6 0.4 ^ ^ ^ 5
Na-free
NaCl 150 150 ^ Na-gluconate ^ ^ ^ K-gluconate ^ ^ ^ NaX ^ ^ ^ NMDG-Cl ^ ^ 150 1 1 1 MgCl2 ^ ^ ^ MgSO4 CaCl2 0.7 ^ 0.7 ^ ^ ^ Ca-(gluconate)2 CaX2 ^ ^ ^ K2 HPO4 1.6 1.6 1.6 0.4 0.4 0.4 KH2 PO4 Na2 HPO4 ^ ^ ^ ^ ^ ^ NaH2 PO4 EGTA ^ 1 ^ glucose 5 5 5 NMDG, N-methyl-D-glucamine. EGTA, ethylene glycol bis (L-aminoethylether)-N,N,NP,NP-tetraacetic acid. X is Br, F, or I. a CaX2 = 0 mM for X3 = F3 because of its low solubility.
ginal cell in the cochlea, has Na -K-Cl3 cotransport activity (Wangemann and Marcus, 1990), (ii) the endocochlear potential is sensitive to loop diuretics (Kusakari et al., 1978a; Kusakari et al., 1978b), and (iii) bumetanide inhibits K secretion from strial marginal cells (Wangemann et al., 1995). Our preliminary study revealed that isolated intact marginal cells shrink appreciably when depleted of extracellular Cl3 and that the shrunken cells' original volume is restored when replenished with Cl3 (Takeuchi and Irimajiri, 1997), suggesting that the volume of marginal cells depends largely on Cl3 transport mechanisms. Although marginal cells in vivo are never exposed to Cl3 -free condition, `Cl removal' is utilized as an experimental procedure to induce isotonic cell shrinkage in this study. We characterize the anionic pathways involved in the changes in cell volume induced by isotonic Cl3 depletion/restoration, and con¢rm the functional activity of Cl3 channels and Na K -Cl3 cotransporters in intact (not patch-clamped) single marginal cells. This report also further strengthens the previous observation of the existence of similarities between vestibular dark cells and strial marginal cells (Wangemann, 1995). 2. Methods
2.1. Cell preparation
Marginal cells were prepared from the inner ear of gerbils as described previously (Takeuchi et al., 1995). Brie£y, cochleae were obtained under anesthesia with
K-free
7.5 Cl 3.6 K
7.5 Cl 72 K
X3
150 ^ ^ ^ ^ 1 ^ 0.7 ^ ^ ^ ^ 1.6 0.4 ^ 5
5.5 142.5 ^ ^ ^ 1 ^ ^ 4 ^ 1.6 0.4 ^ ^ ^ 5
5.5 74.1 68.4 ^ ^ 1 ^ ^ 4 ^ 1.6 0.4 ^ ^ ^ 5
^ ^ ^ 150 ^ ^ 1 ^ ^ 0.7a 1.6 0.4 ^ ^ ^ 5
pentobarbital sodium (50 mg/kg, i.p.). Tissue strips from the stria vascularis freed of the spiral ligament were incubated for 20 min at 23^25³C in `Ca-free' solution (for composition, see Table 1) containing 0.2% papain and kept up to 3 h in cold (8³C) control solution (Table 1) until use. These strips were transferred to a bath mounted on an inverted microscope (TMD 300, Nikon, Tokyo, Japan) and dissected with ¢ne needles under visual control. Single marginal cells retaining their characteristic morphology (i.e., a relatively smooth apical membrane, numerous basal infoldings, and an apically located nucleus) were selected and separated from other dissociated cells. The care and use of animals used in this study were approved by the Kochi Medical School Animal Care and Use Committee. 2.2. Measurement of relative cell volume
The test cell was held at the center of the visual ¢eld by means of a holding pipette. The cell's pro¢le was viewed through Nomarski optics with a 100U objective (NA 1.4) under oil immersion. The bottom of the recording bath was made of a glass coverslip 0.12^0.17 mm thick. Cell images were recorded with a high-resolution black/white TV camera (SSC-M370, Sony, Tokyo, Japan) and a video cassette recorder (S-2200, Sony). Cell width measurements were made, o¡ line, with the aid of an image processor (Argus 10, Hamamatsu Photonics, Hamamatsu, Japan). We estimated relative cell volume from measurements of the width of the cell trunk in sharp focus in essentially the same way as described previously (Takeuchi and Irimajiri, 1996). The rationale behind this
HEARES 2889 28-11-97
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
101
Fig. 1. Estimation of cell volume changes. A : Video-microscopic images of a typical marginal cell undergoing a reversible volume change. The
3;
cell is held by a suction pipette (p) at basal infoldings with its apex turned upwards. Left, control ; center, 90 s after omission of Cl
3
2 min after restoration of Cl
(control solution). Scale bar, 10
Wm.
right,
B : Schematic drawings of the cell under control (left) and test (right) condi-
tions, with the de¢nition of size parameters attached. C : Correlation between the rate of change in width of the cell trunk and that of the basal part. Note that changes in the trunk width elicited by depletion and restoration of bath Cl
3
parallel those of the basal part. D : Similar plots
showing much weaker changes in cell height. In both C and D, dimensional measurements were made with cells just before shrinkage (control), at their maximum shrinkage and after full recovery to their initial volumes. Paired symbols connected by solid lines refer to an identical cell. Di¡erent symbols stand for separate cells. Mean slopes of the connecting lines are 0.91 þ 0.09 (N = 7) for C and 0.14 þ 0.04 (N = 6) for D.
HEARES 2889 28-11-97
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
102
came from the following observations : (i) marginal cells
which has been suggested to retain the normal distribu-
were cylindrical in shape, (ii) changes in cell volume
tion characteristics required for Student's
were found to predominate in the widths of the cell
cor and Cochran, 1989). A level of
trunk and basal infoldings (Fig. 1A) and a good corre-
cepted as signi¢cant.
p
6
t-test
(Snede-
0.05 was ac-
w) of the
lation was found between the change in width (
cell trunk and that of the basal processes (Fig. 1C), (iii) cell height was little a¡ected by changes in cell width (Fig. 1D), suggesting that marginal cells were unlikely to alter cell width without changing cell volume, and (iv)
dissociated
marginal
cells
responded
similarly
to
osmotic challenges in terms of cell width and cell height (data not shown). As illustrated in Fig. 1B, we ¢rst measured the widths of the cell trunk at three separate section levels for the cell bathed in control solution, and then similar measurements were repeated in test solutions
and
volume,
v=vc
also
v/vc ,
after
washout.
Thus,
the
relative
cell
may be de¢ned as
w=wc
2
1
w w1 w2 w3 =3 wc1 wc2 wc3 =3.
wc
and
where
Marginal cells in situ may behave di¡erently since changes in the volume of vestibular dark cells in situ, an
analogue
height
to
marginal
(Wangemann
and
cells,
predominate
Marcus,
1990 ;
in
cell
Wangemann
and Shiga, 1994).
2.3. Measurement of intracellular potential A high input impedance ampli¢er (model 707, WPI, New Haven, CT) was employed. Microelectrodes for conventional
intracellular
recordings
were
fabricated
from borosilicate glass capillaries, having a resistance of 100^200 M
6
when ¢lled with 1 M KCl. The refer-
ence in the bath was a £owing 3 M KCl electrode with a £ow rate of less than 100 nl/min. The tip of the reference electrode was placed downstream the test cell while perfusing the bath solution. Both the intracellular and bath electrodes were connected to the ampli¢er via AgAgCl wires. The criteria for acceptable recordings were : (i) an abrupt change of potential upon impalement, (ii)
6
stable potentials with
2 mV £uctuations for at least
30 s, (iii) a stable input resistance during recording, and (iv) a swift return of potential to its original baseline þ 3 mV upon withdrawal of the microelectrode. Input resistances were monitored by injection of current pulses (
3
0.1
which
nA,
100
ms)
appeared
as
at
a
frequency
voltage
of
de£ections
6
pulses/min,
superimposed
on the potential recordings.
3,
Fig. 2. E¡ects of single omissions of bath Cl
lated with reference to the control value (
2.4. Statistics
3,
N,
Data are expressed as means þ S.E.M. ( Relative
Student's
t-test
3
fore the ¢rst change of solution. A : Cl gluconate
cells).
values after
for a
data
Na , and K
v vc )
cell volume under isotonicity. Relative cell volume ( /
were
logarithmic
number of
compared
using
transformation,
vc )
on
was calcu-
read immediately be-
was replaced by equimolar
as indicated. B : Na was replaced by NMDG ; subse-
3
was restored and Cl quently, Na
3.
was replaced by gluconate
C:
3
K was replaced by Na ; subsequently, K was restored and Cl
was
replaced
each of A^C.
HEARES 2889 28-11-97
by
3.
gluconate
Data
are
means þ S.E.M.
(
N = 6)
for
103
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
2.5. Solutions and chemicals
Compositions of the solutions used are de¢ned in Table 1. Free Ca2 levels for `Cl-free' and `7.5 Cl' were V0.6 mM as determined with a Ca2 electrode. The bath (V0.1 ml) was constantly perfused at 1.5 ml/ min with solutions maintained at 37 þ 1³C. Bumetanide, ouabain, and 4-acetamido-4P-isothiocyanato-stilbene2,2P-disulfonic acid (SITS) were directly dissolved in test solutions. 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB) was dissolved in dimethyl sulfoxide (DMSO). When added to test solutions, this resulted in a DMSO concentration of 0.1%. This concentration of DMSO alone exerted no adverse e¡ect on cell volume ( =10). NPPB was a generous gift from Hoechst (Frankfurt, Germany). To make Na-free solution, Na was replaced by -methyl-D-glucamine (NMDG ). All the solutions were bu¡ered by phosphates (Table 1), and ¢nally adjusted to pH 7.4 at 37³C using a small amount of NaOH, NMDG, or gluconic acid. Ionic replacement was made on an equimolar basis. N
N
3. Results
3.1. Cell shrinkage induced by isotonic replacement of Cl
3,
Na
or K
in the bath
We ¢rst examined to what extent cell volume varies in response to individual omissions of major ions from the perfusate when the osmolality was kept isotonic throughout the experiment. Substitution for bath Cl3 with equimolar amounts of gluconate3 resulted in a rapid decrease in cell volume to 80% in 30 s, followed by a further decrease to 65^70% in 90 s. The decrease
was reversible and reproducible with respect to the time course and the ¢nal level of / c (Fig. 2A). In contrast, isotonic replacement of bath Na by an impermeant cation, -methyl-D-glucamine (NMDG), led to a much smaller reduction in cell volume to 94% (30 s) and to 91% (90 s), as shown in Fig. 2B. The removal of K also elicited a similar and modest shrinkage to 94% (30 s) and to 93% (90 s), as shown in Fig. 2C. When the `Na removal'- or `K removal'-induced shrinkage is compared with subsequent `Cl removal'-induced shrinkage, the additional e¡ect of `Cl removal' is apparent: the cells that had shrunken to 91^93% as a result of Na or K deprivation could further reduce their volumes to 64^67% within 2 min after omission of bath Cl3. This even occurred when Na or K levels had returned to normal (Fig. 2B and C). In order to characterize the `Cl removal'-induced volume decrease by the marginal cell, bath Cl3 was replaced by equimolar concentrations of di¡erent halides (Fig. 3). Cells subjected to Br3 replacement shrank only slightly to 97% in 1.5 min (Fig. 3A). Fluoride substitution resulted in a shrinkage to 87% in 1.5 min (Fig. 3B); and iodide substitution caused a stronger shrinkage to 82% in 1.5 min (Fig. 3C). All of the e¡ects of these halides were, however, smaller than that of gluconate3 , as shown in Fig. 3. The order of potency for these anions with respect to their ability to induce isotonic shrinkage was: gluconate3 s I3 s F3 s Br3 . This order was expected from the ion selectivity sequence of the volume-correlated Cl3 conductance in the marginal cell (Takeuchi and Irimajiri, 1996) (see Section 4). v v
N
3.2. E¡ect of Cl
3 channel blockers, bumetanide, ouabain
and high [K]o on `Cl removal'-induced shrinkage
As shown in Fig. 4A, `Cl removal'-induced cell
Fig. 3. E¡ect of the replacement of Cl3 by Br3 , F3 , or I3 on relative cell volume ( / c ). A^C: Bath Cl3 was replaced by Br3 , F3 , or I3 . After waiting for volume recovery in control solution for 2 min, Cl3 was replaced by gluconate3 (gluc3 ) for comparison. Meansþ S.E.M. ( =6) for each of A^C. v v
N
HEARES 2889 28-11-97
104
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
shrinkage in the presence of 0.2 mM NPPB (to 86% in 2 min) was smaller than the ¢rst `Cl removal'-induced
shrinkage in the absence of NPPB (to 68% in 2 min). SITS (0.5 mM) was essentially ine¡ective (Fig. 4B). The
Fig. 4. E¡ects of blockers and high [K ] on `Cl removal'-induced shrinkage. Relative cell volume (v/v ), as de¢ned in Fig. 2. In A and B, the second trial of Cl3 depletion was made in the presence of NPPB (0.2 mM, N = 6) or SITS (0.5 mM, N = 6) and the resulting degree of shrinkage was compared with that from the ¢rst trial. C: Bath Cl3 was isosmotically reduced to 7.5 mM in the presence (bum) or absence of 10 WM bumetanide (N = 6). D: Cl3 was replaced by equimolar gluconate3 in the presence of 1 mM ouabain (N = 6). E: Cl3 was lowered to 7.5 mM at 72 mM K or 3.6 mM K (N = 7). o
c
HEARES 2889 28-11-97
105
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
Fig. 5. E¡ects of omission of bath Na or K , and the e¡ect of bumetanide on cell volume recovery after `Cl removal'-induced shrinkage. Relative cell volume ( / ) is as de¢ned in Fig. 2. A: Time course of changes in / due to `Cl3 depletion/restoration'. Cells were exposed to Clfree solution for 4.5 min. B, C: Time course of / upon switching to Cl3 restored but Na or K depleted solution. D: Time course of changes in / showing the e¡ect of 10 WM bumetanide on cell volume recovery. Data are meansþ S.E.M. ( = 6) for each of A^D. v vc
v vc
v vc
N
v vc
partial inhibition by NPPB and the ine¡ectiveness of SITS are similar to the reported e¡ects of these drugs on whole-cell Cl3 conductance (Takeuchi and Irimajiri, 1996). We examined the possibility that other transporters might participate in `Cl removal'-induced shrinkage. Bumetanide (10 WM), a speci¢c blocker of the Na K -Cl3 cotransporter (e.g., McRoberts et al., 1982), induced by itself a slight shrinkage to 91% in 2 min. This was followed by a further shrinkage to 67% (2 min) after reducing the bath Cl3 to 7.5 mM in the presence of bumetanide (Fig. 4C). We have chosen this concentration (7.5 mM) for extracellular Cl3 because the binding of bumetanide to the Na -K -Cl3 cotransporter is known to become maximal within the range [Cl] = 5^10 mM (see Section 4). Similarly, ouabain (1 mM) caused a modest shrinkage to 91% in 2 min, followed by a further shrinkage to 64% (2 min) upon deprivation of Cl3 in the presence of ouabain (Fig. 4D). These results indicate that the normal volume of isolated marginal cells is partly regulated by both the Na -K -Cl3 cotransporter and the Na ,K ATPase, and that the main e¥ux pathway(s) responsio
ble for the `Cl removal'-induced shrinkage is essentially independent of the routes mediated by these two transporters. As for the K -Cl3 cotransporter and other transporters dependent on the K electrochemical gradient, we examined their involvement in this shrinkage by comparing the e¡ects of `low-[Cl] / -[K] ' and `low[Cl] / -[K] ' conditions using solutions of `7.5 Cl 72 K' and `7.5 Cl 3.6 K', respectively (Table 1). The experiments presented in Fig. 4E showed no statistically signi¢cant di¡erences between these two challenges with respect to either the maximally shrunken level or the time course, indicating that the K gradient does not a¡ect `Cl removal'-induced shrinkage (see Section 4). o high
o low
o
o
-K -Cl3
3.3. Involvement of the Na
cotransport in
recovery from `Cl removal'-induced shrinkage
In the absence of Cl3, Na or K , the cells that had shrunk after Cl3 depletion were unable to resume their original volume unless all the omitted ions were restored to the bath solution (Fig. 5). As shown in Fig. 5A, the shrunken state persisted for 4.5 min under the
HEARES 2889 28-11-97
106
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
Fig. 6. E¡ects on membrane potential (E ) of isotonically varied anionic conditions. A: Typical traces of alterations in E resulting from the speci¢ed substitutions. Arrow indicates impalement. Arrowheads a^g indicate points of timed measurements to be cited in B. B: Summary of changes in E upon replacement of bath Cl3 by gluconate3 . a^g specify time points as de¢ned in A. Meansþ S.E.M. (N =17). C: Initial change in E following a total replacement of Cl3 by another anion (vE ) normalized to vE of gluconateÿ . m
m
m
m
m
`Cl-free' condition. In the absence of bath Na , cells remained shrunken even when the bath Cl3 was restored (Fig. 5B). When the bath was switched to a solution containing normal levels of both Na and Cl3 but completely lacking in K , the cell volume only partially recovered from 70% to 77% in 60 s (Fig. 5C). Thus, full recovery from the `Cl-free'-induced shrinkage apparently required the simultaneous presence of Na, K and Cl3 in the bath, suggesting the involvement of the Na -K -Cl3 cotransport system in volume recovery. In the presence of 10 WM bumetanide, the cells that had shrunken under the Cl3 depleted condition displayed, upon Cl3 restoration, a partial recovery of their volume from 68% to 75% in 60 s and to 78% in 3 min (Fig. 5D). This was comparable to the level of cell volume recuperation observed under `K-free' recovery conditions (Fig. 5C). Following washout of bumetanide,
m
the v/v returned from 80% to 97% in 2 min (Fig. 5D). These results indicate again that the Na -K -Cl3 cotransporter plays a substantial role in volume recovery. To obtain a clue to the mechanism(s) underlying the bumetanide-insensitive volume recovery noted above, we applied either 4,4P-diisothiocyanatostilbene-2,2P-disulfonic acid (DIDS), amiloride or chlorothiazide (all at 0.1 mM) simultaneously with 10 WM bumetanide to the bath solutions. None of these blockers had any appreciable e¡ect on the bumetanide-insensitive fraction of volume recovery (data, not shown), and the nature of the mechanism(s) responsible was not pursued any further in this study. c
3.4. Changes in intracellular potential upon anion substitution
Based on the strong indication that volume-corre-
HEARES 2889 28-11-97
107
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
lated Cl3 conductance is involved in the `Cl removal'induced shrinkage, we investigated the changes in membrane potential ( m ) that occur in association with anion substitutions using conventional intracellular microelectrodes. Fig. 6A shows a typical cell's response to successive changes of bath anions. A detailed account of the `Cl-free (gluconate3 )'-induced changes in the m ( = 17) (summarized in Fig. 6B) is as follows: (i) the m for the `control' immediately after impalement was 39.6 þ 1.6 mV, (ii) the m for `control' immediately before switching to `Cl-free' was 33.9 þ 0.5 mV, (iii) when replacing bath Cl3 by gluconate3 , the m jumped to +75.0 þ 5.8 mV, (iv) 40 s after switching to gluconate3, the m returned to +2.8 þ 2.6 mV, and remained thereafter at a quasi-steady state, (v) the m 90 s after gluconate3 replacement was +5.8 þ 3.3 mV, and (vi) upon restoration of Cl3 , an abrupt negative shift of m to 320.1 þ2.7 mV occurred, and was followed by a return of the m to 34.5 þ 0.8 mV in 120 s. The initial changes in m upon replacement of Cl3 by halides are summarized in Fig. 6C. The initial change in m following a total replacement of Cl3 by another anion is designated as `v m '. The v m values normalized to those of gluconateÿ replacement were 60.3 þ5.5% for I3 , 40.3 þ 5.0% for F3, and 30.2 þ 0.2% for Br3 (Fig. 6C). These results show that the anion selectivity sequence of the Cl3 conductance responsible for the initial change in m is Cl3 = Br3 s F3 s I3 s gluconate3 , which is the same as that of the volume-correlated Cl3 conductance found in the marginal cell under a voltage clamp (Takeuchi and Irimajiri, 1996). E
E
N
E
E
E
E
E
E
E
E
E
E
E
E
4. Discussion
4.1. Cell shrinkage under isotonic conditions
The present results have clearly demonstrated that isolated, intact (not patch-clamped) marginal cells change their volume in response to changes in ionic conditions. This was especially true for variations in [Cl]o, even in an isotonic milieu. Although `low [Cl]o'induced cell shrinkage has been reported in rabbit renal cortical cells (Macknight, 1985), frog skin mitochondria-rich cells (Spring and Ussing, 1986), and vestibular dark cells (Wangemann and Shiga, 1994), so far the underlying mechanism(s) has not been de¢ned. In general, any reduction in cell volume under apparent isotonic conditions presupposes the presence of certain net e¥uxes of cytosolic solutes accompanied by an obligatory loss of water. Among solutes capable of crossing the plasma membrane, we have examined the contribution of K, Cl3 and Na to the cell volume changes observed. If the requirement for electroneutrality is taken into account, `Cl removal'-induced cell
shrinkage could be explained by either: (i) a combination of a Cl3 channel and a cation channel, (ii) electroneutral transporters such as a Na -K -2Cl3 cotransporter and a K -Cl3 cotransporter, or (iii) a combination of an electrogenic cation pump and anion channels, such as Na ,K-ATPase and a Cl3 channel. Less likely to be involved are substantial contributions of HCO3 3-Cl3 and Na -H exchangers, since our experiments were performed in the nominal absence of HCO3 3. 4.2. Relation between `Cl removal'-induced shrinkage and Cl
3
conductance
The sequence of ion potency in reducing cell volume (gluconate3 s I3 s F3 s Br3 ) is the reverse of the ion selectivity sequence of volume-correlated Cl3 conductance (Takeuchi and Irimajiri, 1996). The above observation is in support of a major role for the Cl3 channel in the `Cl removal'-induced shrinkage, since replacement of bath Cl3 by a less permeant anion species leads to an increased e¥ux of cell Cl3 driven by a steeper, outward Cl3 gradient imposed across the Cl3 channel. A large Cl3 conductance on the basolateral side of marginal cell epithelium has been reported (Wangemann et al., 1995). Slight decreases in / c due to Br3 substitution (Fig. 3A) may be attributed to retarded in£uxes for solutes via the Na-K -Cl3 cotransporter which is known to allow an incomplete replacement of Cl3 with Br3 (Owen and Prastein, 1985). The fact that the whole-cell patch-clamped marginal cells displayed a volume decrease of V35% in 60 s when a gradient of [Cl]i /[Cl]o =140 mM/15 mM was imposed (Takeuchi and Irimajiri, 1996), supports an essential involvement of the Cl3 channel in the observed shrinkage. The partial inhibition of the intact cell's shrinkage by NPPB (Fig. 4A), as well as the ine¡ectiveness of SITS (Fig. 4B), is reminiscent of the reported e¡ects of these drugs on the whole-cell Cl3 conductance of the marginal cell (Takeuchi and Irimajiri, 1996). The inhibition by NPPB at a relatively high concentration (0.2 mM) may be attributed to the inhibition of intracellular metabolism, such as oxidative phosphorylation in mitochondria. Both the major contribution of Cl3 conductance to cell membrane potential (Fig. 6A, B) and its ion selectivity sequence (Fig. 6C) are compatible with previous reports (Wangemann et al., 1995; Takeuchi et al., 1995; Takeuchi and Irimajiri, 1996) and are similar to observations made in vestibular dark cells (Wangemann and Marcus, 1992). Since membrane potential is largely dependent on Cl3 conductance, Cl3 removal markedly depolarizes the marginal cell (Fig. 6). This depolarization in turn should increase the driving force for the e¥ux of cations, most likely K . Although the mechanism(s) underlying the rapid m decrease from the peak value of 75 mV to 3 mV within 40 s after switching to
HEARES 2889 28-11-97
v v
E
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
108
`Cl-free' solution (Fig. 6A, B) was not investigated in
3 Cl
this study, the link between volume
(Takeuchi
and
conductance and cell
Irimajiri,
1996)
could
be
in-
to that of the `control', whereas the K' is much greater. Therefore, if K
vW
for `7.5 Cl 3.6
-Cl
3
cotransport
was the dominant mechanism for the observed cell volume
in cell volume was observed within 30 s of the change of
would be expected to be larger at `7.5 Cl 3.6 K' than
solution (Fig. 2A).
at `7.5 Cl 72 K'. This was not the case, however.
-K -Cl3
4.3. Are the Na
,K -ATPase
porter
as
an
shrinkage,
e¥ux
pathway
substantial
in
inhibition
shrinkage
`low
[Cl] '-induced
shrinkage
Assuming cation
that
e¥ux
also
indicates
that
o
an
isotonic
re-
by K does not a¡ect cell shrinkage.
a
conductive
placement of Na
involved in
-K -Cl3
the
o
induced
`Cl removal'-induced shrinkage ?
Assuming a major role for the Na
then
The ine¡ectiveness of `high [K] ' on the `low [Cl] '-
-Cl3
cotransporter, K
cotransporter and Na
changes,
o
volved in this mechanism, since an apparent decrease
route,
the
pathway
non-selective
functions cation
as
a
channel
cotrans-
found in both apical (Takeuchi et al., 1992) and baso-
the
`low
Cl'-induced
lateral (Takeuchi et al., 1995) membranes is a possible
of
`7.5
Cl'-induced
candidate since this channel does not discriminate be-
shrinkage should occur in the presence of bumetanide.
3 Cl
We have chosen this concentration (7.5 mM) of
tween Na
and K
.
in
It is surprising that the presence of ouabain in the
this particular experiment since bumetanide binding to
control solution caused slight cell shrinkage (Fig. 4D)
the Na
instead of swelling as observed in many other cells (e.g.,
-K -Cl3
cotransporter has been shown to be
o
is set at 5^10 mM (Forbush and
Strange, 1989). Although the mechanism(s) underlying
Palfrey, 1983 ; O'Grady et al., 1987 ; Hedge and Palfrey,
this shrinkage remains unknown, one possibility is that
1992)
inhibition of Na
maximal when [Cl]
and
reported
also
because
between
the
a
close
binding
correlation
of
this
has
blocking
been agent
,K
-ATPase leads to an elevated [Na]
which in turn reduces the driving force for the Na
3
-K
i
-
and the resulting inhibition of cotransport (Haas and
Cl
Forbush, 1986). Actually, bumetanide (10
ages have been reported for the frog bladder epithe-
WM) failed to
o
cotransporter. Similar ouabain-induced cell shrink-
inhibit the `7.5 mM [Cl] '-induced shrinkage (Fig. 4C).
lium (Davis and Finn, 1987) and vestibular dark cells
Thus,
(Wangemann and Marcus, 1990). The shrinkage evoked
Na
-K -Cl3
cotransport
cannot
be
the
major
by
e¥ux pathway responsible for the shrinkage. The weak shrinkage caused by bumetanide alone in the control solution (Fig. 4C, before 7.5 Cl gests that Na
-K -Cl3
3
trial) sug-
cotransport serves as a mecha-
switching
to
`Cl-free'
indicates that the Na
in
of
ouabain
shrinkage.
4.4. Role of the Na
age induced by the `Na-free' (Fig. 2B), `K-free' (Fig.
-K
-Cl
3
cotransporter in recovery
from `Cl removal'-induced shrinkage
2C) and bumetanide (Fig. 4C) also suggests the existence of a common mechanism underlying these three
presence
e¥ux pathway for cations during `Cl removal'-induced
nism for constitutive ion uptake by the marginal cell. The apparent similarity between the degrees of shrink-
the
,K -ATPase cannot be the major
The Na
-K
3
-Cl
cotransporter plays an important
cases of weak shrinkage, i.e., inhibition of solute in£ux-
role in solute transport in many types of epithelial and
es via the Na
non-epithelial cells (for reviews, see Geck and Heinz,
-K -Cl3
cotransporter.
o
The apparent inability of `high [K] ' to counterbal-
o
ance `low [Cl] '-induced shrinkage (Fig. 5C) clearly indicates that etry
can
3 K -Cl
be
cotransport with a 1 :1 stoichiom-
excluded
as
a
major
1986 ; the
Haas, 1989 ;
simultaneous
and
3 Cl
Haas, 1994). The requirement for
presence
of
extracellular
Na
,
K
in the full recovery from `Cl removal'-induced
mechanism(s)
shrinkage (Fig. 5A^C) indicates that the coupled trans-
underlying the `low [Cl] '-induced shrinkage. The ra-
port of these ion species is essential for volume recov-
tionale for this conclusion is as follows. If one assumes
ery. The marked inhibition of volume recovery by bu-
a stoichiometry of 1 K
metanide (Fig. 5D) also supports the notion that the
o
:1
Cl
3
for this cotransporter
(for review, see Lauf et al., 1992), the driving force (
vW)
Na
tive Cl
vW RT lnKi Cli =Ko Clo ;
2
where R and T have their usual meaning and square brackets with subscripts refer to ion the
cell.
Eq.
(2)
implies
that
a
lowering
o
constant by a raised [K] , should not a¡ect
i
i
as the cell's [K] [Cl]
o
vW,
of
is kept
3
channels is unlikely as this is not compatible
, K
and Cl
3
in the bath for full recovery (Fig.
5A^C).
Acknowledgments
as long
remains unchanged. In the experi-
ment shown in Fig. 5C, the
cotransporter contributes substantially to
activities in- or
[Cl] , in conditions where the product [K] [Cl]
o
3
with the requirement for the simultaneous presence of Na
o
-K -Cl
this process. A major involvement of bumetanide-sensi-
acting on it may be given by
outside
vW for `7.5 Cl 72 K' is equal
The authors thank Dr. Philine Wangemann for her critical comments, and Mrs. Takako Ichinowatari for
HEARES 2889 28-11-97
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109 her
secretarial
Grant-in-Aid 9671749)
work. for
from
This
study
Scienti¢c
The
was
Research
Ministry
of
supported (7671864
Education,
by and
Science,
Sports and Culture, Japan.
109
3
in winter £ounder intestine and bovine kidney outer medulla : [ H] bumetanide
binding
and
e¡ects
of
furosemide
analogues.
J. Membr. Biol. 96, 11^18. Owen, S.E., Prastein, M.L., 1985. Na/K/Cl cotransport in cultured human ¢broblasts. J. biol. Chem. 260, 1445^1451. Snedecor, G.W. and Cochran, W.G., 1989. Statistical Methods, 8th ed. Iowa State Press, Ames, IA.
References
Spring, K.R., Ussing, H.H., 1986. The volume of mitochondria-rich cells of frog skin epithelium. J. Membr. Biol. 92, 21^26. Strange, K., 1989. Ouabain-induced cell swelling in rabbit cortical
Davis, W.C., Finn, A.L., 1987. Interactions of sodium transport, cell volume, and calcium in frog urinary bladder. J. Gen. Physiol. 89, 687^702.
branes isolated from dog kidney outer medulla. J. Biol. Chem. 258, 11787^11792. P.,
Heinz,
E.,
1986.
The
Na-K-2Cl
cotransport
system.
Ann. Rev. Physiol. 51, 443^457.
C869^C885. Haas, M., Forbush, B., 1986. [ H]Bumetanide binding to duck red inhibition
of
(Na+K+2Cl)
co-transport.
J. Biol. Chem. 261, 8434^8441.
to the activated Na/K/2Cl cotransporter :
selectivity and kinetic
properties on ion binding sites. J. Membr. Biol. 126, 27^37. Kusakari, J., Ise, I., Comegys, T.H., Thalmann, I., Thalmann, R., 1978a. E¡ect of ethacrynic acid, furosemide, and ouabain upon the endocochlear potential and upon high energy phosphates of the stria vascularis. Laryngoscope 88, 12^37.
tion of the endocochlear potential by the new loop diuretic, bu-
Adragna,
N.C.,
3
H.,
Zade-Oppen,
port : properties and regulation. Am. J. Physiol. 263, C917^C932. Macknight, A.D.C., 1985. The role of anions in cellular volume regulation. P£ugers Arch. 405, S12^S16.
gerbils. J. Membr. Biol. 150, 47^62. S.,
Irimajiri,
A.,
1997.
3
£ux-related
Cl
isotonic
volume
changes by strial marginal cells dissociated from gerbils. Abstr. Assoc. Res. Otolaryngol. 20, 18.
2
Takeuchi, S., Marcus, D.C., Wangemann, P., 1992. Ca nonselective cation, maxi K
3
and Cl
-activated
channels in apical mem-
brane of marginal cells of stria vascularis. Hear. Res. 61, 86^96. Wangemann, P., 1995. Comparison of ion transport mechanisms be-
149^157. Wangemann, P., Liu, J., Marcus, D.C., 1995. Ion transport mechasecretion and the transepithelial voltage
across marginal cells of stria vascularis in vitro. Hear. Res. 84, 19^ 29.
-induced swelling of vestib and Cl3 and inhibited by
Wangemann, P., Marcus, D.C., 1990. K ular dark cells is dependent on Na
piretanide. P£ugers Arch. 416, 262^269.
Marcus, D.C., Shen, Z., 1994. Slowly activating voltage-dependent conductance is apical pathway for
con-
ductance in marginal cells dissociated from the stria vascularis of
nisms responsible for K
Fujise,
A.M.M., Ryu, K.H., Delpire, E., 1992. Erythrocyte K-Cl cotrans-
K
from gerbil stria vascularis. Hear. Res. 83, 89^100.
metanide. Acta Oto-Laryngol. 86, 336^341. J.,
secretion in vestibular dark
tween vestibular dark cells and strial marginal cells. Hear. Res. 90,
Kusakari, J., Kambayashi, J., Ise, I., Kawamoto, K., 1978b. Reduc-
Bauer,
Takeuchi, S., Ando, M., Kozakura, K., Saito, H., Irimajiri, A., 1995.
Takeuchi,
Hedge, R.S., Palfrey, H.C., 1992. Ionic e¡ects on bumetanide binding
P.K.,
channel current and K
Takeuchi, S., Irimajiri, A., 1996. A novel, volume-correlated Cl
3
with
apical IsK
Ion channels in basolateral membrane of marginal cells dissociated
Haas, M., 1994. The Na-K-Cl cotransporters. Am. J. Physiol. 267,
Lauf,
vascularis dissected from guinea pig. Hear. Res. 80, 86^92.
cells. J. Membr. Biol. 156, 25^35.
Haas, M., 1989. Properties and diversity of (Na-K-Cl) cotransporters.
correlation
vated K channel in luminal membrane of marginal cells of stria
Sunose, H., Liu, J., Shen, Z., Marcus, D.C., 1997. cAMP increases
J. Membr. Biol. 91, 97^105.
cells :
NaCl transport by principal cells. J. Membr.
Biol. 107, 249^261. Sunose, H., Ikeda, K., Suzuki, M., Takasaka, T., 1994. Voltage-acti-
3
Forbush, B., Palfrey, H.C., 1983. [ H]bumetanide binding to mem-
Geck,
collecting tubule :
K
secretion in vestibular
dark cells. Am. J. Physiol. 267, C857^C864.
Wangemann, vestibular
P., Marcus, D.C., 1992. The membrane potential of dark
cell
is
controlled
by
a
large
3
Cl
conductance.
Hear. Res. 62, 149^156.
McRoberts, J.A., Erlinger, S., Rindler, M.J., Saier, M.H., 1982. Furosemide-sensitive salt transport in the Madin-Darby canine kidney cell line. J. Biol. Chem. 257, 2260^2266.
Wangemann, P., Shiga, N., 1994. Cell volume control in vestibular dark cells during and after a hyposmotic challenge. Am. J. Physiol. 266, C1046^C1060.
O'Grady, S.M., Palfrey, H.C., Field, M., 1987. Na-K-2Cl cotransport
HEARES 2889 28-11-97
Changes in the volume of marginal cells induced by isotonic `Cl3 depletion/restoration': involvement of the Cl3 channel and Na-K-Cl3 cotransporter Shunji Takeuchi *, Motonori Ando, Akihiko Irimajiri Department of Physiology, Kochi Medical School, Nankoku 783, Japan
Received 3 March 1997; revised 10 June 1997; accepted 18 July 1997
Abstract
Marginal cells constitute the endolymph-facing epithelium responsible for the secretion of endolymph by the stria vascularis in the inner ear. We have studied the possible involvement of Cl3 conductance and Na -K -Cl3 cotransport in the mechanism of changes in cell volume upon isotonic Cl3 depletion/restoration. Changes in cell volume were estimated from video-microscopic images with the aid of an image processor. Marginal cells shrank to 80% of their original volume in 30 s and to 65^70% in 90 s upon total replacement of [Cl]o ( 150 mM) by gluconate3 , and the original volume of the shrunken cells was restored within 2 min after restoration of Cl3 . The order of potency of anions to induce isotonic shrinkage was gluconate3 I3 F3 Br3 . The cell shrinkage caused by Cl3 depletion was partially inhibited by 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB, 0.2 mM), but not by either 4-acetamido-4P-isothiocyanato-stilbene-2,2P-disulfonic acid (SITS, 0.5 mM), bumetanide (10 WM) or ouabain (1 mM). The cell shrinkage caused by a reduction of [Cl]o from 150 mM to 7.5 mM was not affected by [K]o in the range of 3.6 mM to 72 mM. These results suggest that the main efflux pathway(s) responsible for the `Cl removal'-induced shrinkage depends on volumecorrelated Cl3 conductance (Takeuchi and Irimajiri, J. Membrane Biol. 150, 47^62, 1996) and that this pathway(s) is essentially independent of the Na -K -Cl3 cotransporter, the Na ,K -ATPase, and the K -Cl3 cotransporter. With regard to volume recovery after isotonic shrinkage, its critical dependence on the simultaneous presence of Na , K and Cl3 in the bath and its substantial inhibition by bumetanide (10 WM) both indicate a major role for Na -K -Cl3 cotransport. The strong influence on cell volume of solute fluxes working through the Cl3 channel and the Na -K -Cl3 cotransporter implies an essential role for these pathways in the ion transport mechanism(s) of the marginal cell.
V
V
s s s
V
Keywords :
Cell volume; Cl3 channel; Na -K -Cl3 cotransporter; Inner ear; Gerbil
1. Introduction
Several ion transport mechanisms in marginal cells have been reported (for review, see Wangemann, 1995) and similarities in ion transport mechanisms between strial marginal cells and vestibular dark cells have been pointed out (Wangemann, 1995). In addition to the apical IsK channel as a pathway for K secretion (Sunose et al., 1994; Marcus and Shen, 1994; Sunose et al., 1997), the importance of the basolateral Cl3 conductance in the normal function of marginal cells has * Corresponding author. Tel.: +81 (888) 80 2311; Fax: +81 (888) 80 2310.
been suggested by recent electrophysiological studies. These include: (i) marked depolarization of the transepithelial potential across the marginal cell layer upon depletion of basolateral Cl3 (Wangemann et al., 1995), (ii) relatively high density of 80 pS Cl3 channels in the basolateral membrane of marginal cells (Takeuchi et al., 1995), and (iii) a large whole-cell Cl3 conductance in dissociated marginal cells (Takeuchi and Irimajiri, 1996). The basolateral Cl3 conductance can provide a Cl3 e¥ux pathway (Wangemann, 1995). Besides conductive pathways and the Na ,K -ATPase, the Na -K -Cl3 cotransporter is essential in the normal function of stria vascularis since: (i) the dark cell in the vestibular labyrinth, an analogue to the mar-
0378-5955 / 97 / $17.00 ß 1997 Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 7 ) 0 0 1 3 4 - 2
HEARES 2889 28-11-97
100 Table 1 Composition of solutions (in mM) Solution control
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
Ca-free
Cl-free or gluconate3 ^ 150 ^ ^ ^ ^ 1 ^ 4 ^ 1.6 0.4 ^ ^ ^ 5
Na-free
NaCl 150 150 ^ Na-gluconate ^ ^ ^ K-gluconate ^ ^ ^ NaX ^ ^ ^ NMDG-Cl ^ ^ 150 1 1 1 MgCl2 ^ ^ ^ MgSO4 CaCl2 0.7 ^ 0.7 ^ ^ ^ Ca-(gluconate)2 CaX2 ^ ^ ^ K2 HPO4 1.6 1.6 1.6 0.4 0.4 0.4 KH2 PO4 Na2 HPO4 ^ ^ ^ ^ ^ ^ NaH2 PO4 EGTA ^ 1 ^ glucose 5 5 5 NMDG, N-methyl-D-glucamine. EGTA, ethylene glycol bis (L-aminoethylether)-N,N,NP,NP-tetraacetic acid. X is Br, F, or I. a CaX2 = 0 mM for X3 = F3 because of its low solubility.
ginal cell in the cochlea, has Na -K-Cl3 cotransport activity (Wangemann and Marcus, 1990), (ii) the endocochlear potential is sensitive to loop diuretics (Kusakari et al., 1978a; Kusakari et al., 1978b), and (iii) bumetanide inhibits K secretion from strial marginal cells (Wangemann et al., 1995). Our preliminary study revealed that isolated intact marginal cells shrink appreciably when depleted of extracellular Cl3 and that the shrunken cells' original volume is restored when replenished with Cl3 (Takeuchi and Irimajiri, 1997), suggesting that the volume of marginal cells depends largely on Cl3 transport mechanisms. Although marginal cells in vivo are never exposed to Cl3 -free condition, `Cl removal' is utilized as an experimental procedure to induce isotonic cell shrinkage in this study. We characterize the anionic pathways involved in the changes in cell volume induced by isotonic Cl3 depletion/restoration, and con¢rm the functional activity of Cl3 channels and Na K -Cl3 cotransporters in intact (not patch-clamped) single marginal cells. This report also further strengthens the previous observation of the existence of similarities between vestibular dark cells and strial marginal cells (Wangemann, 1995). 2. Methods
2.1. Cell preparation
Marginal cells were prepared from the inner ear of gerbils as described previously (Takeuchi et al., 1995). Brie£y, cochleae were obtained under anesthesia with
K-free
7.5 Cl 3.6 K
7.5 Cl 72 K
X3
150 ^ ^ ^ ^ 1 ^ 0.7 ^ ^ ^ ^ 1.6 0.4 ^ 5
5.5 142.5 ^ ^ ^ 1 ^ ^ 4 ^ 1.6 0.4 ^ ^ ^ 5
5.5 74.1 68.4 ^ ^ 1 ^ ^ 4 ^ 1.6 0.4 ^ ^ ^ 5
^ ^ ^ 150 ^ ^ 1 ^ ^ 0.7a 1.6 0.4 ^ ^ ^ 5
pentobarbital sodium (50 mg/kg, i.p.). Tissue strips from the stria vascularis freed of the spiral ligament were incubated for 20 min at 23^25³C in `Ca-free' solution (for composition, see Table 1) containing 0.2% papain and kept up to 3 h in cold (8³C) control solution (Table 1) until use. These strips were transferred to a bath mounted on an inverted microscope (TMD 300, Nikon, Tokyo, Japan) and dissected with ¢ne needles under visual control. Single marginal cells retaining their characteristic morphology (i.e., a relatively smooth apical membrane, numerous basal infoldings, and an apically located nucleus) were selected and separated from other dissociated cells. The care and use of animals used in this study were approved by the Kochi Medical School Animal Care and Use Committee. 2.2. Measurement of relative cell volume
The test cell was held at the center of the visual ¢eld by means of a holding pipette. The cell's pro¢le was viewed through Nomarski optics with a 100U objective (NA 1.4) under oil immersion. The bottom of the recording bath was made of a glass coverslip 0.12^0.17 mm thick. Cell images were recorded with a high-resolution black/white TV camera (SSC-M370, Sony, Tokyo, Japan) and a video cassette recorder (S-2200, Sony). Cell width measurements were made, o¡ line, with the aid of an image processor (Argus 10, Hamamatsu Photonics, Hamamatsu, Japan). We estimated relative cell volume from measurements of the width of the cell trunk in sharp focus in essentially the same way as described previously (Takeuchi and Irimajiri, 1996). The rationale behind this
HEARES 2889 28-11-97
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
101
Fig. 1. Estimation of cell volume changes. A : Video-microscopic images of a typical marginal cell undergoing a reversible volume change. The
3;
cell is held by a suction pipette (p) at basal infoldings with its apex turned upwards. Left, control ; center, 90 s after omission of Cl
3
2 min after restoration of Cl
(control solution). Scale bar, 10
Wm.
right,
B : Schematic drawings of the cell under control (left) and test (right) condi-
tions, with the de¢nition of size parameters attached. C : Correlation between the rate of change in width of the cell trunk and that of the basal part. Note that changes in the trunk width elicited by depletion and restoration of bath Cl
3
parallel those of the basal part. D : Similar plots
showing much weaker changes in cell height. In both C and D, dimensional measurements were made with cells just before shrinkage (control), at their maximum shrinkage and after full recovery to their initial volumes. Paired symbols connected by solid lines refer to an identical cell. Di¡erent symbols stand for separate cells. Mean slopes of the connecting lines are 0.91 þ 0.09 (N = 7) for C and 0.14 þ 0.04 (N = 6) for D.
HEARES 2889 28-11-97
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
102
came from the following observations : (i) marginal cells
which has been suggested to retain the normal distribu-
were cylindrical in shape, (ii) changes in cell volume
tion characteristics required for Student's
were found to predominate in the widths of the cell
cor and Cochran, 1989). A level of
trunk and basal infoldings (Fig. 1A) and a good corre-
cepted as signi¢cant.
p
6
t-test
(Snede-
0.05 was ac-
w) of the
lation was found between the change in width (
cell trunk and that of the basal processes (Fig. 1C), (iii) cell height was little a¡ected by changes in cell width (Fig. 1D), suggesting that marginal cells were unlikely to alter cell width without changing cell volume, and (iv)
dissociated
marginal
cells
responded
similarly
to
osmotic challenges in terms of cell width and cell height (data not shown). As illustrated in Fig. 1B, we ¢rst measured the widths of the cell trunk at three separate section levels for the cell bathed in control solution, and then similar measurements were repeated in test solutions
and
volume,
v=vc
also
v/vc ,
after
washout.
Thus,
the
relative
cell
may be de¢ned as
w=wc
2
1
w w1 w2 w3 =3 wc1 wc2 wc3 =3.
wc
and
where
Marginal cells in situ may behave di¡erently since changes in the volume of vestibular dark cells in situ, an
analogue
height
to
marginal
(Wangemann
and
cells,
predominate
Marcus,
1990 ;
in
cell
Wangemann
and Shiga, 1994).
2.3. Measurement of intracellular potential A high input impedance ampli¢er (model 707, WPI, New Haven, CT) was employed. Microelectrodes for conventional
intracellular
recordings
were
fabricated
from borosilicate glass capillaries, having a resistance of 100^200 M
6
when ¢lled with 1 M KCl. The refer-
ence in the bath was a £owing 3 M KCl electrode with a £ow rate of less than 100 nl/min. The tip of the reference electrode was placed downstream the test cell while perfusing the bath solution. Both the intracellular and bath electrodes were connected to the ampli¢er via AgAgCl wires. The criteria for acceptable recordings were : (i) an abrupt change of potential upon impalement, (ii)
6
stable potentials with
2 mV £uctuations for at least
30 s, (iii) a stable input resistance during recording, and (iv) a swift return of potential to its original baseline þ 3 mV upon withdrawal of the microelectrode. Input resistances were monitored by injection of current pulses (
3
0.1
which
nA,
100
ms)
appeared
as
at
a
frequency
voltage
of
de£ections
6
pulses/min,
superimposed
on the potential recordings.
3,
Fig. 2. E¡ects of single omissions of bath Cl
lated with reference to the control value (
2.4. Statistics
3,
N,
Data are expressed as means þ S.E.M. ( Relative
Student's
t-test
3
fore the ¢rst change of solution. A : Cl gluconate
cells).
values after
for a
data
Na , and K
v vc )
cell volume under isotonicity. Relative cell volume ( /
were
logarithmic
number of
compared
using
transformation,
vc )
on
was calcu-
read immediately be-
was replaced by equimolar
as indicated. B : Na was replaced by NMDG ; subse-
3
was restored and Cl quently, Na
3.
was replaced by gluconate
C:
3
K was replaced by Na ; subsequently, K was restored and Cl
was
replaced
each of A^C.
HEARES 2889 28-11-97
by
3.
gluconate
Data
are
means þ S.E.M.
(
N = 6)
for
103
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
2.5. Solutions and chemicals
Compositions of the solutions used are de¢ned in Table 1. Free Ca2 levels for `Cl-free' and `7.5 Cl' were V0.6 mM as determined with a Ca2 electrode. The bath (V0.1 ml) was constantly perfused at 1.5 ml/ min with solutions maintained at 37 þ 1³C. Bumetanide, ouabain, and 4-acetamido-4P-isothiocyanato-stilbene2,2P-disulfonic acid (SITS) were directly dissolved in test solutions. 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB) was dissolved in dimethyl sulfoxide (DMSO). When added to test solutions, this resulted in a DMSO concentration of 0.1%. This concentration of DMSO alone exerted no adverse e¡ect on cell volume ( =10). NPPB was a generous gift from Hoechst (Frankfurt, Germany). To make Na-free solution, Na was replaced by -methyl-D-glucamine (NMDG ). All the solutions were bu¡ered by phosphates (Table 1), and ¢nally adjusted to pH 7.4 at 37³C using a small amount of NaOH, NMDG, or gluconic acid. Ionic replacement was made on an equimolar basis. N
N
3. Results
3.1. Cell shrinkage induced by isotonic replacement of Cl
3,
Na
or K
in the bath
We ¢rst examined to what extent cell volume varies in response to individual omissions of major ions from the perfusate when the osmolality was kept isotonic throughout the experiment. Substitution for bath Cl3 with equimolar amounts of gluconate3 resulted in a rapid decrease in cell volume to 80% in 30 s, followed by a further decrease to 65^70% in 90 s. The decrease
was reversible and reproducible with respect to the time course and the ¢nal level of / c (Fig. 2A). In contrast, isotonic replacement of bath Na by an impermeant cation, -methyl-D-glucamine (NMDG), led to a much smaller reduction in cell volume to 94% (30 s) and to 91% (90 s), as shown in Fig. 2B. The removal of K also elicited a similar and modest shrinkage to 94% (30 s) and to 93% (90 s), as shown in Fig. 2C. When the `Na removal'- or `K removal'-induced shrinkage is compared with subsequent `Cl removal'-induced shrinkage, the additional e¡ect of `Cl removal' is apparent: the cells that had shrunken to 91^93% as a result of Na or K deprivation could further reduce their volumes to 64^67% within 2 min after omission of bath Cl3. This even occurred when Na or K levels had returned to normal (Fig. 2B and C). In order to characterize the `Cl removal'-induced volume decrease by the marginal cell, bath Cl3 was replaced by equimolar concentrations of di¡erent halides (Fig. 3). Cells subjected to Br3 replacement shrank only slightly to 97% in 1.5 min (Fig. 3A). Fluoride substitution resulted in a shrinkage to 87% in 1.5 min (Fig. 3B); and iodide substitution caused a stronger shrinkage to 82% in 1.5 min (Fig. 3C). All of the e¡ects of these halides were, however, smaller than that of gluconate3 , as shown in Fig. 3. The order of potency for these anions with respect to their ability to induce isotonic shrinkage was: gluconate3 s I3 s F3 s Br3 . This order was expected from the ion selectivity sequence of the volume-correlated Cl3 conductance in the marginal cell (Takeuchi and Irimajiri, 1996) (see Section 4). v v
N
3.2. E¡ect of Cl
3 channel blockers, bumetanide, ouabain
and high [K]o on `Cl removal'-induced shrinkage
As shown in Fig. 4A, `Cl removal'-induced cell
Fig. 3. E¡ect of the replacement of Cl3 by Br3 , F3 , or I3 on relative cell volume ( / c ). A^C: Bath Cl3 was replaced by Br3 , F3 , or I3 . After waiting for volume recovery in control solution for 2 min, Cl3 was replaced by gluconate3 (gluc3 ) for comparison. Meansþ S.E.M. ( =6) for each of A^C. v v
N
HEARES 2889 28-11-97
104
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
shrinkage in the presence of 0.2 mM NPPB (to 86% in 2 min) was smaller than the ¢rst `Cl removal'-induced
shrinkage in the absence of NPPB (to 68% in 2 min). SITS (0.5 mM) was essentially ine¡ective (Fig. 4B). The
Fig. 4. E¡ects of blockers and high [K ] on `Cl removal'-induced shrinkage. Relative cell volume (v/v ), as de¢ned in Fig. 2. In A and B, the second trial of Cl3 depletion was made in the presence of NPPB (0.2 mM, N = 6) or SITS (0.5 mM, N = 6) and the resulting degree of shrinkage was compared with that from the ¢rst trial. C: Bath Cl3 was isosmotically reduced to 7.5 mM in the presence (bum) or absence of 10 WM bumetanide (N = 6). D: Cl3 was replaced by equimolar gluconate3 in the presence of 1 mM ouabain (N = 6). E: Cl3 was lowered to 7.5 mM at 72 mM K or 3.6 mM K (N = 7). o
c
HEARES 2889 28-11-97
105
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
Fig. 5. E¡ects of omission of bath Na or K , and the e¡ect of bumetanide on cell volume recovery after `Cl removal'-induced shrinkage. Relative cell volume ( / ) is as de¢ned in Fig. 2. A: Time course of changes in / due to `Cl3 depletion/restoration'. Cells were exposed to Clfree solution for 4.5 min. B, C: Time course of / upon switching to Cl3 restored but Na or K depleted solution. D: Time course of changes in / showing the e¡ect of 10 WM bumetanide on cell volume recovery. Data are meansþ S.E.M. ( = 6) for each of A^D. v vc
v vc
v vc
N
v vc
partial inhibition by NPPB and the ine¡ectiveness of SITS are similar to the reported e¡ects of these drugs on whole-cell Cl3 conductance (Takeuchi and Irimajiri, 1996). We examined the possibility that other transporters might participate in `Cl removal'-induced shrinkage. Bumetanide (10 WM), a speci¢c blocker of the Na K -Cl3 cotransporter (e.g., McRoberts et al., 1982), induced by itself a slight shrinkage to 91% in 2 min. This was followed by a further shrinkage to 67% (2 min) after reducing the bath Cl3 to 7.5 mM in the presence of bumetanide (Fig. 4C). We have chosen this concentration (7.5 mM) for extracellular Cl3 because the binding of bumetanide to the Na -K -Cl3 cotransporter is known to become maximal within the range [Cl] = 5^10 mM (see Section 4). Similarly, ouabain (1 mM) caused a modest shrinkage to 91% in 2 min, followed by a further shrinkage to 64% (2 min) upon deprivation of Cl3 in the presence of ouabain (Fig. 4D). These results indicate that the normal volume of isolated marginal cells is partly regulated by both the Na -K -Cl3 cotransporter and the Na ,K ATPase, and that the main e¥ux pathway(s) responsio
ble for the `Cl removal'-induced shrinkage is essentially independent of the routes mediated by these two transporters. As for the K -Cl3 cotransporter and other transporters dependent on the K electrochemical gradient, we examined their involvement in this shrinkage by comparing the e¡ects of `low-[Cl] / -[K] ' and `low[Cl] / -[K] ' conditions using solutions of `7.5 Cl 72 K' and `7.5 Cl 3.6 K', respectively (Table 1). The experiments presented in Fig. 4E showed no statistically signi¢cant di¡erences between these two challenges with respect to either the maximally shrunken level or the time course, indicating that the K gradient does not a¡ect `Cl removal'-induced shrinkage (see Section 4). o high
o low
o
o
-K -Cl3
3.3. Involvement of the Na
cotransport in
recovery from `Cl removal'-induced shrinkage
In the absence of Cl3, Na or K , the cells that had shrunk after Cl3 depletion were unable to resume their original volume unless all the omitted ions were restored to the bath solution (Fig. 5). As shown in Fig. 5A, the shrunken state persisted for 4.5 min under the
HEARES 2889 28-11-97
106
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
Fig. 6. E¡ects on membrane potential (E ) of isotonically varied anionic conditions. A: Typical traces of alterations in E resulting from the speci¢ed substitutions. Arrow indicates impalement. Arrowheads a^g indicate points of timed measurements to be cited in B. B: Summary of changes in E upon replacement of bath Cl3 by gluconate3 . a^g specify time points as de¢ned in A. Meansþ S.E.M. (N =17). C: Initial change in E following a total replacement of Cl3 by another anion (vE ) normalized to vE of gluconateÿ . m
m
m
m
m
`Cl-free' condition. In the absence of bath Na , cells remained shrunken even when the bath Cl3 was restored (Fig. 5B). When the bath was switched to a solution containing normal levels of both Na and Cl3 but completely lacking in K , the cell volume only partially recovered from 70% to 77% in 60 s (Fig. 5C). Thus, full recovery from the `Cl-free'-induced shrinkage apparently required the simultaneous presence of Na, K and Cl3 in the bath, suggesting the involvement of the Na -K -Cl3 cotransport system in volume recovery. In the presence of 10 WM bumetanide, the cells that had shrunken under the Cl3 depleted condition displayed, upon Cl3 restoration, a partial recovery of their volume from 68% to 75% in 60 s and to 78% in 3 min (Fig. 5D). This was comparable to the level of cell volume recuperation observed under `K-free' recovery conditions (Fig. 5C). Following washout of bumetanide,
m
the v/v returned from 80% to 97% in 2 min (Fig. 5D). These results indicate again that the Na -K -Cl3 cotransporter plays a substantial role in volume recovery. To obtain a clue to the mechanism(s) underlying the bumetanide-insensitive volume recovery noted above, we applied either 4,4P-diisothiocyanatostilbene-2,2P-disulfonic acid (DIDS), amiloride or chlorothiazide (all at 0.1 mM) simultaneously with 10 WM bumetanide to the bath solutions. None of these blockers had any appreciable e¡ect on the bumetanide-insensitive fraction of volume recovery (data, not shown), and the nature of the mechanism(s) responsible was not pursued any further in this study. c
3.4. Changes in intracellular potential upon anion substitution
Based on the strong indication that volume-corre-
HEARES 2889 28-11-97
107
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
lated Cl3 conductance is involved in the `Cl removal'induced shrinkage, we investigated the changes in membrane potential ( m ) that occur in association with anion substitutions using conventional intracellular microelectrodes. Fig. 6A shows a typical cell's response to successive changes of bath anions. A detailed account of the `Cl-free (gluconate3 )'-induced changes in the m ( = 17) (summarized in Fig. 6B) is as follows: (i) the m for the `control' immediately after impalement was 39.6 þ 1.6 mV, (ii) the m for `control' immediately before switching to `Cl-free' was 33.9 þ 0.5 mV, (iii) when replacing bath Cl3 by gluconate3 , the m jumped to +75.0 þ 5.8 mV, (iv) 40 s after switching to gluconate3, the m returned to +2.8 þ 2.6 mV, and remained thereafter at a quasi-steady state, (v) the m 90 s after gluconate3 replacement was +5.8 þ 3.3 mV, and (vi) upon restoration of Cl3 , an abrupt negative shift of m to 320.1 þ2.7 mV occurred, and was followed by a return of the m to 34.5 þ 0.8 mV in 120 s. The initial changes in m upon replacement of Cl3 by halides are summarized in Fig. 6C. The initial change in m following a total replacement of Cl3 by another anion is designated as `v m '. The v m values normalized to those of gluconateÿ replacement were 60.3 þ5.5% for I3 , 40.3 þ 5.0% for F3, and 30.2 þ 0.2% for Br3 (Fig. 6C). These results show that the anion selectivity sequence of the Cl3 conductance responsible for the initial change in m is Cl3 = Br3 s F3 s I3 s gluconate3 , which is the same as that of the volume-correlated Cl3 conductance found in the marginal cell under a voltage clamp (Takeuchi and Irimajiri, 1996). E
E
N
E
E
E
E
E
E
E
E
E
E
E
E
4. Discussion
4.1. Cell shrinkage under isotonic conditions
The present results have clearly demonstrated that isolated, intact (not patch-clamped) marginal cells change their volume in response to changes in ionic conditions. This was especially true for variations in [Cl]o, even in an isotonic milieu. Although `low [Cl]o'induced cell shrinkage has been reported in rabbit renal cortical cells (Macknight, 1985), frog skin mitochondria-rich cells (Spring and Ussing, 1986), and vestibular dark cells (Wangemann and Shiga, 1994), so far the underlying mechanism(s) has not been de¢ned. In general, any reduction in cell volume under apparent isotonic conditions presupposes the presence of certain net e¥uxes of cytosolic solutes accompanied by an obligatory loss of water. Among solutes capable of crossing the plasma membrane, we have examined the contribution of K, Cl3 and Na to the cell volume changes observed. If the requirement for electroneutrality is taken into account, `Cl removal'-induced cell
shrinkage could be explained by either: (i) a combination of a Cl3 channel and a cation channel, (ii) electroneutral transporters such as a Na -K -2Cl3 cotransporter and a K -Cl3 cotransporter, or (iii) a combination of an electrogenic cation pump and anion channels, such as Na ,K-ATPase and a Cl3 channel. Less likely to be involved are substantial contributions of HCO3 3-Cl3 and Na -H exchangers, since our experiments were performed in the nominal absence of HCO3 3. 4.2. Relation between `Cl removal'-induced shrinkage and Cl
3
conductance
The sequence of ion potency in reducing cell volume (gluconate3 s I3 s F3 s Br3 ) is the reverse of the ion selectivity sequence of volume-correlated Cl3 conductance (Takeuchi and Irimajiri, 1996). The above observation is in support of a major role for the Cl3 channel in the `Cl removal'-induced shrinkage, since replacement of bath Cl3 by a less permeant anion species leads to an increased e¥ux of cell Cl3 driven by a steeper, outward Cl3 gradient imposed across the Cl3 channel. A large Cl3 conductance on the basolateral side of marginal cell epithelium has been reported (Wangemann et al., 1995). Slight decreases in / c due to Br3 substitution (Fig. 3A) may be attributed to retarded in£uxes for solutes via the Na-K -Cl3 cotransporter which is known to allow an incomplete replacement of Cl3 with Br3 (Owen and Prastein, 1985). The fact that the whole-cell patch-clamped marginal cells displayed a volume decrease of V35% in 60 s when a gradient of [Cl]i /[Cl]o =140 mM/15 mM was imposed (Takeuchi and Irimajiri, 1996), supports an essential involvement of the Cl3 channel in the observed shrinkage. The partial inhibition of the intact cell's shrinkage by NPPB (Fig. 4A), as well as the ine¡ectiveness of SITS (Fig. 4B), is reminiscent of the reported e¡ects of these drugs on the whole-cell Cl3 conductance of the marginal cell (Takeuchi and Irimajiri, 1996). The inhibition by NPPB at a relatively high concentration (0.2 mM) may be attributed to the inhibition of intracellular metabolism, such as oxidative phosphorylation in mitochondria. Both the major contribution of Cl3 conductance to cell membrane potential (Fig. 6A, B) and its ion selectivity sequence (Fig. 6C) are compatible with previous reports (Wangemann et al., 1995; Takeuchi et al., 1995; Takeuchi and Irimajiri, 1996) and are similar to observations made in vestibular dark cells (Wangemann and Marcus, 1992). Since membrane potential is largely dependent on Cl3 conductance, Cl3 removal markedly depolarizes the marginal cell (Fig. 6). This depolarization in turn should increase the driving force for the e¥ux of cations, most likely K . Although the mechanism(s) underlying the rapid m decrease from the peak value of 75 mV to 3 mV within 40 s after switching to
HEARES 2889 28-11-97
v v
E
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109
108
`Cl-free' solution (Fig. 6A, B) was not investigated in
3 Cl
this study, the link between volume
(Takeuchi
and
conductance and cell
Irimajiri,
1996)
could
be
in-
to that of the `control', whereas the K' is much greater. Therefore, if K
vW
for `7.5 Cl 3.6
-Cl
3
cotransport
was the dominant mechanism for the observed cell volume
in cell volume was observed within 30 s of the change of
would be expected to be larger at `7.5 Cl 3.6 K' than
solution (Fig. 2A).
at `7.5 Cl 72 K'. This was not the case, however.
-K -Cl3
4.3. Are the Na
,K -ATPase
porter
as
an
shrinkage,
e¥ux
pathway
substantial
in
inhibition
shrinkage
`low
[Cl] '-induced
shrinkage
Assuming cation
that
e¥ux
also
indicates
that
o
an
isotonic
re-
by K does not a¡ect cell shrinkage.
a
conductive
placement of Na
involved in
-K -Cl3
the
o
induced
`Cl removal'-induced shrinkage ?
Assuming a major role for the Na
then
The ine¡ectiveness of `high [K] ' on the `low [Cl] '-
-Cl3
cotransporter, K
cotransporter and Na
changes,
o
volved in this mechanism, since an apparent decrease
route,
the
pathway
non-selective
functions cation
as
a
channel
cotrans-
found in both apical (Takeuchi et al., 1992) and baso-
the
`low
Cl'-induced
lateral (Takeuchi et al., 1995) membranes is a possible
of
`7.5
Cl'-induced
candidate since this channel does not discriminate be-
shrinkage should occur in the presence of bumetanide.
3 Cl
We have chosen this concentration (7.5 mM) of
tween Na
and K
.
in
It is surprising that the presence of ouabain in the
this particular experiment since bumetanide binding to
control solution caused slight cell shrinkage (Fig. 4D)
the Na
instead of swelling as observed in many other cells (e.g.,
-K -Cl3
cotransporter has been shown to be
o
is set at 5^10 mM (Forbush and
Strange, 1989). Although the mechanism(s) underlying
Palfrey, 1983 ; O'Grady et al., 1987 ; Hedge and Palfrey,
this shrinkage remains unknown, one possibility is that
1992)
inhibition of Na
maximal when [Cl]
and
reported
also
because
between
the
a
close
binding
correlation
of
this
has
blocking
been agent
,K
-ATPase leads to an elevated [Na]
which in turn reduces the driving force for the Na
3
-K
i
-
and the resulting inhibition of cotransport (Haas and
Cl
Forbush, 1986). Actually, bumetanide (10
ages have been reported for the frog bladder epithe-
WM) failed to
o
cotransporter. Similar ouabain-induced cell shrink-
inhibit the `7.5 mM [Cl] '-induced shrinkage (Fig. 4C).
lium (Davis and Finn, 1987) and vestibular dark cells
Thus,
(Wangemann and Marcus, 1990). The shrinkage evoked
Na
-K -Cl3
cotransport
cannot
be
the
major
by
e¥ux pathway responsible for the shrinkage. The weak shrinkage caused by bumetanide alone in the control solution (Fig. 4C, before 7.5 Cl gests that Na
-K -Cl3
3
trial) sug-
cotransport serves as a mecha-
switching
to
`Cl-free'
indicates that the Na
in
of
ouabain
shrinkage.
4.4. Role of the Na
age induced by the `Na-free' (Fig. 2B), `K-free' (Fig.
-K
-Cl
3
cotransporter in recovery
from `Cl removal'-induced shrinkage
2C) and bumetanide (Fig. 4C) also suggests the existence of a common mechanism underlying these three
presence
e¥ux pathway for cations during `Cl removal'-induced
nism for constitutive ion uptake by the marginal cell. The apparent similarity between the degrees of shrink-
the
,K -ATPase cannot be the major
The Na
-K
3
-Cl
cotransporter plays an important
cases of weak shrinkage, i.e., inhibition of solute in£ux-
role in solute transport in many types of epithelial and
es via the Na
non-epithelial cells (for reviews, see Geck and Heinz,
-K -Cl3
cotransporter.
o
The apparent inability of `high [K] ' to counterbal-
o
ance `low [Cl] '-induced shrinkage (Fig. 5C) clearly indicates that etry
can
3 K -Cl
be
cotransport with a 1 :1 stoichiom-
excluded
as
a
major
1986 ; the
Haas, 1989 ;
simultaneous
and
3 Cl
Haas, 1994). The requirement for
presence
of
extracellular
Na
,
K
in the full recovery from `Cl removal'-induced
mechanism(s)
shrinkage (Fig. 5A^C) indicates that the coupled trans-
underlying the `low [Cl] '-induced shrinkage. The ra-
port of these ion species is essential for volume recov-
tionale for this conclusion is as follows. If one assumes
ery. The marked inhibition of volume recovery by bu-
a stoichiometry of 1 K
metanide (Fig. 5D) also supports the notion that the
o
:1
Cl
3
for this cotransporter
(for review, see Lauf et al., 1992), the driving force (
vW)
Na
tive Cl
vW RT lnKi Cli =Ko Clo ;
2
where R and T have their usual meaning and square brackets with subscripts refer to ion the
cell.
Eq.
(2)
implies
that
a
lowering
o
constant by a raised [K] , should not a¡ect
i
i
as the cell's [K] [Cl]
o
vW,
of
is kept
3
channels is unlikely as this is not compatible
, K
and Cl
3
in the bath for full recovery (Fig.
5A^C).
Acknowledgments
as long
remains unchanged. In the experi-
ment shown in Fig. 5C, the
cotransporter contributes substantially to
activities in- or
[Cl] , in conditions where the product [K] [Cl]
o
3
with the requirement for the simultaneous presence of Na
o
-K -Cl
this process. A major involvement of bumetanide-sensi-
acting on it may be given by
outside
vW for `7.5 Cl 72 K' is equal
The authors thank Dr. Philine Wangemann for her critical comments, and Mrs. Takako Ichinowatari for
HEARES 2889 28-11-97
S. Takeuchi et al. / Hearing Research 113 (1997) 99^109 her
secretarial
Grant-in-Aid 9671749)
work. for
from
This
study
Scienti¢c
The
was
Research
Ministry
of
supported (7671864
Education,
by and
Science,
Sports and Culture, Japan.
109
3
in winter £ounder intestine and bovine kidney outer medulla : [ H] bumetanide
binding
and
e¡ects
of
furosemide
analogues.
J. Membr. Biol. 96, 11^18. Owen, S.E., Prastein, M.L., 1985. Na/K/Cl cotransport in cultured human ¢broblasts. J. biol. Chem. 260, 1445^1451. Snedecor, G.W. and Cochran, W.G., 1989. Statistical Methods, 8th ed. Iowa State Press, Ames, IA.
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