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Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea
Brain Research, 614 (1993) 205-211 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
205
BRES 18920
Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea Narinobu
Harada
a,b, A r n e
Ernst
a and Hans
Peter
Zenner
a
a Hearing Research Laboratories, Department of Otolaryngology, University of Tiibingen, Tiibingen (Germany) and b Department of Otolaryngology, Kansai Medical University, Fumizonocho, Moriguchi, Osaka (Japan)
(Accepted 12 January 1993)
Key words: Whole-cell patch clamp technique; Outer hair cell; Fura-2; Hyperpolarization; Hyposmotic activation
The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 + 2.3 mV from the resting membrane potential of -58.5 + 5.9 mV (n = 48). This hyperpolarization was associated with an outward current (97.7_+22.2 pA, n = 15). The hyperpolarization was inhibited by 300 p.M quinine, 5 mN Ba 2+ and increasing the extraceliular K ÷ to 30 mM from 5 mM. In the absence of extracellular Ca 2÷ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50/zM GdCl3, which is known to block stretch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50/zM GdCI 3 also inhibited [Ca2+]i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca2+]i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca 2+ influx. In turn, this [Ca2+ ]i increase leads to an activation of the Ca2+-activated K ÷ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K + efflux.
INTRODUCTION
p r o v i d e e v i d e n c e t h a t i n t r a c e l l u l a r c a l c i u m is i n c r e a s e d by h y p o s m o t i c activation t9'23'2°. R e c e n t l y , we could re-
Previous studies have shown t h a t the m e m b r a n e p o t e n t i a l was h y p e r p o l a r i z e d by a cell swelling d u r i n g h y p o s m o t i c activation in d i f f e r e n t types o f cells t3'16'27.
p o r t on R V D in i s o l a t e d o u t e r hair cells ( O H C s ) o f the g u i n e a pig c o c h l e a d e s p i t e t h e c o n t i n u e d e x p o s u r e to a h y p o t o n i c s o l u t i o n 12. A n i n c r e a s e of t h e i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n ([Ca2+] i) d u r i n g h y p o s m o t i c activation using t h e c a l c i u m sensitive dye fura-2 could also b e d e s c r i b e d in o u r p a p e r le. H o w e v e r , the e l e c t r o physiological r e s p o n s e s o f O H C s to h y p o s m o t i c activation still r e m a i n s to b e e l u c i d a t e d in m o r e detail. In the p r e s e n t study we i n v e s t i g a t e d t h e r e f o r e the c h a n g e s o f t h e r e c e p t o r p o t e n t i a l in O H C s d u r i n g h y p o s m o t i c activation by a whole-cell p a t c h - c l a m p t e c h n i q u e . M o r e o v e r , t h e role o f i n t r a c e l l u l a r c a l c i u m for v o l u m e r e g u l a t i o n in O H C s is discussed.
T h o s e studies also s u g g e s t e d t h a t this h y p e r p o l a r i z a tion is d u e to an i n t r a c e l l u l a r K ÷ efflux t h r o u g h C a z+a c t i v a t e d K ÷ channels. T h e K ÷ effiux d u r i n g hyposm o t i c activation is in p a r a l l e l to the r e g u l a t o r y v o l u m e d e c r e a s e ( R V D ) following t h e initial cell swelling despite t h e c o n t i n u e d e x p o s u r e to a h y p o t o n i c solution in d i f f e r e n t t y p e s o f cells 10't3'14. It has b e e n s u g g e s t e d t h a t this R V D results f r o m t h e loss o f K ÷ a n d C1- associa t e d with a loss o f i n t r a c e l l u l a r w a t e r t°'13'14'26. It could also e v i d e n c e d t h a t t h e activation o f t h e v o l u m e - r e g u latory K ÷ efflux is c o n t r o l l e d by the i n t r a c e l l u l a r Ca 2÷ (refs. 13, 16). T h e h y p o s m o t i c activation is a c c o m p a n i e d by t h e K ÷ effiux which c o n t r i b u t e s to t h e c e l l u l a r h y p e r p o l a r i z a t i o n . It is largely b a s e d on c a a + - a c t i v a t e d K ÷ c h a n n e l s 24'25. A d d i t i o n a l investigations c o u l d also
M A T E R I A L S AND METHODS Isolation of outer hair cells Single outer hair cells (OHCs) were isolated from guinea pig cochleas without enzymatic treatment. The 3rd and 4th turn were
Correspondence: N. Harada, Dept. of Otolaryngology, Kansai Medical University, Fumizonocho 1, Moriguchi, Osaka, Japan. Fax: (81) (6) 992-1055.
206 removed with fine forceps and placed into droplets of Hanks' balanced salt solution (HBSS) (Gibco-Biochrom, Germany) buffered with 5 mN HEPES and adjusted to pH 7.4, 300 m O s m by addition of NaCI. The organ of Corti was further separated by mechanical dissociation and pipetting in fresh HBSS. The isolated O H C s were placed in an experimental chamber coated with C E L L - T A K (Collaborative Research Inc., USA) filled with fresh HBSS for 10 rain. Subsequently, the perfusion of physiological standard solution (PSS) was started. The chamber was filled with PSS up to an internal volume of 500 /zl. Experiments were performed in physiological standard solution (PSS) containing (in mM): NaCI 150, KC1 5, CaCI 2 1, MgCI 2 1, glucose 5, HEPES 10, adjusted to pH 7.4 and 300 m O s m .
Whole-cell patch-clamp recordings Whole cell recordings with patch-clamp pipettes were performed as described by Marty and Neher is. Patch-clamp pipettes were pulled from soda glass by m e a n s of a P-87 micropipette puller (Flaming-Brown, Sutter Instr., USA) and back-filled with KC1 Ringer solution containing (in mM): KCl 140, MgCl 2 2, CaCl 2 1, E G T A 11, HEPES 10, pCa 8, pH 7.2 adjusted with KOH. The input resistances amounted to 3 - 5 MI2. Silver-silver chloride electrodes connected pipette and bath with the patch-clamp amplifier EPC-7 (List-electronic, Darmstadt, Germany). The pipette's voltage was adjusted at - 4 . 1 mV in order to compensate the diffusion potential at the pipette's tip which was measured independently in control experiments. The intracellular potential was monitored by clamping the current ( I ) from the electrode into the cell to I = O. All values were expressed as mean_+standard error of the mean, n denotes the number of cells. The experiments were observed with a CCD video camera (F10, Panasonic, Japan). Video signals were stored on a video tape recorder (U-matic, Sony, Japan). Measurements of [Ca e + ]i The O H C s were loaded with 2 ~zM f u r a - 2 / A M (Calbiochem, USA) for 40 min in HBSS. The fura-2 loaded hair cells were rinsed with PSS. The chamber was m o u n t e d on the stage of an inverted microscope (IM35, Zeiss, Germany) equipped with a × 63 epifluorescence lens (numerical aperture 0.9). [Ca 2+ ]i measurements in isolated O H C s were performed by means of a microscope photometer system (MSP21, Zeiss, Germany) coupled to an inverted microscope. when the [Ca 2+ ]i was increased, the fluorescence intensity was also risen at a wavelength of 340 nm and decreased at 380 nm. A 75 W xenon lamp was employed for fluorescence excitation. After passing through the 340 nm and 380 nm filters, the excitation light beam was reflected by a 395 nm dichroic mirror. Emitted light was monitored after the passage through a 500 n m cut-off filter (a 30 nm band-pass). The fluorescence ratio (340 n m / 3 8 0 nm) was calculated and transferred to [Ca 2+ ]i by an in vitro calibration procedure II using the following formula:
ings, the fluorescence was observed from the emitted light. After the fluorescence remained stable for at least 3 min, the recording was started. The whole-cell recordings and [Ca 2÷ ]i measurements were performed as described above.
Solutions applied The hypotonic solution was obtained by a reduction of NaCI in the PSS from 150 m M to 120 m M NaC1 at pH 7.4. The osmolarity of this hypotonic solution ranged from 250 m O s m to 253 mOsm. In a CaZ+-free solution, the PSS or hypotonic solution was prepared by omitting CaCI 2, but by addition of 1 m M E G T A . To prevent a depletion of Ca z+ from internal Ca 2+ stores, the Ca2+-free solution was exchanged just prior to experiment. In the 30 m M KCI isotonic and hypotonic solution, NaCI was replaced by KC1 in the PSS and the hypotonic solution. Barium (Sigma, USA), Quinine (Sigma, USA) and GdCI 3 (Aldrich, Germany) were added to both PSS and the hypotonic solution. The osmolarity of each solution was measured by an osmometer (Osmomat 030, Gonotec, USA). Cells were superfused at a flow rate of 1 m l / m i n . Separate controls were superfused with PSS only (n = 7) to monitor a possible influence of the perfusion pressure on the cells' electrophysiology and size. All experiments were performed at room temperature (20-22°C). RESULTS
Hyperpolarization of OHCs by hyposmotic activation The OHCs had a resting membrane potential of -58.5 _+ 5.9 mV (n = 48). Controls superfused with PSS only did not change Significantly their membrane potential. The membrane potential of each cell (n = 48) was hyperpolarized by a cell swelling during hyposmotic activation (250 mOsm). Fig. 1A shows the typical electrophysiological response of the OHCs to hyposmotic activation. The amplitude of hyperpolarization
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Simultaneous measurements of membrane potential and [Ca 2 + ]i For the simultaneous m e a s u r e m e n t s of m e m b r a n e potential and [Ca:+ ]i changes, the pipette solution was prepared without CaCI 2 and E G T A in order to prevent artifacts. The pipette was back-filled with 150 m M KCI, 2 m M MgClz, 10 m M HEPES, pH 7.4. The O H C s were loaded with 2 p,M f u r a - 2 / A M and rinsed with PSS when breaking through the cell m e m b r a n e for further whole-cell record-
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Fig. 1. Electrical responses of outer hair cells to hyposmotic activation (250 mOsm). A: changes of the m e m b r a n e potential under current clamp mode (clamped at zero current). Hyposmotic activation induced membrane hyperpolarization from the resting potential of - 6 3 mV to - 7 1 mV. B: hyposmotic activation induced an outward current. The cell was held under voltage clamp at - 60 m V and zero current potential was - 5 8 mV. At the arrow, the perfusion of hypotonic solution and physiological standard solution (PSS) was started.
207 amounted to 6.6 _+ 2.3 mV (n = 48). This hyperpolarization usually occurred in a time-dependent manner after a lag period of 10-20 s, reached a steady state within 30-40 s, and lasted for several minutes (at least 2 min) when the exposure to a hypotonic solution was continued. Thereafter, a slow repolarization occurred. In 6 out of 48 cells (12%), this hyperpolarization was followed by a depolarization during hyposmotic activation. The amplitude of depolarization was 6.3_+ 2.7 mV. The amplitude of the hyperpolarization induced by hyposmotic activation were almost the same when repeating the measurement for a second time after 60120 s. The amplitude of the second hyperpolarization was 98.9 + 4.3% of the first response (n = 12). Thus, the effect was reversible and repeatable. Hyposmotic activation also induced an outward current under voltage-clamp (Fig. 1B). The peak amplitude of the outward current was 97.7 _+ 22.2 pA (n = 15). In order to investigate the effect of a reduced NaC1 in the hypotonic solution, an isotonic solution was prepared by the addition of mannitol. When the isotonic solution was replaced by the hypotonic solution, the membrane potential was also hyperpolarized. This indicates that the hyperpolarization during hyposmotic activation is not due to the reduction of NaC1 in the hypotonic solution but simply due to the observed cell swelling. Effect o f quinine and Ba 2 + on hyperpolarization Fig. 2 shows the typical response to hyposmotic activation in the presence of 300/.~M quinine which is a blocker of CaZ+-activated K + channels 3. The hyperpolarization during hyposmotic activation was inhibited by quinine (n = 9). The amplitude of hyperpolarization was reduced by quinine to 13% of the controls (n = 9). 5 mM Ba 2+ which also blocks Ca2+-activated K + channels 8 led to a reduced hyperpolarization upon hyposmotic activation to 29% of the controls (n = 7). Effects o f extracellular Ca 2 + In 6 cells, the hyperpolarization was completely blocked in the absence of extracellular Ca 2+ which was achieved by addition of 1 mM EGTA. Fig. 3 shows a typical response by hyposmotic activation in the absence of extracellular Ca 2+. In this cell, the depolarization was preserved while the hyperpolarization was abolished in the absence of extracellular Ca 2+. However, one cell still showed the hyperpolarization even in the absence of extracellular Ca 2+. The amplitude of the hyperpolarization in the absence of extracellular Ca z+ was reduced to 27% of the control response in this cell.
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Fig. 3. Effect of extracellular Ca2+ on hyperpolarization during hyposmotic activation. The hyperpolarization was completely inhibited in the absence of extracellular Ca 2+ (1 mM EGTA). Note the depolarization was induced even in the absence of extracellular Ca2+. At the arrow, the perfusion of each solution was started.
208
Effects of extracellular K +
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The influence of an increase in extracellular K ÷ on the hyperpolarization during hyposmotic activation was also investigated. When the PSS was replaced by the 30 mM KC1 solution (300 mOsm) after the control measurement, the membrane potential was depolarized to - 2 8 . 2 + 7.4 mV from the resting potential of - 5 0 . 5 + 7.5 mV. In parallel, a slight cell swelling was observed (n = 7). After stable electrophysiological recordings with persisting cell swelling, the 30 mM KC1 solution was replaced by a 30 mM KC1 hypotonic solution (250 mOsm). After the application of the 30 mM KCI hypotonic solution, the same magnitude of cell swelling was observed as compared to controls. However, the hyperpolarization during hyposmotic activation in the presence of 30 mM extracellular K ÷ was inhibited (Fig. 4). The amplitude of the hyperpolarization was reduced to 21% of the control response (n = 7).
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Fig. 5. Effect of gadolinium (50 /zM) on hyperpolarization during hyposmotic activation. T h e effect of gadolinium was reversible. The hyperpolarization recovered after a washout. At the arrow, the perfusion of each solution was started.
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examined the effects of gadolinium on the hyperpolarization during hyposmotic activation. 5 0 / x M GdC13 reduced the response to 30% of the controls (n = 10). In 5 out of 10 cells, very stable giga-seals were established so that the gadolinium superfusion could be repeated several times. It was found that the inhibition of hyperpolarization by 50 /zM GdCI3 was reversible, but the amplitude of hyperpolarization recovered to 88% of the control. Fig. 5 shows the typical electrophysiological response by hyposmotic activation in the presence of 50 tzM GdC13. The hyperpolarization was reversibly inhibited in this cell. We also examined the effects of 50 /xM GdCI 3 on [Ca2+]i changes in the OHCs during hyposmotic activation using the calcium sensitive dye fura-2. The OHC was characterized by an increase of [Ca2+] i during hyposmotic activation (n = 11) as previously described in more detail t2. Fig. 6A outlines a typical response to hyposmotic activation in PSS. Hyposmotic activation
209 produced a [Ca2+] i increase up to a peak of 241 nM from a resting level of 110 nM. This [Ca2+] i increase was also accompanied by a cell shortening and swelling. In the presence of 50 tzM GdCI 3, the increase of [CaZ+] i was significantly inhibited ( P < 0.01, n = 15) although the cell swelling was constantly observed in those cells. Fig. 6B shows the typical response in the presence of 50 t~M GdC13. [Ca2+]i was increased to 64 nM from the resting level of 60 nM. The amplitude of [Ca2+]i increase in the presence of 5 0 / ~ M GdCI 3 was 25 + 23 nM (n = 15) while the amplitude of [Ca2+] i increase during the control measurements was 177 + 89 nM (n = 11). An excessive cell swelling was observed in some cells during fura-2 measurements. However, these cells easily ruptured. This rupture of the cell m e m b r a n e led to a rapid loss of all of the indicator from the cytoplasm to the extracellular space and resulted in a sudden decrease of the fluorescence at both 340 nm and 380 nm. These cells were excluded from the present study.
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Fig. 7. Simultaneous measurements of membrane potential and [Ca2+]i changes during hyposmotic activation. The resting membrane potential was -66 mV at zero current. Hyposmotic activation induced a 12 mV hyperpolarization (B). Simultaneous [Ca2+]i increase was also observed in this cell (A).
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In 6 cells we could measure simultaneously changes of m e m b r a n e potential and [CaZ+] i using the combined methods of whole-cell recordings and fura-2 measurements. Fig. 7 shows the successive m e a s u r e m e n t of the m e m b r a n e potential and [Ca2+]i changes during hyposmotic activation. [Ca2+]i was increased to a peak of 289 nM from the resting level of 152 nM which was accompanied by a hyperpolarization to - 7 8 m V from the resting potential of - 6 6 inV.
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Fig. 6. Typical time course of changes of [Caz+ ]i during hyposmotic activation in the presence and the absence of gadolinium (50/zM). Control and test measurements were made with the cells from the same cochlea.
The present findings suggest that a cell swelling induced by hyposmotic activation can induce a hyperpolarization of O H C s in guinea pig cochlea. Previous whole-cell patch-clamp studies on other cell types have demonstrated that the hyperpolarization could be induced by a cell swelling during hyposmotic activation 6'13'16. It was hypothesized that the hyperpolarization results in the activation of CaZ+-activated K + channels in different types of cells 6'24'25. In our study,
210 the hyperpolarization of OHCs during hyposmotic activation was inhibited by quinine, Ba 2+ and the increase of extracellular K +. The hyperpolarization accompanied by a [Ca2+]i increase could be observed using the combined methods of whole-cell recordings and fura-2 measurements in parallel. In essence, our data strongly suggest that the hyperpolarization of OHCs during hyposmotic activation relies on a K + effiux by the activation of CaZ+-activated K + channels. It was also reported that a Ca 2+ influx from the extracellular space which activates K + channels is essential for the regulatory volume decrease (RVD) during hyposmotic activation 2"6'24. In a previous study, the existence of RVD in OHCs despite the continued exposure to a hypotonic solution could be described in detail 12. It could also be demonstrated that hyposmotic activation leads to an increase of [Ca2+]i which largely depends on extracellular Ca 2+ as well as the RVD in OHCs 12. Therefore, we have postulated that Ca 2+ also thoroughly controls volume regulation in OHCs. However, only 76% of the cells in our previous study showed RVD during hyposmotic activation 12. Therefore, additional data are required to fully understand the mechanism of RVD in the OHCs during hyposmotic activation. Recent eletrophysiological studies showed that the stretching of the cell membrane by hyposmotic activation causes an influx of Ca 2+ through stretch-activated non-specific cation channels which is followed by the activation of Ca2+-activated K + channels 2'2°. Indeed, Okada et al. 2° showed that a [Ca2+]i increase and a Ca 2+ current during hyposmotic activation could be abolished by GdC13 which is a specific blocker of stretch-activated channels 7'2s. Our findings that GdC13 blocked [Ca2+]i increase and the hyperpolarization during hyposmotic activation are line with the study cited before. However, one cell of our study was still hyperpolarized even in the absence of extracellular Ca 2+. This result indicates the possibility of a Ca 2+ release from internal stores. On the other hand, we could not detect a significant [Ca2+]i increase in the absence of extracellular Ca 2+ during hyposmotic activation by fura-2 measurements 12. In cultured rabbit kidney proximal tubule cells, K + currents in the absence of extracellular Ca 2+ were described by wholecell recordings 16, but a [Ca2+]i increase using fura-2 measurements during hyposmotic activation could not be evidenced 23. One possible reason might be a [Ca2+]~ increase only in the region of the subcellular membrane, e.g. the lateral cisternae 21, of the O H C membrane. In turn, this could activate K + channels which is not detectable by the imaging system used. The same seemingly holds true for the cultured rabbit proximal
tubule cells 23. The other possible reason might be that a small number of K + channels may be directly activated by a stretching of the cell membrane without dependency to Ca 2+. Recently, two types of stretchactivated channels in the lateral cell membrane of the guinea pig OHCs have been characterized 5. It was suggested that one of these channels may have a permeability to K + (ref. 5). Therefore, a cell swelling by hyposmotic activation may also activate directly this type of stretch-activated channels. However, the activation of hyperpolarization in OHCs in response to hyposmotic activation was clearly a time-dependent phenomenon. The lag period was about 10-20 s. The time-course suggests that the hyperpolarization may be mediated by intracellular messengers such as Ca 2+ instead of a direct activation by membrane stretch. These two hypotheses need to be further evidenced. The other type of a stretch-activated channel in the O H C membrane is a non-specific cation channel with a permeability to Ca 2+ (ref. 5). Another study showed that a C-type K + channel which is activated by Ca 2+ is localized in the lateral cell membrane of OHCs 9. Thus, we conclude that a cell swelling by hyposmotic activation opens stretch-activated non-specific cation channels which allow a Ca 2+ influx into the cell. Thereafter, a [Ca2+]~ increase may activate C-type K + channels which hyperpolarize OHCs by a K + efflux. 6 out of 48 cells showed the described depolarization after the initial hyperpolarization. Other cells (42/48) were usually slowly repolarized after the hyperpolarization. These results are in general agreement with previous studies 6"13A6'27. The depolarization was preserved even in the absence of extracellular Ca 2+ which was also found earlier 13. Authors suggest that the subsequent depolarization may be based on a C1effiux which is Ca-independent. However, not all OHCs showed the depolarization. Therefore, further singlechannel or pharmacological investigations are required to fully elucidate the role of the CI- channel activation in OHC during hyposmotic activation. The most interesting finding of the present study is the hyperpolarization induced by stretch of the cell membrane which contributes to motility and, hence, tuning of the OHC. The hyperpolarization of the OHCs is followed by an elongation of the cellular body 1,22,29 in contrast to other non-sensory cells such as the proximal tubule. Santos-Sacchi et al. 22 speculated that the mechanical response of the OHCs to actively amplify the travelling wave 3° is widely dependent on the membrane potential rather than the transmembrane current. Therefore, we described the changes of the receptor potential in the OHCs under current-clamp conditions.
211 The OHC motility is essential to provide a feedback in the fine tuning of the cochlea4. The resulting OHC length could tune the basilar membrane by bringing about a shift in its mean position. Thereby, the responsiveness of the cochlear partition is improved 17. It seems to be likely that a stretch of the OHC membrane is physiological during sound stimulation. Thus, the hyperpolarization induced by membrane stretch may participate to the control of the OHC motility and fine tuning, respectively. Under pathophysiological conditions, any change of the inner ear fluid volume or composition can possibly provoke such a membrane stretch and hyperpolarization with the well-known clinical findings of tinnitus, hearing loss and vertigo, respectively 15,31. Acknowledgements.
We would like to thank Professor F. Lang, Department of Physiology, University of Tiibingen, for his helpful comments during this work. This work was supported by a Leibniz grant of the DFG to H.P.Z. REFERENCES 1 Ashmore, J.F., A fast motile response in guinea-pig outer hair cells. The cellular basis of the cochlear amplifier, J. PhysioL, 338 (1987) 323-347. 2 Cristensen, O., Mediation of cell volume regulation by Ca 2+ influx through stretch-activated channels, Nature, 330 (1987) 6668. 3 Cook, N.S. and Haylett, D.G., Effects of apamin, quinine and neuromuscular blockers on calcium-activated potassium channels in guinea-pig hepatocytes, J. PhysioL, 358 (1985) 373-394. 4 Dallos, P. and Corey, M.E., The role of outer hair cell motility in cochlear tuning, Curr. Opinions NeurobioL, 1 (1991) 215-220. 5 Ding, J.P., Salvi, R.J. and Sachs, F., Stretch-activated ion channels in guinea pig outer hair cells, Hearing Res., 56 (1991) 18-28. 6 Dube, L. Parent, L. and Save, R., Hypotonic shock activates a max K ÷ channel in primary cultured proximal tubule cells, Am. J. Physiol., 259 (1990) F348-F356. 7 Filipovic, D. and Sakin, H., A calcium-permeable stretch-activated cation channel in renal proximal tubule, Am. J. PhysioL, 260 (1991) Fl19-129. 8 Gitter, A.H., Beyenbach, K.W., Christine, C.W., Gross, P., Minuth, W.W. and Fr6mter, E., High-conductance K ÷ channel in apical membranes of principal cells cultured from renal cortical collecting duct anlagen, Pfliigers Arch., 408 (1987) 282-290. 9 Gitter, A.H., Fr6mter, E. and Zenner, H.P., C-Type potassium channels in the lateral cell membrane of guinea-pig outer hair cells, Hearing Res., 60 (1992) 13-19 10 Grinstein, S., Dupre, A. and Rothstein, A., Volume regulation by human lymphocytes, role of calcium, J. Gen. PhysioL, 79 (1982) 849-868. 11 Gynkiewcz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2" indicators with greatly improved fluorescence properties, Z Biol. Chem., 260 (1985) 3440-3450.
12 Harada, N., Ernst, A., Zenner, H.P., Intracellular calcium rises by hyposmotic activation of guinea pig outer hair cells, Hearing Res., in press. 13 Hazama, A. and Okada, Y., Ca 2÷ sensitivity of volume-regulatory K ÷ and CI- channels in cultured human epithelial cells, J. Physiol., 402 (1988) 687-702. 14 Hoffman, E.K., Simonsen, L.O. and Lambert, I.H., Volume-induced increase of K ÷ and CI- permeabilities in Ehrlich tumor ceils. Role of internal Ca 2÷, J. Membrane Biol., 78 (1984) 211-222. 15 Horner, K.C., Old theme and new reflections: hearing impairment associated with endolymphatic hydrops, Hearing Res., 52 (1991) 147-156. 16 Kawahara, K., Ogawa, A. and Suzuki, M., Hyposmotic activation of Ca-activated K channels in cultured rabbit kidney proximal tubule cells, Am. J. Physiol., 260 (1991) F27-F33. 17 LePage, E.L., Frequency-dependent self-induced bias of the basilar membrane and its potential for controlling sensitivity and tuning in the mammalian cochlea, J. Acoust. Soc. Am., 82 (1987) 139-154. 18 Marty, A. and Neher, E., Tight-seal whole-cell recording. In B. Sakman and E. Neher (Eds.), Single Channel Recordings, Plenum Press, 1983, New York. 19 McCarty, N.A. and O'Neil, R.G., Calcium-dependent control of volume regulation in renal proximal tubule cells: I. Swellingactivated Ca 2÷ entry and release, J. Membrane Biol., 123 (1991) 149-160. 20 Okada, Y., Hazama, A. and Yuan, W., Stretch-induced activation of Ca2+-permeable ion channels is involved in the volume regulation of hypotonically swollen epithelial cells, Neurosci. Res., Suppl. 12 (1990) $5-S13. 21 Saito, K., Fine structure of the sensory epithelium of guinea pig organ of corti: subsurface cisternae and lamellar bodies in the outer hair cells, Cell Tiss. Res., 229 (1983) 467-481. 22 Santos-Sacchi, J. and Dilger, J.P., Whole cell currents and mechanical responses of isolated outer hair cells, Hearing Res., 35 (1988) 143-150. 23 Suzuki, M., Kawahara, K., Ogawa, A. and Morita, T., [Ca 2÷ ]i rises via G-protein during regulatory volume decrease in rabbit proximal tubule cells, Am. J. Physiol., 258 (1990) F690-F696. 24 Taniguchi, J. and Guggio, W.B., Membrane stretch: a physiological stimulator of Ca2+-activated K channels in thick ascending limb, Am. Z Physiol., 257 (1989) F347-F352. 25 Ubl, J., Murer, H. and Kolb, H.A., Hypotonic shock evokes opening of Ca2+-activated K channels in opossum kidney cells, Pfliigers Arch., 412 (1988) 551-553. 26 V61kl, H. and Lang, F., Ionic requirement for regulatory cell volume decrease in renal straight proximal tubules, Pfliigers Arch., 412 (1988) 1-6. 27 Welling, P.A. and O'Neil, R.G., Cell swelling activates basolateral membrane C1 and K conductances in rabbit proximal tubule, Am. J. Physiol., 258 (1990) F951-F962. 28 Yang, X.C. and Sachs, F., Block of stretch activated ion channels in Xenopus oocytes by gadolinium and calcium ions, Science, 243 (1989) 1068-1071. 29 Zenner, H.P., Zimmermann, U. and Gitter, A.H., Fast motility of isolated mammalian auditory sensory cells, Biochem. Biophys. Res. Commun., 49 (1987) 304-308. 30 Zenner, H.P. and Ernst, A., Sound processing by ac and dc movements of cochlea outer hair cells, Prog. Brain Res., in press. 31 Zenner, H.P. and Ernst, A., Three models of tinnitus, Arch. Otorhinolaryngol., in press.
205
BRES 18920
Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea Narinobu
Harada
a,b, A r n e
Ernst
a and Hans
Peter
Zenner
a
a Hearing Research Laboratories, Department of Otolaryngology, University of Tiibingen, Tiibingen (Germany) and b Department of Otolaryngology, Kansai Medical University, Fumizonocho, Moriguchi, Osaka (Japan)
(Accepted 12 January 1993)
Key words: Whole-cell patch clamp technique; Outer hair cell; Fura-2; Hyperpolarization; Hyposmotic activation
The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 + 2.3 mV from the resting membrane potential of -58.5 + 5.9 mV (n = 48). This hyperpolarization was associated with an outward current (97.7_+22.2 pA, n = 15). The hyperpolarization was inhibited by 300 p.M quinine, 5 mN Ba 2+ and increasing the extraceliular K ÷ to 30 mM from 5 mM. In the absence of extracellular Ca 2÷ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50/zM GdCl3, which is known to block stretch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50/zM GdCI 3 also inhibited [Ca2+]i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca2+]i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca 2+ influx. In turn, this [Ca2+ ]i increase leads to an activation of the Ca2+-activated K ÷ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K + efflux.
INTRODUCTION
p r o v i d e e v i d e n c e t h a t i n t r a c e l l u l a r c a l c i u m is i n c r e a s e d by h y p o s m o t i c activation t9'23'2°. R e c e n t l y , we could re-
Previous studies have shown t h a t the m e m b r a n e p o t e n t i a l was h y p e r p o l a r i z e d by a cell swelling d u r i n g h y p o s m o t i c activation in d i f f e r e n t types o f cells t3'16'27.
p o r t on R V D in i s o l a t e d o u t e r hair cells ( O H C s ) o f the g u i n e a pig c o c h l e a d e s p i t e t h e c o n t i n u e d e x p o s u r e to a h y p o t o n i c s o l u t i o n 12. A n i n c r e a s e of t h e i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n ([Ca2+] i) d u r i n g h y p o s m o t i c activation using t h e c a l c i u m sensitive dye fura-2 could also b e d e s c r i b e d in o u r p a p e r le. H o w e v e r , the e l e c t r o physiological r e s p o n s e s o f O H C s to h y p o s m o t i c activation still r e m a i n s to b e e l u c i d a t e d in m o r e detail. In the p r e s e n t study we i n v e s t i g a t e d t h e r e f o r e the c h a n g e s o f t h e r e c e p t o r p o t e n t i a l in O H C s d u r i n g h y p o s m o t i c activation by a whole-cell p a t c h - c l a m p t e c h n i q u e . M o r e o v e r , t h e role o f i n t r a c e l l u l a r c a l c i u m for v o l u m e r e g u l a t i o n in O H C s is discussed.
T h o s e studies also s u g g e s t e d t h a t this h y p e r p o l a r i z a tion is d u e to an i n t r a c e l l u l a r K ÷ efflux t h r o u g h C a z+a c t i v a t e d K ÷ channels. T h e K ÷ effiux d u r i n g hyposm o t i c activation is in p a r a l l e l to the r e g u l a t o r y v o l u m e d e c r e a s e ( R V D ) following t h e initial cell swelling despite t h e c o n t i n u e d e x p o s u r e to a h y p o t o n i c solution in d i f f e r e n t t y p e s o f cells 10't3'14. It has b e e n s u g g e s t e d t h a t this R V D results f r o m t h e loss o f K ÷ a n d C1- associa t e d with a loss o f i n t r a c e l l u l a r w a t e r t°'13'14'26. It could also e v i d e n c e d t h a t t h e activation o f t h e v o l u m e - r e g u latory K ÷ efflux is c o n t r o l l e d by the i n t r a c e l l u l a r Ca 2÷ (refs. 13, 16). T h e h y p o s m o t i c activation is a c c o m p a n i e d by t h e K ÷ effiux which c o n t r i b u t e s to t h e c e l l u l a r h y p e r p o l a r i z a t i o n . It is largely b a s e d on c a a + - a c t i v a t e d K ÷ c h a n n e l s 24'25. A d d i t i o n a l investigations c o u l d also
M A T E R I A L S AND METHODS Isolation of outer hair cells Single outer hair cells (OHCs) were isolated from guinea pig cochleas without enzymatic treatment. The 3rd and 4th turn were
Correspondence: N. Harada, Dept. of Otolaryngology, Kansai Medical University, Fumizonocho 1, Moriguchi, Osaka, Japan. Fax: (81) (6) 992-1055.
206 removed with fine forceps and placed into droplets of Hanks' balanced salt solution (HBSS) (Gibco-Biochrom, Germany) buffered with 5 mN HEPES and adjusted to pH 7.4, 300 m O s m by addition of NaCI. The organ of Corti was further separated by mechanical dissociation and pipetting in fresh HBSS. The isolated O H C s were placed in an experimental chamber coated with C E L L - T A K (Collaborative Research Inc., USA) filled with fresh HBSS for 10 rain. Subsequently, the perfusion of physiological standard solution (PSS) was started. The chamber was filled with PSS up to an internal volume of 500 /zl. Experiments were performed in physiological standard solution (PSS) containing (in mM): NaCI 150, KC1 5, CaCI 2 1, MgCI 2 1, glucose 5, HEPES 10, adjusted to pH 7.4 and 300 m O s m .
Whole-cell patch-clamp recordings Whole cell recordings with patch-clamp pipettes were performed as described by Marty and Neher is. Patch-clamp pipettes were pulled from soda glass by m e a n s of a P-87 micropipette puller (Flaming-Brown, Sutter Instr., USA) and back-filled with KC1 Ringer solution containing (in mM): KCl 140, MgCl 2 2, CaCl 2 1, E G T A 11, HEPES 10, pCa 8, pH 7.2 adjusted with KOH. The input resistances amounted to 3 - 5 MI2. Silver-silver chloride electrodes connected pipette and bath with the patch-clamp amplifier EPC-7 (List-electronic, Darmstadt, Germany). The pipette's voltage was adjusted at - 4 . 1 mV in order to compensate the diffusion potential at the pipette's tip which was measured independently in control experiments. The intracellular potential was monitored by clamping the current ( I ) from the electrode into the cell to I = O. All values were expressed as mean_+standard error of the mean, n denotes the number of cells. The experiments were observed with a CCD video camera (F10, Panasonic, Japan). Video signals were stored on a video tape recorder (U-matic, Sony, Japan). Measurements of [Ca e + ]i The O H C s were loaded with 2 ~zM f u r a - 2 / A M (Calbiochem, USA) for 40 min in HBSS. The fura-2 loaded hair cells were rinsed with PSS. The chamber was m o u n t e d on the stage of an inverted microscope (IM35, Zeiss, Germany) equipped with a × 63 epifluorescence lens (numerical aperture 0.9). [Ca 2+ ]i measurements in isolated O H C s were performed by means of a microscope photometer system (MSP21, Zeiss, Germany) coupled to an inverted microscope. when the [Ca 2+ ]i was increased, the fluorescence intensity was also risen at a wavelength of 340 nm and decreased at 380 nm. A 75 W xenon lamp was employed for fluorescence excitation. After passing through the 340 nm and 380 nm filters, the excitation light beam was reflected by a 395 nm dichroic mirror. Emitted light was monitored after the passage through a 500 n m cut-off filter (a 30 nm band-pass). The fluorescence ratio (340 n m / 3 8 0 nm) was calculated and transferred to [Ca 2+ ]i by an in vitro calibration procedure II using the following formula:
ings, the fluorescence was observed from the emitted light. After the fluorescence remained stable for at least 3 min, the recording was started. The whole-cell recordings and [Ca 2÷ ]i measurements were performed as described above.
Solutions applied The hypotonic solution was obtained by a reduction of NaCI in the PSS from 150 m M to 120 m M NaC1 at pH 7.4. The osmolarity of this hypotonic solution ranged from 250 m O s m to 253 mOsm. In a CaZ+-free solution, the PSS or hypotonic solution was prepared by omitting CaCI 2, but by addition of 1 m M E G T A . To prevent a depletion of Ca z+ from internal Ca 2+ stores, the Ca2+-free solution was exchanged just prior to experiment. In the 30 m M KCI isotonic and hypotonic solution, NaCI was replaced by KC1 in the PSS and the hypotonic solution. Barium (Sigma, USA), Quinine (Sigma, USA) and GdCI 3 (Aldrich, Germany) were added to both PSS and the hypotonic solution. The osmolarity of each solution was measured by an osmometer (Osmomat 030, Gonotec, USA). Cells were superfused at a flow rate of 1 m l / m i n . Separate controls were superfused with PSS only (n = 7) to monitor a possible influence of the perfusion pressure on the cells' electrophysiology and size. All experiments were performed at room temperature (20-22°C). RESULTS
Hyperpolarization of OHCs by hyposmotic activation The OHCs had a resting membrane potential of -58.5 _+ 5.9 mV (n = 48). Controls superfused with PSS only did not change Significantly their membrane potential. The membrane potential of each cell (n = 48) was hyperpolarized by a cell swelling during hyposmotic activation (250 mOsm). Fig. 1A shows the typical electrophysiological response of the OHCs to hyposmotic activation. The amplitude of hyperpolarization
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Rmin, R . . . . Kd, Fo/E~ values were determined to be 0.096, 2.171, 220 and 4.47, respectively, using a E G T A - C a 2+ buffer solution containing 25 /xM fura-2 pentapotassium salt. Each fluorescence signal was also focused on a SIT (silicon intensifier target) camera (C2400, H a m a m a t s u , Japan). The video signal from the camera was stored on a video tape recorder (U-matic, Sony, Japan). The recorded images on the video tape were subsequently digitized by a digital image processing system (DVS 3000, Hamamatsu, Japan). Photobleaching during m e a s u r e m e n t and autofluorescence were negligible in the present study.
Simultaneous measurements of membrane potential and [Ca 2 + ]i For the simultaneous m e a s u r e m e n t s of m e m b r a n e potential and [Ca:+ ]i changes, the pipette solution was prepared without CaCI 2 and E G T A in order to prevent artifacts. The pipette was back-filled with 150 m M KCI, 2 m M MgClz, 10 m M HEPES, pH 7.4. The O H C s were loaded with 2 p,M f u r a - 2 / A M and rinsed with PSS when breaking through the cell m e m b r a n e for further whole-cell record-
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Fig. 1. Electrical responses of outer hair cells to hyposmotic activation (250 mOsm). A: changes of the m e m b r a n e potential under current clamp mode (clamped at zero current). Hyposmotic activation induced membrane hyperpolarization from the resting potential of - 6 3 mV to - 7 1 mV. B: hyposmotic activation induced an outward current. The cell was held under voltage clamp at - 60 m V and zero current potential was - 5 8 mV. At the arrow, the perfusion of hypotonic solution and physiological standard solution (PSS) was started.
207 amounted to 6.6 _+ 2.3 mV (n = 48). This hyperpolarization usually occurred in a time-dependent manner after a lag period of 10-20 s, reached a steady state within 30-40 s, and lasted for several minutes (at least 2 min) when the exposure to a hypotonic solution was continued. Thereafter, a slow repolarization occurred. In 6 out of 48 cells (12%), this hyperpolarization was followed by a depolarization during hyposmotic activation. The amplitude of depolarization was 6.3_+ 2.7 mV. The amplitude of the hyperpolarization induced by hyposmotic activation were almost the same when repeating the measurement for a second time after 60120 s. The amplitude of the second hyperpolarization was 98.9 + 4.3% of the first response (n = 12). Thus, the effect was reversible and repeatable. Hyposmotic activation also induced an outward current under voltage-clamp (Fig. 1B). The peak amplitude of the outward current was 97.7 _+ 22.2 pA (n = 15). In order to investigate the effect of a reduced NaC1 in the hypotonic solution, an isotonic solution was prepared by the addition of mannitol. When the isotonic solution was replaced by the hypotonic solution, the membrane potential was also hyperpolarized. This indicates that the hyperpolarization during hyposmotic activation is not due to the reduction of NaC1 in the hypotonic solution but simply due to the observed cell swelling. Effect o f quinine and Ba 2 + on hyperpolarization Fig. 2 shows the typical response to hyposmotic activation in the presence of 300/.~M quinine which is a blocker of CaZ+-activated K + channels 3. The hyperpolarization during hyposmotic activation was inhibited by quinine (n = 9). The amplitude of hyperpolarization was reduced by quinine to 13% of the controls (n = 9). 5 mM Ba 2+ which also blocks Ca2+-activated K + channels 8 led to a reduced hyperpolarization upon hyposmotic activation to 29% of the controls (n = 7). Effects o f extracellular Ca 2 + In 6 cells, the hyperpolarization was completely blocked in the absence of extracellular Ca 2+ which was achieved by addition of 1 mM EGTA. Fig. 3 shows a typical response by hyposmotic activation in the absence of extracellular Ca 2+. In this cell, the depolarization was preserved while the hyperpolarization was abolished in the absence of extracellular Ca 2+. However, one cell still showed the hyperpolarization even in the absence of extracellular Ca 2+. The amplitude of the hyperpolarization in the absence of extracellular Ca z+ was reduced to 27% of the control response in this cell.
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Fig. 3. Effect of extracellular Ca2+ on hyperpolarization during hyposmotic activation. The hyperpolarization was completely inhibited in the absence of extracellular Ca 2+ (1 mM EGTA). Note the depolarization was induced even in the absence of extracellular Ca2+. At the arrow, the perfusion of each solution was started.
208
Effects of extracellular K +
>
The influence of an increase in extracellular K ÷ on the hyperpolarization during hyposmotic activation was also investigated. When the PSS was replaced by the 30 mM KC1 solution (300 mOsm) after the control measurement, the membrane potential was depolarized to - 2 8 . 2 + 7.4 mV from the resting potential of - 5 0 . 5 + 7.5 mV. In parallel, a slight cell swelling was observed (n = 7). After stable electrophysiological recordings with persisting cell swelling, the 30 mM KC1 solution was replaced by a 30 mM KC1 hypotonic solution (250 mOsm). After the application of the 30 mM KCI hypotonic solution, the same magnitude of cell swelling was observed as compared to controls. However, the hyperpolarization during hyposmotic activation in the presence of 30 mM extracellular K ÷ was inhibited (Fig. 4). The amplitude of the hyperpolarization was reduced to 21% of the control response (n = 7).
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Effects of gadolinium on the hyperpolarization and [ Cae +]i Gadolinium is described to block stretch-activated non-specific cation channels with a permeability to Ca z+ (refs. 7, 28). In the following experiments, we
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Fig. 5. Effect of gadolinium (50 /zM) on hyperpolarization during hyposmotic activation. T h e effect of gadolinium was reversible. The hyperpolarization recovered after a washout. At the arrow, the perfusion of each solution was started.
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examined the effects of gadolinium on the hyperpolarization during hyposmotic activation. 5 0 / x M GdC13 reduced the response to 30% of the controls (n = 10). In 5 out of 10 cells, very stable giga-seals were established so that the gadolinium superfusion could be repeated several times. It was found that the inhibition of hyperpolarization by 50 /zM GdCI3 was reversible, but the amplitude of hyperpolarization recovered to 88% of the control. Fig. 5 shows the typical electrophysiological response by hyposmotic activation in the presence of 50 tzM GdC13. The hyperpolarization was reversibly inhibited in this cell. We also examined the effects of 50 /xM GdCI 3 on [Ca2+]i changes in the OHCs during hyposmotic activation using the calcium sensitive dye fura-2. The OHC was characterized by an increase of [Ca2+] i during hyposmotic activation (n = 11) as previously described in more detail t2. Fig. 6A outlines a typical response to hyposmotic activation in PSS. Hyposmotic activation
209 produced a [Ca2+] i increase up to a peak of 241 nM from a resting level of 110 nM. This [Ca2+] i increase was also accompanied by a cell shortening and swelling. In the presence of 50 tzM GdCI 3, the increase of [CaZ+] i was significantly inhibited ( P < 0.01, n = 15) although the cell swelling was constantly observed in those cells. Fig. 6B shows the typical response in the presence of 50 t~M GdC13. [Ca2+]i was increased to 64 nM from the resting level of 60 nM. The amplitude of [Ca2+]i increase in the presence of 5 0 / ~ M GdCI 3 was 25 + 23 nM (n = 15) while the amplitude of [Ca2+] i increase during the control measurements was 177 + 89 nM (n = 11). An excessive cell swelling was observed in some cells during fura-2 measurements. However, these cells easily ruptured. This rupture of the cell m e m b r a n e led to a rapid loss of all of the indicator from the cytoplasm to the extracellular space and resulted in a sudden decrease of the fluorescence at both 340 nm and 380 nm. These cells were excluded from the present study.
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Fig. 7. Simultaneous measurements of membrane potential and [Ca2+]i changes during hyposmotic activation. The resting membrane potential was -66 mV at zero current. Hyposmotic activation induced a 12 mV hyperpolarization (B). Simultaneous [Ca2+]i increase was also observed in this cell (A).
300
control 250
200
Therefore the indicator substance can also be applied to reliably test the viability of the isolated OHC.
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Simultaneous measurements of membrane potential and [ Cae +]i changes during hyposmotic activation
100
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In 6 cells we could measure simultaneously changes of m e m b r a n e potential and [CaZ+] i using the combined methods of whole-cell recordings and fura-2 measurements. Fig. 7 shows the successive m e a s u r e m e n t of the m e m b r a n e potential and [Ca2+]i changes during hyposmotic activation. [Ca2+]i was increased to a peak of 289 nM from the resting level of 152 nM which was accompanied by a hyperpolarization to - 7 8 m V from the resting potential of - 6 6 inV.
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Fig. 6. Typical time course of changes of [Caz+ ]i during hyposmotic activation in the presence and the absence of gadolinium (50/zM). Control and test measurements were made with the cells from the same cochlea.
The present findings suggest that a cell swelling induced by hyposmotic activation can induce a hyperpolarization of O H C s in guinea pig cochlea. Previous whole-cell patch-clamp studies on other cell types have demonstrated that the hyperpolarization could be induced by a cell swelling during hyposmotic activation 6'13'16. It was hypothesized that the hyperpolarization results in the activation of CaZ+-activated K + channels in different types of cells 6'24'25. In our study,
210 the hyperpolarization of OHCs during hyposmotic activation was inhibited by quinine, Ba 2+ and the increase of extracellular K +. The hyperpolarization accompanied by a [Ca2+]i increase could be observed using the combined methods of whole-cell recordings and fura-2 measurements in parallel. In essence, our data strongly suggest that the hyperpolarization of OHCs during hyposmotic activation relies on a K + effiux by the activation of CaZ+-activated K + channels. It was also reported that a Ca 2+ influx from the extracellular space which activates K + channels is essential for the regulatory volume decrease (RVD) during hyposmotic activation 2"6'24. In a previous study, the existence of RVD in OHCs despite the continued exposure to a hypotonic solution could be described in detail 12. It could also be demonstrated that hyposmotic activation leads to an increase of [Ca2+]i which largely depends on extracellular Ca 2+ as well as the RVD in OHCs 12. Therefore, we have postulated that Ca 2+ also thoroughly controls volume regulation in OHCs. However, only 76% of the cells in our previous study showed RVD during hyposmotic activation 12. Therefore, additional data are required to fully understand the mechanism of RVD in the OHCs during hyposmotic activation. Recent eletrophysiological studies showed that the stretching of the cell membrane by hyposmotic activation causes an influx of Ca 2+ through stretch-activated non-specific cation channels which is followed by the activation of Ca2+-activated K + channels 2'2°. Indeed, Okada et al. 2° showed that a [Ca2+]i increase and a Ca 2+ current during hyposmotic activation could be abolished by GdC13 which is a specific blocker of stretch-activated channels 7'2s. Our findings that GdC13 blocked [Ca2+]i increase and the hyperpolarization during hyposmotic activation are line with the study cited before. However, one cell of our study was still hyperpolarized even in the absence of extracellular Ca 2+. This result indicates the possibility of a Ca 2+ release from internal stores. On the other hand, we could not detect a significant [Ca2+]i increase in the absence of extracellular Ca 2+ during hyposmotic activation by fura-2 measurements 12. In cultured rabbit kidney proximal tubule cells, K + currents in the absence of extracellular Ca 2+ were described by wholecell recordings 16, but a [Ca2+]i increase using fura-2 measurements during hyposmotic activation could not be evidenced 23. One possible reason might be a [Ca2+]~ increase only in the region of the subcellular membrane, e.g. the lateral cisternae 21, of the O H C membrane. In turn, this could activate K + channels which is not detectable by the imaging system used. The same seemingly holds true for the cultured rabbit proximal
tubule cells 23. The other possible reason might be that a small number of K + channels may be directly activated by a stretching of the cell membrane without dependency to Ca 2+. Recently, two types of stretchactivated channels in the lateral cell membrane of the guinea pig OHCs have been characterized 5. It was suggested that one of these channels may have a permeability to K + (ref. 5). Therefore, a cell swelling by hyposmotic activation may also activate directly this type of stretch-activated channels. However, the activation of hyperpolarization in OHCs in response to hyposmotic activation was clearly a time-dependent phenomenon. The lag period was about 10-20 s. The time-course suggests that the hyperpolarization may be mediated by intracellular messengers such as Ca 2+ instead of a direct activation by membrane stretch. These two hypotheses need to be further evidenced. The other type of a stretch-activated channel in the O H C membrane is a non-specific cation channel with a permeability to Ca 2+ (ref. 5). Another study showed that a C-type K + channel which is activated by Ca 2+ is localized in the lateral cell membrane of OHCs 9. Thus, we conclude that a cell swelling by hyposmotic activation opens stretch-activated non-specific cation channels which allow a Ca 2+ influx into the cell. Thereafter, a [Ca2+]~ increase may activate C-type K + channels which hyperpolarize OHCs by a K + efflux. 6 out of 48 cells showed the described depolarization after the initial hyperpolarization. Other cells (42/48) were usually slowly repolarized after the hyperpolarization. These results are in general agreement with previous studies 6"13A6'27. The depolarization was preserved even in the absence of extracellular Ca 2+ which was also found earlier 13. Authors suggest that the subsequent depolarization may be based on a C1effiux which is Ca-independent. However, not all OHCs showed the depolarization. Therefore, further singlechannel or pharmacological investigations are required to fully elucidate the role of the CI- channel activation in OHC during hyposmotic activation. The most interesting finding of the present study is the hyperpolarization induced by stretch of the cell membrane which contributes to motility and, hence, tuning of the OHC. The hyperpolarization of the OHCs is followed by an elongation of the cellular body 1,22,29 in contrast to other non-sensory cells such as the proximal tubule. Santos-Sacchi et al. 22 speculated that the mechanical response of the OHCs to actively amplify the travelling wave 3° is widely dependent on the membrane potential rather than the transmembrane current. Therefore, we described the changes of the receptor potential in the OHCs under current-clamp conditions.
211 The OHC motility is essential to provide a feedback in the fine tuning of the cochlea4. The resulting OHC length could tune the basilar membrane by bringing about a shift in its mean position. Thereby, the responsiveness of the cochlear partition is improved 17. It seems to be likely that a stretch of the OHC membrane is physiological during sound stimulation. Thus, the hyperpolarization induced by membrane stretch may participate to the control of the OHC motility and fine tuning, respectively. Under pathophysiological conditions, any change of the inner ear fluid volume or composition can possibly provoke such a membrane stretch and hyperpolarization with the well-known clinical findings of tinnitus, hearing loss and vertigo, respectively 15,31. Acknowledgements.
We would like to thank Professor F. Lang, Department of Physiology, University of Tiibingen, for his helpful comments during this work. This work was supported by a Leibniz grant of the DFG to H.P.Z. REFERENCES 1 Ashmore, J.F., A fast motile response in guinea-pig outer hair cells. The cellular basis of the cochlear amplifier, J. PhysioL, 338 (1987) 323-347. 2 Cristensen, O., Mediation of cell volume regulation by Ca 2+ influx through stretch-activated channels, Nature, 330 (1987) 6668. 3 Cook, N.S. and Haylett, D.G., Effects of apamin, quinine and neuromuscular blockers on calcium-activated potassium channels in guinea-pig hepatocytes, J. PhysioL, 358 (1985) 373-394. 4 Dallos, P. and Corey, M.E., The role of outer hair cell motility in cochlear tuning, Curr. Opinions NeurobioL, 1 (1991) 215-220. 5 Ding, J.P., Salvi, R.J. and Sachs, F., Stretch-activated ion channels in guinea pig outer hair cells, Hearing Res., 56 (1991) 18-28. 6 Dube, L. Parent, L. and Save, R., Hypotonic shock activates a max K ÷ channel in primary cultured proximal tubule cells, Am. J. Physiol., 259 (1990) F348-F356. 7 Filipovic, D. and Sakin, H., A calcium-permeable stretch-activated cation channel in renal proximal tubule, Am. J. PhysioL, 260 (1991) Fl19-129. 8 Gitter, A.H., Beyenbach, K.W., Christine, C.W., Gross, P., Minuth, W.W. and Fr6mter, E., High-conductance K ÷ channel in apical membranes of principal cells cultured from renal cortical collecting duct anlagen, Pfliigers Arch., 408 (1987) 282-290. 9 Gitter, A.H., Fr6mter, E. and Zenner, H.P., C-Type potassium channels in the lateral cell membrane of guinea-pig outer hair cells, Hearing Res., 60 (1992) 13-19 10 Grinstein, S., Dupre, A. and Rothstein, A., Volume regulation by human lymphocytes, role of calcium, J. Gen. PhysioL, 79 (1982) 849-868. 11 Gynkiewcz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2" indicators with greatly improved fluorescence properties, Z Biol. Chem., 260 (1985) 3440-3450.
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