Delayed shortening and shrinkage of cochlear outer hair cells SUM10 OHNISHI, MITSUYOSHI HARA, MASAFUMI INOUE, TOSHIO TADAMI KUMAZAWA, AK10 MINATO, AND CHIYOKO INAGAKI Departments of Pharmacology and Department of Chemistry,
and Otolaryngology, Kansai Medical University, Osaka 570; Kyoto Pharmaceutical University, Kyoto 607, Japan
Ohnishi, Sumio, Mitsuyoshi Hara, Masafumi Inoue, Toshio Yamashita, Tadami Kumazawa, Akio Minato, and Chiyoko Inagaki. Delayed shortening and shrinkage of cochlear outer hair cells. Am. J. Physiol. 263 (Cell Physiol. 32): C1088-C1095, 1992.-Slow shortening of cochlear outer hair cells has been speculated to modify cochlear sensitivity. Tetanic electrical field stimulation of isolated outer hair cells from guinea pigs shortened the cells for 2-3 min. Electrical stimulation reduced cell length and volume (-13.5 t 1.5 and -37.3 t 3.0% of initial values, respectively, n = 16) and decreased the intracellular Cl- concentration. Cytochalasin B (100 PM) inhibited electrical stimulation-induced shortening but not volume reduction. The following chemicals or manipulations inhibited the responses: 10 PM furosemide, 0.1 mM 4,4’diisothiocyanostilbene-2,2’-disulfonic acid (DIDS), 1 mM anthracene-9-carboxylic acid (AC9), 25 mM tetraethylammonium, 2.3 PM charybdotoxin (ChTX), 250 nM o-conotoxin, and Ca”+-free medium. These findings suggest that both electrical stimulation-induced shortening and shrinkage of outer hair cells result not only from an actin-mediated contractile force, but also from Cl- efflux through furosemide-, DIDS-, and AC9sensitive Cl- channels, and K+ efflux through ChTX-sensitive K+ channels. volume regulation; chloride channels; potassium furosemide; anthracene-9-carboxylic acid
channels;
OF CORTI in the mammalian cochlea contains two types of specialized auditory sensory cells, the inner and outer hair cells, which differ from each other in function and structure. While the inner hair cells are the primary transducers of afferent acoustic information, the outer hair cells are thought to alter the micromechanics of the basilar membrane through their motile properties (8). Isolated outer hair cells in vitro have at least two types of motile properties: fast motility and slow motility. Fast motility follows electrical field stimulation in audible frequencies and is regarded as a possible mechanism to allow sharp frequency tuning and increased sensitivity of transduction. Slow motility is assumed to be able to affect the posture of the basilar membrane in vivo, which is important for keeping good sensitivity or desensitizing to intense sound stimuli (37). Undesirable acoustic overstimulation induces a temporary threshold shift, a protective loss of frequency selectivity and sensitivity lasting a few minutes. The precise mechanism of temporary threshold shift in mammals is not clear. However, physiological and morphological in vivo studies suggest the contraction and shrinkage of outer hair cells concomitant with temporary threshold shift (2, 22). We recently found that tetanic electrical field stimulation shortened the isolated outer hair cells with concomitant decreases in cell volume (26). In general, cells regulate their volume against anisosmotic stress-in-
THE ORGAN
Cl088
0363-6143/92
YAMASHITA,
duced water loss or gain by modulating Na+, K+, and Cltransport mechanisms (12, 14,36). This study describes the ionic mechanisms of the electrical stimulation-induced slow shortening in cochlear outer hair cells. MATERIALS
AND METHODS
Preparation of outer hair cells. Guinea pigs under light ether anesthesia were decapitated, and the apical 2.5 turns of organ of Corti were dissected and immersed in modified Hanks’ solution containing (in mM) 136.9 NaCl, 5.37 KCl, 1.26 CaCL,, 0.81 MgSO*, 0.34 Na2HP04, 0.44 KH2P04, and 5.55 glucose (buffered to pH 7.4 with 5 mM N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid-NaOH). The outer hair cells were then isolated mechanically with or without incubation in 3 mg/ml Dispase (Godo Shusei) and were settled on a cover slip coated with poly-L-lysine. The cells were incubated for 60 min with 10 mM N-(6-methoxyquinolyl)acetoethyl ester (MQAE), a Cl-sensitive fluorescent dye, in modified Hanks’ solution and were thoroughly rinsed with dye-free modified Hanks’ solution. Drugs were dissolved in the same solution and applied by total replacement of the medium. All experiments were performed at room temperature. Electrical stimulation. Tetanic electrical field stimulation (typically 3- to 20-mA, 50-Hz, l-ms-width rectangular pulses for 30 s) was applied using a pair of platinum wire electrodes with a tip distance of l-2 mm. Inspection and fluorometric measurement of intracellular Clconcentration. Outer hair cells were inspected on an inverted microscope with fluorometric equipment and video camera recorder (IMT-2; Olympus; a 50-W xenon lamp, a 355- to 365-nm band-pass excitation filter, a 455-nm dichroic mirror, a 450- to 460-nm band-pass emission filter, and a silicon intensifier target video camera). Cell length and volume were estimated from microscopic images assuming rotational symmetry about the longitudinal cellular axis. Cellular MQAE fluorescence intensity, which correlates inversely to intracellular Cl- concentrawas continuously recorded with a night vision tion ([Cl-],), video camera-based window photometer. The recording window always covered the whole cell for the sake of recording only the total fluorescence intensity but not the fluorescence density of any small spot. Excitation light was occasionally shut for monitoring the drift of background levels derived from the recording system. The fluorescence intensity and the reactivity to stimulation were kept constant even under the continuous illumination without any other consideration against bleaching or cell injury. The traces were typical of 3-16 experiments with similar results. Values shown are means t SE; n indicates the number of observations on different cells. Statistical significance was determined by Student’s t test. The effect of drugs was described by the ratio of stimulation-induced change in the presence of drugs to that before the drug application, and the ratio was compared with that of consecutive control responses without drugs. To determine the resting value of [Cl-Ii, the initial MQAE fluorescence intensity of the cell was recorded, and the cell was then incubated with medium containing 10 PM valinomycin, 5
$2.00 Copyright 0 1992 The American Physiological
Society
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DELAYED
SHORTENING
PM nigericin, and 10PM tributyltin to minimize the transmembrane Cl- gradient (34). Increasing concentrations of Cl- were then addedin a stepwisemanner. The resting [Cl-Ii corresponding to the initial fluorescenceintensity wasestimated from the calibration curve of fluorescenceintensity against log [Cl-] for each cell. Materials. MQAE was synthesizedaccording to the methods of Verkman et al. (35). o-Conotoxin GVIA waspurchasedfrom Peptide Institute (Osaka,Japan). Charybdotoxin wasfrom Receptor ResearchChemicals(Cooksville, MD). Other chemicals were of the highest quality commercially available. RESULTS
In isolated outer hair cells, tetanic field electrical stimulation in 6-mA, 50-Hz, 1-ms-width rectangular pulses for 30 s elicited shortening and volume reduction which before TS
during shrinkage
after recovery
OF OUTER
Cl089
HAIR CELLS
began 20-40 s after the onset of stimulation and lasted 2-3 min (Fig. 1). The cell length and volume recovered almost completely to the dimensions seen before stimulation. The shortening was always delayed following the beginning of stimulation, sometimes occurring after stimulation had ended. Some cells showed two-stage responses; additional shortening appeared just before the recovery. It was possible to repeat the delayed shortening several times at an interval of 4-5 min without any changes in response pattern. When repeated, stimulationinduced shortening and volume reduction were 91.9 k 2.6 and 91.7 + 2.7%, respectively, of those in an immediately Table 1. Effects of various electrical stimulation conditions on cellular MQAE fluorescence and cell length Fluorescence Frequency, HZ
$kh Ins
Duration, S
Total, Ill8
Increased! arbitrary units
Shortening, %
Contn 01 Group 1
50 1.0 30 1,500 67 17.2 50 1.0 10 500 6 0.0 50 1.0 20 1,000 63 17.2 Group 2 9 1.0 30 270 0 0.0 22 1.0 30 660 10 0.0 30 1.0 30 900 68 17.2 30 0.5 450 2 0.0 10pm Group 3 ,.__(\, 50 0.5 ;i 750 67 17.2 .:,. Group 4 50 0.3 30 450 1 0.0 Fig. 1. Shorteningof a guineapigcochlearouterhair cell,accompanied 70 0.3 30 630 0 0.0 by cellvolumereduction,inducedby tetanicelectricalstimulation(TS). 90 0.3 30 810 71 17.2 TS (6-mA, 50-Hz, 1-ms-widthrectangularpulsesfor 30 s) wasapplied 130 0.3 30 1,170 76 17.2 to cellson an inverted microscope. Both cell length and width were An isolated outer hair cell loaded with N-(6-methoxyquinolyl)acetodecreased during stimulation. Cell length (pm) and estimated volume ethyl ester (MQAE) was stimulated under different stimulation dura(pl) were 53.2 and 2.9 before TS, 35.7 and 0.8 during TS, and 53.2 and tion conditions (group I), different frequency conditions (group 2), or 2.9 after recovery, respectively. different pulse-width and frequency conditions (groups 3 and 4). Stimulation current was fixed at 18 mA. Total fluorescence indicates total time of current flow (frequency x pulse width x duration). In all groups, stimuli with a total time of more than the threshold of - 700 ms induced 1007 changes in fluorescence intensity and cell length. Four experiments were & carried out, and a typical set of data from 1 experiment is shown. 89 Approximate thresholds of other cells were 600, 750, and 900 ms.
I
-l----J 60=zf
8 I 408
before
1OOpM cytochalasin
B 100-5
802
I
ITS 0
1
2
I I 3
time (mid
Fig. 2. Changes induced by electrical stimulation in cell length, volume, and intracellular N-(6-methoxyquinolyl)acetoethyl ester (MQAE) fluorescence intensity. An outer hair cell was incubated in 10 mM MQAE, a W-sensitive fluorescent probe, washed, and electrically stimulated under a fluorometric recording. An increase in fluorescence intensity indicates a decrease in intracellular chloride concentration. Bar, tetanic electrical stimulation (TS) period. Typical data of 16 experiments are presented. Mean changes in celllength,volume,andfluorescence intensity were -13.5 k 1.5, -37.3 + 3.0, and +27.4 + 3.0% of the values before stimulation, respectively.
E 8
TS, 0 1
2 ; time (mid Fig. 3. Effects of cytochalasin B (100 PM) on electrical stimulationinduced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 4 experiments with similar responses are shown. Initial resting cell length and volume did not change after treatment with cytochalasin B only. Mean percentages of electrical stimulation-induced changes in cell length and volume relative to response before application of cytochalasin B were 7.6 + 4.9% (n = 4, P < 0.01, as compared with control response, 91.9 + 2.6%, n = 111) and 83.0 + 7.7%, respectively.
f
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Cl090
DELAYED
SHORTENING
preceding response (n = 111). These values were used as control in the statistical test for drug effects. Even after repeated shortening, the cells were still osmotically reactive throughout the 0.5- to 12-h experimental period, as tested by hypotonic medium-induced cell swelling and shortening (data not shown). To determine the role of ionic movement in the shortening and shrinkage of the cells, changes in [Cl-Ii were continuously monitored using MQAE. The resting [Cl-Ii was estimated to be 8.3 + 0.9 mM (n = 6) using the calibration curve prepared as described in MATERIALS AND METHODS. Electrical stimulation elicited an initial steep decrease in [Cl-Ii (an increase in MQAE fluorescence intensity) synchronously with cell shortening and volume reduction, usually followed by a gradual increase in [Cl-Ii, prestimulated levels being abruptly recovered in 2-3 min (Fig. 2). The latency in initial steep decrease in [Cl-]; and that in cellular shortening, or the latency in abrupt recovery and that in elongation, were strongly correlated under different stimulation conditions (r = 0.946 or 0.994, respectively, n = 14 in 6 different cells), suggesting their close relationship. Some cells showing two-step shortening tended to sustain or even augment the decrease in [Cl-], during the additional shortening. Various conditions of tetanic electrical stimulation were tested for [Cl-Ii (MQAE fluorescence changes) (Table 1). The changes in [Cl-Ii and cell length occurred in an all-or-none fashion; no changes were elicited by weak stimuli, whereas responses elicited by stimuli stronger than the threshold of -700 ms in the total time were almost the same. Despite the small variations in fluorescence changes among stimuli, no graded responses in length were observed. Thereafter, cells were stimulated under conditions of 3- to 20-mA, 50-Hz, 1-ms-width rectangular pulses for 30 s. Such electrical stimulation always shortened the cell length concomitantly with reduction in cell volume. Actinlike immunoreactivity has been demonstrated in the lateral wall and cytoplasm of outer hair cells as well as cuticular plate and stereocilia, and its role on the motility before
TS
during
shrinkage
OF OUTER HAIR CELLS
was discussed (31). Depolymerization of actin fiber by cytochalasin B (100 PM) completely inhibited the cell shortening induced by tetanic electrical stimulation, but only partly inhibited the decreases in [Cl-Ii and cell volume (Fig. 3). In this case the cell diameter but not the length was diminished by stimulation (Fig. 4). Thus the longitudinal shortening in delayed response to electrical stimulation appeared to be mediated by actin fibers. A Cl- transport inhibitor, furosemide (10 PM), reversibly and dose dependently inhibited the delayed shortening and fluorescence changes (Figs. 5A and 6); the inhiA
before
I
TS
Fig. 4. Tetanic electrical stimulation (TS)-induced shrinkage of an outer hair cell treated with cytochalasin B. An isolated outer hair cell was loaded with MQAE, treated with 100 FM cytochalasin B, and electrically stimulated. Fluorescence image before TS was photographed with longer exposure time than that during shrinkage to illustrate the changes in cell shape rather than those in fluorescence intensity. Note that cell length was slightly changed, but diameter was visibly decreased. Mean changes in length and diameter were -1.4 + 1.0 and -18.1 -I 6.3%, respectively (n = 4).
TS
0
1
2
,
0
B ‘;“OOT
7
.
0
’
2
DIDS
recovery
I-
TS
TS
0
1
2
0
before
TS
1 time (min)
1mM
7
2
0
. 1
2
AC9
r-
TS 0
2 (minf
IOOpM
before
TS
1 time
-;-looT
I
recovery
furosemide
\1
C
1 Opm
10pM
1
2 time
-s---l0 (min)
8 1
2
Fig. 5. Effects of furosemide (10 PM, A), DIDS (0.1 mM; B), and anthracene-9-carboxylic acid (AC9, 1 mM, C) on electrical stimulationinduced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 3 to 8 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before drug application were 14.6 + 12.4% with furosemide (n = 8, P < 0.01, as compared with control response, 91.9 + 2.6%, n = ill), 0.0 + 0.0% with DIDS (n = 3, P < O.Ol), and 0.0 + 0.0% with AC9 (n = 3, P < 0.01). Fluorescence changes in the presence of DIDS and AC9 could not be recorded because of strong fluorescence of these drugs.
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DELAYED
M
SHORTENING
OF OUTER
HAIR CELLS
A
before
25mM tetraethylammonium
I
’
107 [furosemide]
recovery
: fluorescence
. -8 10
Cl091
0
(M)
Fig. 6. Concentration-dependent inhibition by furosemide of electrical stimulation-induced changes in MQAE fluorescence, cell length, and volume of outer hair cells is shown. Changes of fluorescence, length, and volume at indicated concentrations of furosemide relative to those before treatment with furosemide are plotted.
bition of changes in length and volume was in an all-ornone fashion. The approximate inhibitory constant value in fluorescence responses was 0.3 PM. The anion channel blockers 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS, 0.1 mM) and anthracene-9-carboxylic acid (AC9, 1 mM) also completely inhibited the motor responses (Fig. 5, B and C), although changes in [Cl-Ii could not be determined because of their strong fluorescence. Thus electrical stimulation-induced changes in [Cl-Ii appeared to be due to Cl- flux across cell membranes through furosemide-, DIDS-, and ACS-sensitive anion transporters or channels, such transport being essential to cellular motility. To determine the role of cationic flow in the cellular shortening, K+ and Na+ channel blockers were tested. The fluorescence changes and shortening disappeared after treatment with 25 mM tetraethylammonium (TEA) and 2.3 PM charybdotoxin (Fig. 7, A and B), suggesting that Ca2+-activated K+ channels of large unit conductance (BK channels) (21) carry K+ flux. In the presence of 4-aminopyridine (5 mM, Fig. 7C), apamine (2.5 PM, data not shown), and tetrodotoxin (1 PM, data not shown), electrical stimulation still induced the shortening and fluorescence changes, suggesting that the activity of delayed-rectifier K+ channels, Ca2+-activated K+ channels of small unit conductance, and fast Na+ channels is not essential in this process. In outer hair cells, Ca2+-activated K+ conductance is predominant and mainly responsible for the resting membrane potential (1, 17). Additional increase in K+ conductance generated by 10 PM valinomycin neither changed the resting [Cl-]; without Cl- channel stimulation (data not shown) nor masked the electrical stimulation-induced changes in [Cl-],, but overcame the inhibitory effects of a K+ channel blocker, TEA, on the shortening (data not shown). Furosemide still inhibited the delayed responses in the presence of valinomycin (Fig. 8). Thus anion and cation effluxes operate through independently regulated channels. When outer hair cells were incubated in Ca2+-free medium for 5 min or treated with w-conotoxin (250 nM), a blocker of N- and L-type Ca 2+ channels (27), no changes
12-o
I
i
I
‘TS
1
12 time Mn)
B
before
2.3pM charybdotoxin
recovery
B dp 80 I
time (min)
C
. .L--r ..,... :;:
4-w
i TS 0
I
1
’
12-77-7 time (min)
Fig. 7. Effects of tetraethylammonium (25 mM; A), charybdotoxin (2.3 PM; B) and 4-aminopyridine (4-AP, 5 mM; C) on electrical stimulationinduced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 2 to 4 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before drug application were 7.2 t 7.2% with tetraethylammonium (n = 3, P < 0.01, as compared with control response, 91.9 t 2.6%, n = ill), 0.0 t 0.0% with charybdotoxin (n = 4, P < O.Ol), and 89.4 k 5.7% with 4-AP (n = 3).
in [Cl-Ii, cell length, or volume were elicited by the electrical stimulation (Fig. 9, A and B), while in the presence of nicardipine (10 PM), a blocker of L-type Ca2+ channels, normal responses were observed (Fig. 9C). Thus the cell shortening and decrease in [Cl-Ii depend on the influx of the extracellular Ca2+ through o-conotoxin-sensitive Ca2+ channels .
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Cl092
DELAYED before
SHORTENING
OF OUTER
HAIR CELLS
1 OpM valinomycin + 1OpM furosemide
1 OpM valinomycin
100; E 3 80 >o 5 60 o 8
I i
*
i
, ;
Fig. 8. Inhibition by furosemide (10 pM) in the presence of valinomycin (10 PM) of electrical stimulation-induced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 3 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before application of drugs were 100.0 t 0.0% with valinomycin and 0.0 t 0.0% with valinomycin + furosemide (P < 0.01, as compared with control response, 91.9 k 2.6%, n = 111).
TS 0
. 1
. 2
TS 0 1 time (min)
. m 2
TS 0
DISCUSSION
Tetanic electrical field stimulation is not a physiological stimulation. In general, however, current flow through excitable cells driven by electric field locally depolarizes the cell membrane and results in excitation of the cells. Although excitability of outer hair cells is not clear, the shrinkage is probably mediated by an increase in intracellular Ca2+ concentration ( [ Ca2+] i). Cochlear outer hair cells are innervated by efferent fibers, whose most likely neurotransmitter, acetylcholine, also reportedly raises [ Ca2+]; (16). Thus strong or repetitive stimulation of efferent fibers may raise [Ca2+]i with cell shortening and shrinkage. Furthermore, the time course of the electrical stimulation-induced shortening of outer hair cells is similar to that of physiological temporary threshold shift, probably with cell shortening (2, 22). When we take these into account, electrical field stimulation may simulate some physiological stimulation followed by a chain of physiological events in outer hair cells. Tetanic electrical field stimulation of isolated outer hair cells reduced cell length, volume, and [Cl-Ii (Figs. 1 and 2). When stimulation intensity was varied, the cell length and volume were reduced in all-or-none fashion, but [Cl-Ii partly decreased with a subthreshold stimulation. Osmotic force induced by changes in [Cl-Ii seems to induce scarcely visible changes in cell length and volume unless it exceeds a definite threshold. Some factors may be accumulated during the latency period between stimulation and shortening. In many types of cells, swelling induced by a hypotonic environment initiates compensatory loss of KC1 and an osmotically obliged water loss, resulting in reshrinking toward normal size (12, E), i.e., regulatory volume decrease (RVD). RVD in epithelial cells depends on Clefflux through conductive anion channels (14, 36), K+ efflux through TEA- or charybdotoxin-sensitive Ca2+activated K+ channels (32,33), and extracellular Ca2+ (3, 5). No RVD following osmotically induced motility has
. 1
.. 2
been reported in outer hair cells. However, the close similarities in the profiles of channels involved in RVD and in the electrical stimulation-induced shortening and volume decrease in outer hair cells suggest common mechanisms underlying these two phenomena. In contrast to the tetanic electrical stimulation-induced slow shortening and shrinkage in this report, electrically induced fast movements of outer hair cells are reportedly dependent solely on membrane potential but neither on Ca2+ currents sensitive to Cd2+ nor K+ currents sensitive to TEA and Ba2+ (29). Thus the fast and slow motilities seem to be quite different from each other in the mechanisms for induction. Electrically stimulated Cl- transport was characterized by the inhibition profiles with furosemide, DIDS, and AC9. Although furosemide, a loop diuretic, is known to be an inhibitor of the Na+-K+-2Clcotransporter (ll), this drug’s block of Cl- conductance other than the electroneutral cotransport has also been reported in various cells (7, 18, 25). On the other hand, DIDS and AC9 inhibit some conductive Cl- channels (4, 30), but not Na+-K+2Cl- cotransporters (11, 13). Thus furosemide, as well as DIDS and AC9, probably inhibited Cl- channels responsible for electrical stimulation-induced delayed volume changes. The involvement of Ca2+ in the Cl- channel opening mechanism is likely but was not examined in this study. The ionic currents and channels of hair cells reported previously are summarized in Table 2. The presence of Ca2+-activated K+ channels (1, 17, 29) and high-threshold Ca2+ channels (24, 29) has been reported. Among them, TEA-sensitive Ca2+-activated K+ channels and high-threshold (presumably N or L type) Ca2+ channels appear to be involved in the mechanisms of electrical stimulation-induced shortening. Isolated Cl- channels were reported but not characterized enough to be sensitive to specific blockers (10). In this study, furosemidesensitive Cl- channels were first suggested to exist in
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DELAYED Ca2+-free+2mM
0
1
SHORTENING
OF OUTER HAIR CELLS
Cl093
EGTA
iI I 4 2 3 time
250nM
before
0 -conotoxin
time
recovery
Fig. 9. Effects of Ca 2+-free medium [with 2 mM ethylene glycol-his@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA); A], o-conotoxin GVIA (250 nM; B), and nicardipine (10 PM; C) on electrical stimulation-induced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 3 or 4 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before application of Ca2+-free medium or drugs were 0.0 k 0.0% with Ca2+-free medium (n = 3, P < 0.01, as compared with control response, 91.9 t 2.6%, n = ill), 0.0 t 0.0% with o-conotoxin GVIA (n = 3, P < O.Ol), and 100.0 k 0.0% with nicardipine (n. = 4).
(mid
1OpM nicardipine
2
a-:
TS
3 0 time (mid
1
2
1
3
outer hair cells and to be involved in the electrical stimulation-induced shortening. Macroscopic Cl- currents have not been reported. It may have been ignored as a leakage current, or some addition .a1 factors other than Ca2+ en try or voltage step may be required for the activation of the Cl- channels. The electrical stimulation-induced changes in cell volume and [Cl-], were both sensitive to Cl- and K+ channel block. Such apparent coupling of K+ and Cl- fluxes can be explained by the occurrence of conductive effluxes. With the assumption that the resting [Cl-Ii is higher than that of Nernst’s distribution and that the stimulation activates conductive Cl- channels, the inward current carried by Cl- efflux depolarizes the cell membranes, resulti ng in a reductio n of electromoti ve force for Clefflux and an increase in electromotive force for cation efflux. Unless the conductive K+ efflux through BK chan-
nels carrying the outward current neutralizes the Cl- current , no measura ble changes in [Cl-Ii will occur, and the reverse would also be true. Massive electrically coupled anion and cation effluxes supply the driving force for osmotic water efflux. Accordin g to the above-mentioned discussion, both of K + and Cl- conductances are not necessarily needed to be primarily activated by electrical stimulation. Even at the resting range of [Ca2+]i (65 nM), Ca2+-activated K+ conductance is reportedly dominant in outer hair cells (1). Because additional increase in K+ conductance by valinomycin failed to change the resting [Cl-Ii and electrical stimulation-induced changes in [Cl-], (Fig. 8), K+ conductance in outer hair cells appears to be high enough to allow free movement of K+ driven by el.ectromotive force, and Cl- conductance is assumed to be so low as to limit the Cl- and electrically coupled K+ movements.
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Cl094
DELAYED
Table 2. Reported
ionic currents
Current Carrier
SHORTENING
and channels
OF OUTER
Unit Conductance, PS
Whole Ca2+ Inward rectifier Time and voltage High threshold
cell current
dependent
Ba2+,
TEA,
quinidine,
nifedipine
cs+ Cd”+
Acetylcholine ATP Ca2+
Reference No.
Inhibitor
Cd2+,
Quinidine, Isolated
K’ K’ ClMonovalent
CELLS
in cochlear outer hair cells
Activator/ Feature
K+ K+ K’ Ca”+ Cations Cations Cations
HAIR
channels in excised 45, 240 20, 149, 200 22, 40
We thank Kansai Medical University Laboratory Center and Animal Center for providing experimental instruments and animals. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan, the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and Uehara Memorial Foundation. Address for renrint reauests: C. Inagaki. Dent. of Pharmacologv.
Mg2+,
Kansai Japan. Received
phentolamine
patches
cations
It has been reported that an increase in cellular free Ca2+ induced by a Ca2+ ionophore induces elongation, narrowing, and shrinkage in volume in isolated cochlear outer hair cells (6). The authors claimed that actin-mediated Ca2+-dependent radial forces overcame the turgor pressure and extruded water out of the cells, and that this resulted in volume reduction and elongation. The electrical tetanic stimulation-induced delayed shortening in the present study appeared to occur through quite contradictory processes; actin-mediated contractile forces potentially shortened the cell, concomitant with volume decrease. Because TEA did not inhibit the Ca2+ ionophoreinduced elongation of the cell (6), increased [Ca2+]i alone or Ca2+ entry through a nonphysiological route of Ca2+ ionophore may not be sufficient to achieve full responses of electrical stimulation-induced volume changes with the activation of Ca2+ -activated K+ channels. Because electrical stimulation-induced cell shortening did not occur when K+ or Cl- channels were blocked, these channel activities appeared to be needed for actin fiber contraction. The stimulation-induced entry of Ca2+ may be facilitated by increases in K+ and Cl- conductances. Furthermore, physiologically available Ca2+ for contraction of actin fibers may be increased by the increased intracellular concentration of Ca2+ with dehydration, or by facilitated Ca2+ diffusion by intracellular water flow. Clinical doses of furosemide occasionally induce hearing disorders especially in the course of renal disease (28). Such an effect is usually explained by reduced endocochlear potential (20); however, the inhibition of delayed shortening by this drug may also be involved in the mechanisms of furosemide-induced hearing disorders, probably through the loss of protective contractions to intense sound pressure, resulting in the cellular damage. The delayed shortening induced by tetanic electrical stimulation appears to be a useful model for studying the physiological functions of slow motility of cochlear outer hair cells.
tubocurarine,
1, 17, 29 1 17 24, 29 17 9 23
Medical 28 May
1 10 10 19
Ca2+, diltiazem
University,
Fumizono-cho
1992; accepted
in final
1, Moriguchi, form
30 July
Osaka
570,
1992.
REFERENCES 1. Ashmore, J. F., and R. W. Meech. Ionic basis of membrane potential in outer hair cells of guinea pig cochlea. Nature Lond. 322: 368-371, 1986. 2. Cody, A. R., and I. J. Russell. Acoustically induced hearing loss: intracellular studies in the guinea pig cochlea. Hear. Res. 35: 59-70, 1988. 3. Davis, C. W., and A. L. Finn. Cell volume regulation in frog urinary bladder. Federation Proc. 44: 2520-2525, 1985. 4. Diener, M., W. Rummel, P. Mestres, and B. Lindemann. Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat. J. Membr. Biol. 108: 21-30, 1989. 5. Dube, L., L. Parent, and R. Sauve. Hypotonic shock activates a maxi K+ channel in primary cultured proximal tubule cells. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F358-F359, 1990. D., G. Zajic, and J. Schacht. Increasing intracellular 6. Dulon, free calcium induces circumferential contractions in isolated cochlear outer hair cells. J. Neurosci. 10: 1388-1397, 1990. 7. Evans, M. G., A. Marty, Y. P. Tan, and A. Trautmann. Blockade of Ca-activated Cl conductance by furosemide in rat lacrimal glands. Pfluegers Arch. 406: 65-68, 1986. 8. Flock, A. Do sensory cells in the ear have a motile function? Prog. Brain Res. 74: 297-304, 1988. P. A., and B. W. Murrow. Cholinergic inhibition of 9. Fuchs, short (outer) hair cells of chick’s cochlea. J. Neurosci. 12: 800-809, 1992. 10. Gitter, A. H., H. P. Zenner, and E. Fromter. Membrane potential and ion channels in isolated outer hair cells of guinea pig cochlea. ORL Base1 48: 68-75, 1986. 11. Haas, M., W. F. Schmidt III, and T. J. McManus. Catecholamine-stimulated ion transport in duck red cells. J. Gen. Physiol. 80: 125--147, 1982. 12. Hall, A. C., L. Bianchini, and J. C. Ellory. Pathways for cell volume regulation via potassium and chloride loss. In: Ion Transport, edited by D. Keeling and C. Benham. New York: Academic, 1989, p. 217-235. G. L., and W. J. Betz. Evidence for active chloride 13. Harris, accumulation in normal and denervated rat lumbrical muscle. J. Gen. Physiol. 90: 127-144, 1987. 14. Hazama, A., and Y. Okada. Ca2+ sensitivity of volume-regulatory K+ and Cl- channels in cultured human epithelial cells. J. Physiol. Lond. 402: 687-702, 1988. 15. Hazama, A., and Y. Okada. Biphasic rises in cytosolic free Ca2+ in association with activation of K+ and Cl- conductance during the regulatory volume decrease in cultured human epithe
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lial cells. Pfluegers Arch. 416: 710-714, 1990. 16. Housley, G. D., and J. F. Ashmore. Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea. Proc. R. Sot. Lond. B Biol. Sci. 244: 161-167, 1991. 17. Housley, G. D., and J. F. Ashmore. Ionic currents of outer hair cells isolated from the guinea-pig cochlea. J. Physiol. Lond. 448: 73-98, 1992. 18. Katchmann, A. N., and E. Zeimal. Ionic mechanisms of the rapid (nicotinic) phase of acetylcholine responses in identified Planobarius corneus neurons. Brain Res. 241: 95-103, 1982. 19. Kolesnikov, S. S., T. I. Rebrik, A. B. Zhainazarov, G. A. Tavartkiladze, and G. R. Kalamkalov. A cyclic-AMPgated conductance in cochlear hair cells. FEBS Lett. 290: 167-170, 1991. 20. Kusakari, J., I. Ise, T. H. Comegys, I. Thalmann, and R. Thalmann. Effect of ethacrynic acid, furosemide, and ouabain upon the endolymphatic potential and upon high energy phosphates of the stria vascularis. Laryngoscope 88: 12-37, 1978. 21. Latorre, R., A. Oberhauser, P. Labarca, and 0. Alvarez. Varieties of calcium-activated potassium channels. Annu. Rev. Physiol. 51: 385-399, 1989. 22. Mulroy, M. J., R. F. Fromm, and S. Curtis. Changes in the synaptic region of auditory hair cells during noise-induced ternporary threshold shift. Hear. Res. 49: 79-88, 1990. 23. Nakagawa, T., N. Akaike, T. Kimitsuki, S. Komume, and T. Arima. ATP-induced current in isolated outer hair cells of guinea pig cochlea. J. Neurophysiol. 63: 1068-1074, 1990. 24. Nakagawa, T., S. Kakehata, N. Akaike, S. Komume, T. Takasaka, and T. Uemura. Calcium channel in isolated outer hair cells of guinea pig cochlea. Neurosci. Lett. 125: 81-84, 1991. 25. Nicoll, R. A. The blockade of GABA mediated responses in the frog spinal cord by ammonium ions and furosemide. J. Physiol. Lond. 283: 121-132, 1978. 26. Ohnishi, S., M. Hara, T. Yamashita, T. Kumazawa, A. Minato, and C. Inagaki. Calciumand chloride-dependent
OF OUTER
27.
28. 29.
30.
31.
32.
33.
34
35
36
37
HAIR
CELLS
Cl095
shortening of cochlear outer hair cells (Abstract). Jpn. J. Pharmacol. 55: 92P, 1991. Porzig, H. Pharmacological modulation of voltage-dependent calcium channels in intact cells. Rev. Physiol. Biochem. Pharmacol. 114: 209-262, 1990. Quick, C. A. Permanent deafness associated with furosemide administration. Ann. Otol. 84: 94-101, 1975. Santos-Sacchi, J., and J. P. Dilger. Whole cell currents and mechanical responses of isolated outer hair cells. Hear. Res. 35: 143-150, 1988. Schmid, A., G. Burckhardt, and H. Goegelein. Single channels in endosomal vesicle preparations from rat kidney cortex. J. Membr. Biol. 111: 265-275, 1989. Slepecky, N., M. Ulfendhl, and A. Flock. Effects of caffeine and tetracaine on outer hair cell shortening suggest intracellular calcium involvement. Hear. Res. 32: 11-22, 1988. Strange, K. Volume regulation following Na+ pump inhibition in CCT principal cells: apical K+ loss. Am. J. Physiol. 258 (Renal FZuid Electrolyte PhysioZ. 27): F372-F340, 1990. Taut, M., S. L. Maout, and P. Poujeol. Fluorescent videomicroscopy study of regulatory volume decrease in primary culture of rabbit proximal convoluted tubule. Biochim. Biophys. Acta 1052: 278-284, 1990. Verkman, A. S. Development and biological applications of chloride-sensitive fluorescent indicators. Am. J. Physiol. 259 (Cell Physiol. 28): C375-C388, 1990. Verkman, A. S., M. C. Sellers, A. C. Chao, T. Leung, and R. Ketcham. Synthesis and characterization of improved chloride-sensitive fluorescent indicators. Anal. Biochem. 178: 355-361, 1989. Welling, P. A., and R. G. O’Neil. Cell swelling activates basolateral membrane Cl and K conductances in rabbit proximal tubule. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F951-F962, 1990. Zenner, H. P., W. Arnold, and A. H. Gitter. Outer hair cells as fast and slow cochlear amplifiers with a bidirectional trasduction cycle. Acta Oto-Zaryngo!. 105: 457-462, 1988.
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and Otolaryngology, Kansai Medical University, Osaka 570; Kyoto Pharmaceutical University, Kyoto 607, Japan
Ohnishi, Sumio, Mitsuyoshi Hara, Masafumi Inoue, Toshio Yamashita, Tadami Kumazawa, Akio Minato, and Chiyoko Inagaki. Delayed shortening and shrinkage of cochlear outer hair cells. Am. J. Physiol. 263 (Cell Physiol. 32): C1088-C1095, 1992.-Slow shortening of cochlear outer hair cells has been speculated to modify cochlear sensitivity. Tetanic electrical field stimulation of isolated outer hair cells from guinea pigs shortened the cells for 2-3 min. Electrical stimulation reduced cell length and volume (-13.5 t 1.5 and -37.3 t 3.0% of initial values, respectively, n = 16) and decreased the intracellular Cl- concentration. Cytochalasin B (100 PM) inhibited electrical stimulation-induced shortening but not volume reduction. The following chemicals or manipulations inhibited the responses: 10 PM furosemide, 0.1 mM 4,4’diisothiocyanostilbene-2,2’-disulfonic acid (DIDS), 1 mM anthracene-9-carboxylic acid (AC9), 25 mM tetraethylammonium, 2.3 PM charybdotoxin (ChTX), 250 nM o-conotoxin, and Ca”+-free medium. These findings suggest that both electrical stimulation-induced shortening and shrinkage of outer hair cells result not only from an actin-mediated contractile force, but also from Cl- efflux through furosemide-, DIDS-, and AC9sensitive Cl- channels, and K+ efflux through ChTX-sensitive K+ channels. volume regulation; chloride channels; potassium furosemide; anthracene-9-carboxylic acid
channels;
OF CORTI in the mammalian cochlea contains two types of specialized auditory sensory cells, the inner and outer hair cells, which differ from each other in function and structure. While the inner hair cells are the primary transducers of afferent acoustic information, the outer hair cells are thought to alter the micromechanics of the basilar membrane through their motile properties (8). Isolated outer hair cells in vitro have at least two types of motile properties: fast motility and slow motility. Fast motility follows electrical field stimulation in audible frequencies and is regarded as a possible mechanism to allow sharp frequency tuning and increased sensitivity of transduction. Slow motility is assumed to be able to affect the posture of the basilar membrane in vivo, which is important for keeping good sensitivity or desensitizing to intense sound stimuli (37). Undesirable acoustic overstimulation induces a temporary threshold shift, a protective loss of frequency selectivity and sensitivity lasting a few minutes. The precise mechanism of temporary threshold shift in mammals is not clear. However, physiological and morphological in vivo studies suggest the contraction and shrinkage of outer hair cells concomitant with temporary threshold shift (2, 22). We recently found that tetanic electrical field stimulation shortened the isolated outer hair cells with concomitant decreases in cell volume (26). In general, cells regulate their volume against anisosmotic stress-in-
THE ORGAN
Cl088
0363-6143/92
YAMASHITA,
duced water loss or gain by modulating Na+, K+, and Cltransport mechanisms (12, 14,36). This study describes the ionic mechanisms of the electrical stimulation-induced slow shortening in cochlear outer hair cells. MATERIALS
AND METHODS
Preparation of outer hair cells. Guinea pigs under light ether anesthesia were decapitated, and the apical 2.5 turns of organ of Corti were dissected and immersed in modified Hanks’ solution containing (in mM) 136.9 NaCl, 5.37 KCl, 1.26 CaCL,, 0.81 MgSO*, 0.34 Na2HP04, 0.44 KH2P04, and 5.55 glucose (buffered to pH 7.4 with 5 mM N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid-NaOH). The outer hair cells were then isolated mechanically with or without incubation in 3 mg/ml Dispase (Godo Shusei) and were settled on a cover slip coated with poly-L-lysine. The cells were incubated for 60 min with 10 mM N-(6-methoxyquinolyl)acetoethyl ester (MQAE), a Cl-sensitive fluorescent dye, in modified Hanks’ solution and were thoroughly rinsed with dye-free modified Hanks’ solution. Drugs were dissolved in the same solution and applied by total replacement of the medium. All experiments were performed at room temperature. Electrical stimulation. Tetanic electrical field stimulation (typically 3- to 20-mA, 50-Hz, l-ms-width rectangular pulses for 30 s) was applied using a pair of platinum wire electrodes with a tip distance of l-2 mm. Inspection and fluorometric measurement of intracellular Clconcentration. Outer hair cells were inspected on an inverted microscope with fluorometric equipment and video camera recorder (IMT-2; Olympus; a 50-W xenon lamp, a 355- to 365-nm band-pass excitation filter, a 455-nm dichroic mirror, a 450- to 460-nm band-pass emission filter, and a silicon intensifier target video camera). Cell length and volume were estimated from microscopic images assuming rotational symmetry about the longitudinal cellular axis. Cellular MQAE fluorescence intensity, which correlates inversely to intracellular Cl- concentrawas continuously recorded with a night vision tion ([Cl-],), video camera-based window photometer. The recording window always covered the whole cell for the sake of recording only the total fluorescence intensity but not the fluorescence density of any small spot. Excitation light was occasionally shut for monitoring the drift of background levels derived from the recording system. The fluorescence intensity and the reactivity to stimulation were kept constant even under the continuous illumination without any other consideration against bleaching or cell injury. The traces were typical of 3-16 experiments with similar results. Values shown are means t SE; n indicates the number of observations on different cells. Statistical significance was determined by Student’s t test. The effect of drugs was described by the ratio of stimulation-induced change in the presence of drugs to that before the drug application, and the ratio was compared with that of consecutive control responses without drugs. To determine the resting value of [Cl-Ii, the initial MQAE fluorescence intensity of the cell was recorded, and the cell was then incubated with medium containing 10 PM valinomycin, 5
$2.00 Copyright 0 1992 The American Physiological
Society
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SHORTENING
PM nigericin, and 10PM tributyltin to minimize the transmembrane Cl- gradient (34). Increasing concentrations of Cl- were then addedin a stepwisemanner. The resting [Cl-Ii corresponding to the initial fluorescenceintensity wasestimated from the calibration curve of fluorescenceintensity against log [Cl-] for each cell. Materials. MQAE was synthesizedaccording to the methods of Verkman et al. (35). o-Conotoxin GVIA waspurchasedfrom Peptide Institute (Osaka,Japan). Charybdotoxin wasfrom Receptor ResearchChemicals(Cooksville, MD). Other chemicals were of the highest quality commercially available. RESULTS
In isolated outer hair cells, tetanic field electrical stimulation in 6-mA, 50-Hz, 1-ms-width rectangular pulses for 30 s elicited shortening and volume reduction which before TS
during shrinkage
after recovery
OF OUTER
Cl089
HAIR CELLS
began 20-40 s after the onset of stimulation and lasted 2-3 min (Fig. 1). The cell length and volume recovered almost completely to the dimensions seen before stimulation. The shortening was always delayed following the beginning of stimulation, sometimes occurring after stimulation had ended. Some cells showed two-stage responses; additional shortening appeared just before the recovery. It was possible to repeat the delayed shortening several times at an interval of 4-5 min without any changes in response pattern. When repeated, stimulationinduced shortening and volume reduction were 91.9 k 2.6 and 91.7 + 2.7%, respectively, of those in an immediately Table 1. Effects of various electrical stimulation conditions on cellular MQAE fluorescence and cell length Fluorescence Frequency, HZ
$kh Ins
Duration, S
Total, Ill8
Increased! arbitrary units
Shortening, %
Contn 01 Group 1
50 1.0 30 1,500 67 17.2 50 1.0 10 500 6 0.0 50 1.0 20 1,000 63 17.2 Group 2 9 1.0 30 270 0 0.0 22 1.0 30 660 10 0.0 30 1.0 30 900 68 17.2 30 0.5 450 2 0.0 10pm Group 3 ,.__(\, 50 0.5 ;i 750 67 17.2 .:,. Group 4 50 0.3 30 450 1 0.0 Fig. 1. Shorteningof a guineapigcochlearouterhair cell,accompanied 70 0.3 30 630 0 0.0 by cellvolumereduction,inducedby tetanicelectricalstimulation(TS). 90 0.3 30 810 71 17.2 TS (6-mA, 50-Hz, 1-ms-widthrectangularpulsesfor 30 s) wasapplied 130 0.3 30 1,170 76 17.2 to cellson an inverted microscope. Both cell length and width were An isolated outer hair cell loaded with N-(6-methoxyquinolyl)acetodecreased during stimulation. Cell length (pm) and estimated volume ethyl ester (MQAE) was stimulated under different stimulation dura(pl) were 53.2 and 2.9 before TS, 35.7 and 0.8 during TS, and 53.2 and tion conditions (group I), different frequency conditions (group 2), or 2.9 after recovery, respectively. different pulse-width and frequency conditions (groups 3 and 4). Stimulation current was fixed at 18 mA. Total fluorescence indicates total time of current flow (frequency x pulse width x duration). In all groups, stimuli with a total time of more than the threshold of - 700 ms induced 1007 changes in fluorescence intensity and cell length. Four experiments were & carried out, and a typical set of data from 1 experiment is shown. 89 Approximate thresholds of other cells were 600, 750, and 900 ms.
I
-l----J 60=zf
8 I 408
before
1OOpM cytochalasin
B 100-5
802
I
ITS 0
1
2
I I 3
time (mid
Fig. 2. Changes induced by electrical stimulation in cell length, volume, and intracellular N-(6-methoxyquinolyl)acetoethyl ester (MQAE) fluorescence intensity. An outer hair cell was incubated in 10 mM MQAE, a W-sensitive fluorescent probe, washed, and electrically stimulated under a fluorometric recording. An increase in fluorescence intensity indicates a decrease in intracellular chloride concentration. Bar, tetanic electrical stimulation (TS) period. Typical data of 16 experiments are presented. Mean changes in celllength,volume,andfluorescence intensity were -13.5 k 1.5, -37.3 + 3.0, and +27.4 + 3.0% of the values before stimulation, respectively.
E 8
TS, 0 1
2 ; time (mid Fig. 3. Effects of cytochalasin B (100 PM) on electrical stimulationinduced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 4 experiments with similar responses are shown. Initial resting cell length and volume did not change after treatment with cytochalasin B only. Mean percentages of electrical stimulation-induced changes in cell length and volume relative to response before application of cytochalasin B were 7.6 + 4.9% (n = 4, P < 0.01, as compared with control response, 91.9 + 2.6%, n = 111) and 83.0 + 7.7%, respectively.
f
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Cl090
DELAYED
SHORTENING
preceding response (n = 111). These values were used as control in the statistical test for drug effects. Even after repeated shortening, the cells were still osmotically reactive throughout the 0.5- to 12-h experimental period, as tested by hypotonic medium-induced cell swelling and shortening (data not shown). To determine the role of ionic movement in the shortening and shrinkage of the cells, changes in [Cl-Ii were continuously monitored using MQAE. The resting [Cl-Ii was estimated to be 8.3 + 0.9 mM (n = 6) using the calibration curve prepared as described in MATERIALS AND METHODS. Electrical stimulation elicited an initial steep decrease in [Cl-Ii (an increase in MQAE fluorescence intensity) synchronously with cell shortening and volume reduction, usually followed by a gradual increase in [Cl-Ii, prestimulated levels being abruptly recovered in 2-3 min (Fig. 2). The latency in initial steep decrease in [Cl-]; and that in cellular shortening, or the latency in abrupt recovery and that in elongation, were strongly correlated under different stimulation conditions (r = 0.946 or 0.994, respectively, n = 14 in 6 different cells), suggesting their close relationship. Some cells showing two-step shortening tended to sustain or even augment the decrease in [Cl-], during the additional shortening. Various conditions of tetanic electrical stimulation were tested for [Cl-Ii (MQAE fluorescence changes) (Table 1). The changes in [Cl-Ii and cell length occurred in an all-or-none fashion; no changes were elicited by weak stimuli, whereas responses elicited by stimuli stronger than the threshold of -700 ms in the total time were almost the same. Despite the small variations in fluorescence changes among stimuli, no graded responses in length were observed. Thereafter, cells were stimulated under conditions of 3- to 20-mA, 50-Hz, 1-ms-width rectangular pulses for 30 s. Such electrical stimulation always shortened the cell length concomitantly with reduction in cell volume. Actinlike immunoreactivity has been demonstrated in the lateral wall and cytoplasm of outer hair cells as well as cuticular plate and stereocilia, and its role on the motility before
TS
during
shrinkage
OF OUTER HAIR CELLS
was discussed (31). Depolymerization of actin fiber by cytochalasin B (100 PM) completely inhibited the cell shortening induced by tetanic electrical stimulation, but only partly inhibited the decreases in [Cl-Ii and cell volume (Fig. 3). In this case the cell diameter but not the length was diminished by stimulation (Fig. 4). Thus the longitudinal shortening in delayed response to electrical stimulation appeared to be mediated by actin fibers. A Cl- transport inhibitor, furosemide (10 PM), reversibly and dose dependently inhibited the delayed shortening and fluorescence changes (Figs. 5A and 6); the inhiA
before
I
TS
Fig. 4. Tetanic electrical stimulation (TS)-induced shrinkage of an outer hair cell treated with cytochalasin B. An isolated outer hair cell was loaded with MQAE, treated with 100 FM cytochalasin B, and electrically stimulated. Fluorescence image before TS was photographed with longer exposure time than that during shrinkage to illustrate the changes in cell shape rather than those in fluorescence intensity. Note that cell length was slightly changed, but diameter was visibly decreased. Mean changes in length and diameter were -1.4 + 1.0 and -18.1 -I 6.3%, respectively (n = 4).
TS
0
1
2
,
0
B ‘;“OOT
7
.
0
’
2
DIDS
recovery
I-
TS
TS
0
1
2
0
before
TS
1 time (min)
1mM
7
2
0
. 1
2
AC9
r-
TS 0
2 (minf
IOOpM
before
TS
1 time
-;-looT
I
recovery
furosemide
\1
C
1 Opm
10pM
1
2 time
-s---l0 (min)
8 1
2
Fig. 5. Effects of furosemide (10 PM, A), DIDS (0.1 mM; B), and anthracene-9-carboxylic acid (AC9, 1 mM, C) on electrical stimulationinduced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 3 to 8 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before drug application were 14.6 + 12.4% with furosemide (n = 8, P < 0.01, as compared with control response, 91.9 + 2.6%, n = ill), 0.0 + 0.0% with DIDS (n = 3, P < O.Ol), and 0.0 + 0.0% with AC9 (n = 3, P < 0.01). Fluorescence changes in the presence of DIDS and AC9 could not be recorded because of strong fluorescence of these drugs.
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DELAYED
M
SHORTENING
OF OUTER
HAIR CELLS
A
before
25mM tetraethylammonium
I
’
107 [furosemide]
recovery
: fluorescence
. -8 10
Cl091
0
(M)
Fig. 6. Concentration-dependent inhibition by furosemide of electrical stimulation-induced changes in MQAE fluorescence, cell length, and volume of outer hair cells is shown. Changes of fluorescence, length, and volume at indicated concentrations of furosemide relative to those before treatment with furosemide are plotted.
bition of changes in length and volume was in an all-ornone fashion. The approximate inhibitory constant value in fluorescence responses was 0.3 PM. The anion channel blockers 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS, 0.1 mM) and anthracene-9-carboxylic acid (AC9, 1 mM) also completely inhibited the motor responses (Fig. 5, B and C), although changes in [Cl-Ii could not be determined because of their strong fluorescence. Thus electrical stimulation-induced changes in [Cl-Ii appeared to be due to Cl- flux across cell membranes through furosemide-, DIDS-, and ACS-sensitive anion transporters or channels, such transport being essential to cellular motility. To determine the role of cationic flow in the cellular shortening, K+ and Na+ channel blockers were tested. The fluorescence changes and shortening disappeared after treatment with 25 mM tetraethylammonium (TEA) and 2.3 PM charybdotoxin (Fig. 7, A and B), suggesting that Ca2+-activated K+ channels of large unit conductance (BK channels) (21) carry K+ flux. In the presence of 4-aminopyridine (5 mM, Fig. 7C), apamine (2.5 PM, data not shown), and tetrodotoxin (1 PM, data not shown), electrical stimulation still induced the shortening and fluorescence changes, suggesting that the activity of delayed-rectifier K+ channels, Ca2+-activated K+ channels of small unit conductance, and fast Na+ channels is not essential in this process. In outer hair cells, Ca2+-activated K+ conductance is predominant and mainly responsible for the resting membrane potential (1, 17). Additional increase in K+ conductance generated by 10 PM valinomycin neither changed the resting [Cl-]; without Cl- channel stimulation (data not shown) nor masked the electrical stimulation-induced changes in [Cl-],, but overcame the inhibitory effects of a K+ channel blocker, TEA, on the shortening (data not shown). Furosemide still inhibited the delayed responses in the presence of valinomycin (Fig. 8). Thus anion and cation effluxes operate through independently regulated channels. When outer hair cells were incubated in Ca2+-free medium for 5 min or treated with w-conotoxin (250 nM), a blocker of N- and L-type Ca 2+ channels (27), no changes
12-o
I
i
I
‘TS
1
12 time Mn)
B
before
2.3pM charybdotoxin
recovery
B dp 80 I
time (min)
C
. .L--r ..,... :;:
4-w
i TS 0
I
1
’
12-77-7 time (min)
Fig. 7. Effects of tetraethylammonium (25 mM; A), charybdotoxin (2.3 PM; B) and 4-aminopyridine (4-AP, 5 mM; C) on electrical stimulationinduced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 2 to 4 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before drug application were 7.2 t 7.2% with tetraethylammonium (n = 3, P < 0.01, as compared with control response, 91.9 t 2.6%, n = ill), 0.0 t 0.0% with charybdotoxin (n = 4, P < O.Ol), and 89.4 k 5.7% with 4-AP (n = 3).
in [Cl-Ii, cell length, or volume were elicited by the electrical stimulation (Fig. 9, A and B), while in the presence of nicardipine (10 PM), a blocker of L-type Ca2+ channels, normal responses were observed (Fig. 9C). Thus the cell shortening and decrease in [Cl-Ii depend on the influx of the extracellular Ca2+ through o-conotoxin-sensitive Ca2+ channels .
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 9, 2019.
Cl092
DELAYED before
SHORTENING
OF OUTER
HAIR CELLS
1 OpM valinomycin + 1OpM furosemide
1 OpM valinomycin
100; E 3 80 >o 5 60 o 8
I i
*
i
, ;
Fig. 8. Inhibition by furosemide (10 pM) in the presence of valinomycin (10 PM) of electrical stimulation-induced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 3 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before application of drugs were 100.0 t 0.0% with valinomycin and 0.0 t 0.0% with valinomycin + furosemide (P < 0.01, as compared with control response, 91.9 k 2.6%, n = 111).
TS 0
. 1
. 2
TS 0 1 time (min)
. m 2
TS 0
DISCUSSION
Tetanic electrical field stimulation is not a physiological stimulation. In general, however, current flow through excitable cells driven by electric field locally depolarizes the cell membrane and results in excitation of the cells. Although excitability of outer hair cells is not clear, the shrinkage is probably mediated by an increase in intracellular Ca2+ concentration ( [ Ca2+] i). Cochlear outer hair cells are innervated by efferent fibers, whose most likely neurotransmitter, acetylcholine, also reportedly raises [ Ca2+]; (16). Thus strong or repetitive stimulation of efferent fibers may raise [Ca2+]i with cell shortening and shrinkage. Furthermore, the time course of the electrical stimulation-induced shortening of outer hair cells is similar to that of physiological temporary threshold shift, probably with cell shortening (2, 22). When we take these into account, electrical field stimulation may simulate some physiological stimulation followed by a chain of physiological events in outer hair cells. Tetanic electrical field stimulation of isolated outer hair cells reduced cell length, volume, and [Cl-Ii (Figs. 1 and 2). When stimulation intensity was varied, the cell length and volume were reduced in all-or-none fashion, but [Cl-Ii partly decreased with a subthreshold stimulation. Osmotic force induced by changes in [Cl-Ii seems to induce scarcely visible changes in cell length and volume unless it exceeds a definite threshold. Some factors may be accumulated during the latency period between stimulation and shortening. In many types of cells, swelling induced by a hypotonic environment initiates compensatory loss of KC1 and an osmotically obliged water loss, resulting in reshrinking toward normal size (12, E), i.e., regulatory volume decrease (RVD). RVD in epithelial cells depends on Clefflux through conductive anion channels (14, 36), K+ efflux through TEA- or charybdotoxin-sensitive Ca2+activated K+ channels (32,33), and extracellular Ca2+ (3, 5). No RVD following osmotically induced motility has
. 1
.. 2
been reported in outer hair cells. However, the close similarities in the profiles of channels involved in RVD and in the electrical stimulation-induced shortening and volume decrease in outer hair cells suggest common mechanisms underlying these two phenomena. In contrast to the tetanic electrical stimulation-induced slow shortening and shrinkage in this report, electrically induced fast movements of outer hair cells are reportedly dependent solely on membrane potential but neither on Ca2+ currents sensitive to Cd2+ nor K+ currents sensitive to TEA and Ba2+ (29). Thus the fast and slow motilities seem to be quite different from each other in the mechanisms for induction. Electrically stimulated Cl- transport was characterized by the inhibition profiles with furosemide, DIDS, and AC9. Although furosemide, a loop diuretic, is known to be an inhibitor of the Na+-K+-2Clcotransporter (ll), this drug’s block of Cl- conductance other than the electroneutral cotransport has also been reported in various cells (7, 18, 25). On the other hand, DIDS and AC9 inhibit some conductive Cl- channels (4, 30), but not Na+-K+2Cl- cotransporters (11, 13). Thus furosemide, as well as DIDS and AC9, probably inhibited Cl- channels responsible for electrical stimulation-induced delayed volume changes. The involvement of Ca2+ in the Cl- channel opening mechanism is likely but was not examined in this study. The ionic currents and channels of hair cells reported previously are summarized in Table 2. The presence of Ca2+-activated K+ channels (1, 17, 29) and high-threshold Ca2+ channels (24, 29) has been reported. Among them, TEA-sensitive Ca2+-activated K+ channels and high-threshold (presumably N or L type) Ca2+ channels appear to be involved in the mechanisms of electrical stimulation-induced shortening. Isolated Cl- channels were reported but not characterized enough to be sensitive to specific blockers (10). In this study, furosemidesensitive Cl- channels were first suggested to exist in
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DELAYED Ca2+-free+2mM
0
1
SHORTENING
OF OUTER HAIR CELLS
Cl093
EGTA
iI I 4 2 3 time
250nM
before
0 -conotoxin
time
recovery
Fig. 9. Effects of Ca 2+-free medium [with 2 mM ethylene glycol-his@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA); A], o-conotoxin GVIA (250 nM; B), and nicardipine (10 PM; C) on electrical stimulation-induced changes in cell length, volume, and MQAE fluorescence of outer hair cells. Bar, tetanic electrical stimulation (TS) period. Typical data from 3 or 4 experiments with similar responses are shown. Mean percentages of electrical stimulation-induced changes in cell length relative to response before application of Ca2+-free medium or drugs were 0.0 k 0.0% with Ca2+-free medium (n = 3, P < 0.01, as compared with control response, 91.9 t 2.6%, n = ill), 0.0 t 0.0% with o-conotoxin GVIA (n = 3, P < O.Ol), and 100.0 k 0.0% with nicardipine (n. = 4).
(mid
1OpM nicardipine
2
a-:
TS
3 0 time (mid
1
2
1
3
outer hair cells and to be involved in the electrical stimulation-induced shortening. Macroscopic Cl- currents have not been reported. It may have been ignored as a leakage current, or some addition .a1 factors other than Ca2+ en try or voltage step may be required for the activation of the Cl- channels. The electrical stimulation-induced changes in cell volume and [Cl-], were both sensitive to Cl- and K+ channel block. Such apparent coupling of K+ and Cl- fluxes can be explained by the occurrence of conductive effluxes. With the assumption that the resting [Cl-Ii is higher than that of Nernst’s distribution and that the stimulation activates conductive Cl- channels, the inward current carried by Cl- efflux depolarizes the cell membranes, resulti ng in a reductio n of electromoti ve force for Clefflux and an increase in electromotive force for cation efflux. Unless the conductive K+ efflux through BK chan-
nels carrying the outward current neutralizes the Cl- current , no measura ble changes in [Cl-Ii will occur, and the reverse would also be true. Massive electrically coupled anion and cation effluxes supply the driving force for osmotic water efflux. Accordin g to the above-mentioned discussion, both of K + and Cl- conductances are not necessarily needed to be primarily activated by electrical stimulation. Even at the resting range of [Ca2+]i (65 nM), Ca2+-activated K+ conductance is reportedly dominant in outer hair cells (1). Because additional increase in K+ conductance by valinomycin failed to change the resting [Cl-Ii and electrical stimulation-induced changes in [Cl-], (Fig. 8), K+ conductance in outer hair cells appears to be high enough to allow free movement of K+ driven by el.ectromotive force, and Cl- conductance is assumed to be so low as to limit the Cl- and electrically coupled K+ movements.
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Cl094
DELAYED
Table 2. Reported
ionic currents
Current Carrier
SHORTENING
and channels
OF OUTER
Unit Conductance, PS
Whole Ca2+ Inward rectifier Time and voltage High threshold
cell current
dependent
Ba2+,
TEA,
quinidine,
nifedipine
cs+ Cd”+
Acetylcholine ATP Ca2+
Reference No.
Inhibitor
Cd2+,
Quinidine, Isolated
K’ K’ ClMonovalent
CELLS
in cochlear outer hair cells
Activator/ Feature
K+ K+ K’ Ca”+ Cations Cations Cations
HAIR
channels in excised 45, 240 20, 149, 200 22, 40
We thank Kansai Medical University Laboratory Center and Animal Center for providing experimental instruments and animals. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan, the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and Uehara Memorial Foundation. Address for renrint reauests: C. Inagaki. Dent. of Pharmacologv.
Mg2+,
Kansai Japan. Received
phentolamine
patches
cations
It has been reported that an increase in cellular free Ca2+ induced by a Ca2+ ionophore induces elongation, narrowing, and shrinkage in volume in isolated cochlear outer hair cells (6). The authors claimed that actin-mediated Ca2+-dependent radial forces overcame the turgor pressure and extruded water out of the cells, and that this resulted in volume reduction and elongation. The electrical tetanic stimulation-induced delayed shortening in the present study appeared to occur through quite contradictory processes; actin-mediated contractile forces potentially shortened the cell, concomitant with volume decrease. Because TEA did not inhibit the Ca2+ ionophoreinduced elongation of the cell (6), increased [Ca2+]i alone or Ca2+ entry through a nonphysiological route of Ca2+ ionophore may not be sufficient to achieve full responses of electrical stimulation-induced volume changes with the activation of Ca2+ -activated K+ channels. Because electrical stimulation-induced cell shortening did not occur when K+ or Cl- channels were blocked, these channel activities appeared to be needed for actin fiber contraction. The stimulation-induced entry of Ca2+ may be facilitated by increases in K+ and Cl- conductances. Furthermore, physiologically available Ca2+ for contraction of actin fibers may be increased by the increased intracellular concentration of Ca2+ with dehydration, or by facilitated Ca2+ diffusion by intracellular water flow. Clinical doses of furosemide occasionally induce hearing disorders especially in the course of renal disease (28). Such an effect is usually explained by reduced endocochlear potential (20); however, the inhibition of delayed shortening by this drug may also be involved in the mechanisms of furosemide-induced hearing disorders, probably through the loss of protective contractions to intense sound pressure, resulting in the cellular damage. The delayed shortening induced by tetanic electrical stimulation appears to be a useful model for studying the physiological functions of slow motility of cochlear outer hair cells.
tubocurarine,
1, 17, 29 1 17 24, 29 17 9 23
Medical 28 May
1 10 10 19
Ca2+, diltiazem
University,
Fumizono-cho
1992; accepted
in final
1, Moriguchi, form
30 July
Osaka
570,
1992.
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