Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro

HBIRII RIZ.mtCH

ELSEVIER

Hearing Research 84 (1995) 19-29

Ion transport mechanisms responsible for K + secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro Philine Wangemann a,*, Jianzhong Liu

a,b,

Daniel C. Marcus h

a Cell Physiology Laboratory, Boystown National Research Hospital, 555 North 30th Street, Omaha, NE 68131, USA b Biophysics Laboratory, Boystown National Research Hospital, Omaha, NE, USA Received 28 June 1994; revised 6 November 1994; accepted 20 December 1994

Abstract It has long been accepted that marginal cells of stria vascularis are involved in the generation of the endocochlear potential and the secretion of K +. The present study was designed to provide evidence for this hypothesis and for a cell model proposed to explain K + secretion and the generation of the endocochlear potential. Stria vascularis from the cochlea of the gerbil was isolated and mounted into a micro-Ussing chamber such that the apical and basolateral membrane of marginal cells could be perfused independently. In this preparation, the transepithelial voltage (Vt) and resistance (R t) were measured across marginal cells and the resulting equivalent short circuit current (Isc) was calculated (Isc = l't/Rt). Further, K + secretion (JK+probe) was measured with a K+-selective vibrating probe in the vicinity of the apical membrane. In the absence of extrinsic chemical driving forces, when both sides of the marginal cell epithelium were bathed with a perilymph-like solution, V~ was 8 mV (apical side positive), R t was 10 ohm-cm 2 and Isc was 850 ~ A / c m 2 ( N = 27). JK+,probe was outwardly directed from the apical membrane and reversibly inhibited by basolateral bumetanide, a blocker of the N a + / C 1 - / K + cotransporter. On the basolateral but not apical side, ouabain and bumetanide each caused a decline of ~ and an increase of R t suggesting the presence of the Na,K-ATPase and the N a + / C I - / K + cotransporter in the basolateral membrane, The responses to [C1-] steps demonstrated a significant C I - conductance in the basolateral membrane and a small C1conductance in the paracellular pathway or the apical memblane. The responses to [Na +] steps demonstrated no significant Na + conductance in the basolateral membrane and a small Na + or nonselective cation conductance in the apical membrane or paracellular pathway. The responses to [K +] steps demonstrated a large K + conductance in the apical membrane. Apical application of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and basolateral elevation of K + caused an increase in Vt and a decrease in Rt consistent with stimulation of the apical K + conductance. Similar observations have been made in vestibular dark cells, which suggest that strial marginal cells and vestibular dark cells are homologous and transport ions by the same pathways. Taken together, these observations are incompatible with a model for the generation of the endocochlear potential which ascribes the entire potential to the strial marginal cells [Offner et al. (1987) Hear. Res. 29, 117-124]. However, the data are compatible with the notion that marginal cells contribute not more than a few mV to the endocochlear potential as suggested by Salt et al. [Laryngoscope 97, 984-991, 1987].

Keywords: Stria vascularis; Cochlea; Marginal cells; Vibrating probe; Micro-Ussing chamber

1. Introduction T h e t r a n s d u c t i o n o f s o u n d into n e r v e i m p u l s e s in t h e c o c h l e a is d e p e n d e n t o n a s t a n d i n g c u r r e n t ( J o h n -

Reprint requests and correspondence to Philine Wangemann, Ph.D. * Corresponding author. Fax: (402) 498-6638; email: [email protected]. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 3 7 8 - 5 9 5 5 ( 9 5 ) 0 0 0 0 9 - 7

s t o n e a n d Sellick, 1972) w h i c h is t h o u g h t to b e g e n e r a t e d by stria vascularis, a m u l t i l a y e r e d s t r u c t u r e which rests o n t h e spiral l i g a m e n t in t h e l a t e r a l wall of t h e cochlea. I n d i r e c t e v i d e n c e s u p p o r t s t h e c o n c e p t t h a t stria vascularis g e n e r a t e s t h e e n d o c o c h l e a r p o t e n t i a l o f a b o u t 80 m V a n d s e c r e t e s K + f r o m p e r i l y m p h into e n d o l y m p h ( T a s a k i a n d S p y r o p o u l o s , 1959; K o n i s h i et al., 1978). E n d o l y m p h is an u n u s u a l e x t r a c e l l u l a r fluid (similar in t h e c a t i o n c o n t e n t to s o l u t i o n 7, T a b l e 1)

20

P. Wangemann et aL /Hearing Research 84 (1995) 19-29

Table 1 Solutions (mM) Solution 1 NaCI Na-G KCI NMDG-CI MgCI2 MgSO4 CaCI 2 Ca-G 2 K2HPO 4 KH2PO 4

Glucose

150.0 1.0 0.7 1.6 0.4 5.0

2

3

100

15.0 128.6 135.0 . . 21.4 1.0 1.0 0.7 0.7 1.6 1.6 0.4 0.4 5.0 5.0

50.0 1.0 0.7 1.6 0.4 5.0

4

5

6

-

7 1.0

. 150.0 1.0 4.0 1.6 0.4 5.0

. 150 1.0 0.7 1.6 0.4 5.0

151.4 0.03 0.03 1.6 0.4 -

G, gluconate. whereas perilymph is similar to most other extracellular fluids (similar in the cation content to solution 1). Stria vascularis consists of an epithelial and a nonepithelial barrier which enclose the intrastrial compartment. T h e ionic composition of this intrastrial comp a r t m e n t is t h o u g h t to be similar to perilymph (Salt et al., 1987; Ikeda and Morizono, 1989). T h e barrier between e n d o l y m p h and the intrastrial c o m p a r t m e n t is f o r m e d by an epithelial m o n o l a y e r of marginal cells. T h e barrier between the intrastrial c o m p a r t m e n t and perilymph is f o r m e d by basal cells, which are not epithelial cells but nevertheless are c o n n e c t e d to each o t h e r by tight junctions (Jahnke, 1975). Even t h o u g h the concept that stria vascularis generates the e n d o c o c h l e a r potential and secretes K + is well accepted, little is known about the cellular mechanisms involved. Two models designed to explain the generation of the e n d o c o c h l e a r potential and the secretion of K + have recently b e e n presented. C o m m o n to t h e m is the N a , K - A T P a s e in the basolateral m e m b r a n e of marginal cells which has been localized there at an unusually high density (Kerr et al., 1982; I w a n o et al., 1989). Both models assume that marginal ceils secrete K + and that the apical m e m b r a n e potential of marginal cells, which in vivo is up to 10 m V positive with respect to scala media, contributes to the electrochemical driving force for K + secretion (Offner et al., 1987; Ikeda and Morizono, 1989). However, the models differ in the cellular location of the m e c h a n i s m for the generation of the e n d o c o c h l e a r potential. T h e 'single cell m o d e l ' assumes that the basolateral m e m b r a n e of marginal cells is primarily Na + conductive and that this N a + c o n d u c t a n c e generates a positive basolateral m e m b r a n e potential which is the source for the positive e n d o c o c h l e a r potential (Offner et al., 1987). In contrast, t h e ' t w o cell m o d e l ' assumes that the molecular m e c h a n i s m for the generation of the e n d o c o c h l e a r potential is a K + c o n d u c t a n c e localized in the inner m e m b r a n e of basal cells (Salt et al., 1987) or in the putative functional extension of this m e m b r a n e by intermediate cells (Schulte and Steel, 1994). T h e involve-

m e n t of marginal cells in the generation of the endocochlear potential would, according to this model, be limited to the m a i n t e n a n c e of the low K + concentration in the intrastrial c o m p a r t m e n t (Salt et al., 1987). The c o n c e p t that marginal cells transport K + but that the mechanism for the generation of the endocochlear potential is located in a n o t h e r cell type provides the basis to hypothesize that ion transport mechanisms are similar in strial marginal cells and vestibular dark cells. Vestibular dark cells are known to secrete K +, however, prior to the 'two cell model', ion transport mechanisms leading to K + secretion were assumed to be fundamentally different from those in the stria vascularis since there is no equivalent of the large positive e n d o c o c h l e a r potential in the vestibular labyrinth. T h e aim of the present study was to test the hypothesis that strial marginal cells a) secrete K +, b) generate a positive transepithelial potential u n d e r symmetrical conditions, and c) respond to ion substitutions and inhibitors in ways similar to vestibular dark cells. A subset of the results has b e e n presented at a recent meeting (Marcus et al., 1994).

2. Methods 2.1. Preparation

Gerbils were anesthetized with pentobarbital sodium (50 m g / k g i.p.) and decapitated u n d e r a protocol approved by the Creighton University Animal Care and Use Committee. Stria vascularis was dissected at 4 ° C from the cochlea of the inner ear and transferred as a flat sheet into a micro-Ussing c h a m b e r where experiments were c o n d u c t e d at 37 °C. T h e aperture of the micro-Ussing c h a m b e r was 80 /xm and the apical and basolateral side of the epithelium were perfused independently (Marcus et al., 1987; Marcus et al., 1994). For all experiments involving basolateral solution changes, spiral ligament was removed from stria vascularis (Fig. 1A) whereas for some experiments involving apical solution changes, spiral ligament was left attached (Fig. 1B). No enzymatic treatment was used for micro dissection. R e m o v a l of spiral ligament greatly improved the accessibility of the basolateral m e m b r a n e of marginal cells to the basolateral perfusate as evident from the observations that the effects of ouabain and b u m e t a n i d e were complete within 6 and 8 min in the presence of the spiral ligament (insets to Fig. 2 A and 3A) and within 3 and 2.5 min when spiral ligament had b e e n removed. 2.2. Solutions

T h e compositions of solutions are listed in Table 1. All solutions were titrated to p H 7.4.

P. Wangemannet al./ Hearing Research 84 (1995) 19-29

AIB Basolateral Perfusion

u,

Vt (mV)

I II~.~'A-'~2~?'~". FC

I l I , ~r~~_~L.j_.X

10-

IC _

Basolateral

MC

Perfusion

v

0-

Fig. ]. Diagram of stria vascularis mounted in the mJero-Ussing chamber. (A) Stria vascularis without spiral ligament consists only of one resistive barrier, the marginal cell epithelium. (B) Stria vascularis with spiral ligament consists of two resistive barriers, the marginal cell epithelium and the basal cell layer. Note, that the basolateral perfusate has in both preparations access to the intrastrial space since the intrastrial space has been opened laterally. Compromising the basal cell layer as shown in (A) however, markedly improves the access of the basolateral perfusate to the basolateral membrane of marginal cells. FC, fibrocytes; BC, basal cells; IC, intermediate ceils; MC, marginal cells.

2.3. Measurement of the transepithelial uoltage (Pt) and resistance (R t) S t r i a v a s c u l a r i s w a s m o u n t e d in t h e m i c r o - U s s i n g c h a m b e r by s e a l i n g t h e a p i c a l m e m b r a n e o f m a r g i n a l cells to t h e a p e r t u r e . C h a n g e s in t h e a p i c a l a n d b a s o l a t e r a l p e r f u s a t e w e r e c o m p l e t e w i t h i n 1 s. Vt a n d R t w e r e m e a s u r e d as d e s c r i b e d e a r l i e r ( M a r c u s et al., 1987; M a r c u s et al., 1994). Briefly, Vt w a s m e a s u r e d w i t h c a l o m e l e l e c t r o d e s c o n n e c t e d to t h e c h a m b e r via

-

,A

rt

(mv)

:

~

4rn|n

_lrnln

6-

v

E

:

4-

Ia u m ~

E ~5-

1 " Apical Perfusion

8

21

bl:J eurnetonide

J

Fig. 3. Effect of basolateral bumetanide (50 ~M) on (A) the transepithelial voltage Vt and (B) the transepithelial resistance R t of strial marginal cells in a preparation of stria vascularis without spiral ligament. Insert: Effect of basolateral bumetanide (50/xM) on Vt of strial marginal cells in a preparation of stria vascularis with spiral ligament. Note the difference in time course.

f l o w i n g 1 M KC1 b r i d g e s . T r a n s e p i t h e l i a l c u r r e n t p u l s e s w e r e p a s s e d via A g / A g C 1 wires. S a m p l e - a n d - h o l d circ u i t r y w a s u s e d to o b t a i n a signal p r o p o r t i o n a l to R t f r o m t h e v o l t a g e r e s p o n s e to t h e c u r r e n t p u l s e s (50 n A f o r 34 ms at 0.3 H z ) . Vt a n d R t w e r e r e c o r d e d o n a 2 - p e n c h a r t r e c o r d e r . R e p r e s e n t a t i v e t r a c e s w e r e eit h e r d i g i t i z e d m a n u a l l y f r o m p e n - r e c o r d i n g s (Figs. 2, 3, 5, 7 - 9 , 11) o r by d i r e c t A / D c o n v e r s i o n (Figs. 4, 6, 10). T h e e q u i v a l e n t s h o r t c i r c u i t c u r r e n t (Isc) w a s o b t a i n e d a c c o r d i n g to O h m ' s l a w ( V t / R t) f r o m m e a s u r e m e n t s o f Vt a n d R t w h e n t h e e p i t h e l i u m w a s b a t h e d o n b o t h sides w i t h s o l u t i o n s o f i d e n t i c a l i o n i c c o m p o s i t i o n . Fit

O

2-

In

O-

bl:J

Ouabain

J

~ 200 !

._o.vm

l°tB 9 Fig. 2. Effect of basolateral ouabain (10 -3 M) on (A) the transepithelial voltage I/t and (B) the transepithelial resistance R t of strial marginal cells in a preparation of stria vascularis without spiral ligament. Insert: Effect of basolateral ouabain (10 - 3 M) o n lit of strial marginal cells in a preparation of stria vascularis with spiral ligament. Note the difference in time course.

i

~

÷ v

100 bl:l Bumetonlde I Fig. 4. Effect of basolateral bumetanide (50 #M) on K + secretion measured as K + gradient Jr+proOe in the vicinity of the apical membrane of strial marginal cells in a preparation of stria vascularis without spiral ligament.

P. Wangemann et al. / Hearing Research 84 (1995) 19-29

22

b,:l

loo ,,,M Mo iii

U94.0420A STRNAB S P 5

i ~~

Fig. 5. Effect of basolateral [Na + ] steps from 150 to 100 m M on (A) the transepithelial voltage Vt and (B) the transepithelial resistance R, of strial marginal cells in a preparation of stria vascularis without spiral ligament.

was corrected for liquid junction potentials in the text and Figures.

2.4. Measurement of the K + gradient (JK+,probe) in the vicinity of the apical m e m b r a n e of strial marginal cells Stria vascularis without spiral ligament was mounted in the micro-Ussing chamber with the perilymphatic side against the aperture. The apical side of stria vascularis was not perfused but bathed with solution 1 while the perfusate of the perilymphatic side was exchanged about 10 t i m e s / s . JK+,probe was measured with the vibrating probe as described previously (Marcus and Shipley, 1994). The vibrating probe was located 20-240 /xm over the apical m e m b r a n e of the epithelium such that the signal under control conditions was > 30 times the noise level at background which was obtained at a position > 1 m m away from the tissue. JK+,probe w a s measured as the voltage dif-

35

ference between two points near the apical m e m b r a n e by vibrating (amplitude: 30/xm; 0.3 Hz) a K+-selective microelectrode along the axis normal to the plane of the tissue. Microelectrodes (O.D.: 4 /xm) were pulled from borosilicate glass capillaries (O.D.: 1.5 mm) and silanized with dimethyldichlorosilane. Tips contained a column of K+-selective ligand (#60398, Fluka Chemical, Ronkonkoma, NY) about 150 /xm long and the electrode was backfilled with 100 m M KC1 and 0.5% agar. The reference was A g / A g C I with a bridge of 3 M NaC1 and 3% agar. Electrodes were only used if the slope was at least 56 m V / d e c a d e in 10 and 100 m M KCI solutions. The contribution of the voltage gradient produced by the transepithelial electric current was less than about 8% of the voltage gradient observed at the K+-selective electrode near the tissue under control conditions; as in previous studies, no corrections were made for this component of the signal (Marcus and Shipley, 1994). Effects in response to solution changes were expressed as relative changes since the signal was not calibrated in terms of absolute K + flux at the surface of the epithelium. The calibration procedure of the vibrating ion-selective probe in terms of absolute flux is not yet on firm ground because a variable and poorly understood 'efficiency factor' must be employed (Kiihtreiber and Jaffe, 1990).

2.5. Data presentation and statistics Data are given as average _+ standard error of the mean. The number of observations ( N ) is equal to the number of epithelial samples. For statistical analysis of paired samples, averages of original data were compared using Student's t-test. Differences were assumed to be significant when P < 0.05.

A

30

3. Results

25 E 2O

~15 10 5

bl: [

15 rnM CI

I

12

E O

I

"-"

8 6

4 Fig. 6. Effect of basolateral [Cl- ] steps from 153 to 15 m M on (A) the transepithelial voltage Vt and (B) the transepithelial resistance R t of strial marginal cells in a preparation of stria vascularis without spiral ligament.

In order to ascribe measurements of Vt and the associated Is~ solely to active transport processes, it is necessary to bathe the tissue with identical solutions on both sides. Strial marginal cell epithelium was therefore perfused on both sides with solution 1 unless stated otherwise. U n d e r these conditions stria vascularis with and without spiral ligament generated a lumen-side positive transepithelial potential. Without spiral ligament, Vt was 7.6 _+ 0.6 mV, R, was 9.9 +_ 0.9 ohm-cm 2 and the resulting I,c was 849 + 68 / x A / c m 2 ( N = 27). Significantly different results were obtained when spiral ligament was present. I,I, was 17.5 _+ 1.8 mV, R t was 16.0 _+ 1.0 ohm-cm e and the resulting Isc was 1260 -t- 1 7 4 / x A / c m 2 ( N = 29). Further, K + secretion was demonstrated in stria vascularis without spiral ligament as a K + gradient in the vicinity of the apical membrane.

P. Wangemann et al./ Hearing Research 84 (1995) 19-29

23

15

3.1. Effect o f ouabain on Vt, R t and I~

1 mTn

Addition of 1 m M ouabain, an inhibitor of the Na,K-ATPase, to the basolateral perfusate of stria vascularis without spiral ligament caused a significant decrease in Vt from 6.5 ___1.3 to 0.46 + 0.14 m V and no change in R t (13.0_+ 2.7 versus 14.4 + 3.4 ohm-cm2; N = 7). The decrease in Vt was partially reversible. A representative experiment is shown in Fig. 2. Basolateral ouabain caused a significant decrease of Isc from 564 _+ 85 to 44 _+ 20 / x A / c m 2 ( N = 7) within 2.9 + 0.4 min. Qualitatively similar observations were made in stria vascularis with spiral ligament, however, the decline of Vt was significantly slower, it occurred within 6 :t: 1 min ( N = 7) and the a significant increase in R t from 12.1 + 1.1 to 16.1 + 2.8 ohm-cm2; N = 7) was observed (inset in Fig. 2). An initial increase of Vt which was observed in both the presence and absence of spiral ligament (although only shown in the inset of Fig. 2) remains unexplained. Addition of 1 m M ouabain to the apical perfusate of stria vascularis with spiral ligament had no effect on Vt o r R t ( N - 1 , data not shown). 3.2. Effect o f bumetanide on Vt, R t, Isc and JK+,probe

Addition of 5 0 / x M bumetanide, an inhibitor of the N a + / C I - / K + cotransporter, to the basolateral perfusate of stria vascularis without spiral ligament significantly decreased Vt from 7.3 + 1.3 to 0.48 _+ 0.50 m V and significantly increased R t from 9.4 + 1.8 to 10.8 _+ 2.3 ohm-cm 2 ( N = 6), which resulted in an inhibition of I,c from 824 + 148 to 81 + 6 2 / z A / c m 2 within 2.5 _+ 0.3 min ( N = 6). A representative experiment is shown in Fig. 3. The initial increase in Vt and decrease in R t remain unexplained. Qualitatively similar observations were made in stria vascularis with spiral ligament, however, the decline of Vt was significantly slower occurring within 8 _+ 2 min ( N = 3). Addition of 5 0 / x M bumetanide to the apical perfusate of stria vascularis with spiral ligament had no significant effect on Vt and R t (10.0 _+ 1.6 versus 10.0 + 1.6 m V and 14.1 _+ 1.6 versus 14 + 1.6 ohm-cm 2, N = 6). In a second series of experiments stria vascularis without spiral ligament was perfused only on the basolateral side with solution l. Addition of 50 /xM bumetanide to the basolateral perfusate significantly decreased JK+,probe to a level which was a factor 0.18 + 0.14 of the control value of 430 _+ 243 A/zV ( N = 4). A representative experiment is shown in Fig. 4.

A

E

"-" 1 0 >-

bl: ~ " ~

5

Fig. 7. Effect of basolateral IK + ] steps from 3.6 to 25 mM on (A) the transepithelial voltage V~ and (B) the transepithelial resistance R t of strial marginal cells in a preparation of stria vascularis without spiral ligament.

sient increase of Vt from 8.1 _+ 1.0 to 9.1 _+ 1.2 m V after which Vt relaxed to 7.8 _+ 1.1 m V which is not significantly different from the initial value. No significant change of R t w a s observed (8.1 _+ 0.6 versus 8.0 _+ 0.6 ohm-cm2; N = 7; Fig. 5). 3.4. Effect o f basolateral [Cl -] steps on Vt and R t

A decrease of the [C1-] from 153 to 15 m M (solution 3) in the basolateral perfusate of stria vascularis without spiral ligament caused a significant but transient increase in Vt from 7.0_+ 0.5 to 30.3 + 1.6 mV after which Vt relaxed to 12.6_+ 0.3 m V and R t increased from 7.0 _+ 0.4 to 11.2 _+ 0.8 ohm-cm 2 ( N = 7). A representative experiment is shown in Fig. 6. 3.5. Effect o f basolateral [K +] steps on Vt and R t

An increase of the [K +] from 3.6 to 25 m M (solution 4) in the basolateral perfusate of stria vascularis without spiral ligament caused a transient increase of V, from 9.7 + 0.7 to 13.0 + 0.7 m V during which R t decreased significantly from 8.6 _+ 0.9 to 6.3 ___0.7 ohmcm 2 ( N = 6). The transient increase in Vt which occured when the [K +] was reduced from 25 to 3.6 m M remains unexplained. A representative experiment is shown in Fig. 7.

3.3. Effect o f basolateral [Na +] steps on Vt and R t

3.6. Effect o f apical [Na +] steps on V~ and R,

A decrease of the [Na ÷] from 150 to 100 m M (solution 2) in the basolateral perfusate of stria vascularis without spiral ligament caused a small but tran-

A decrease of the [Na +] from 150 to 0 m M (solution 5) in the apical perfusate of stria vascularis with spiral ligament significantly increased Vt from 23.3 + 4.6 to

24

P. Wangemann et al. /Hearing Research 84 (1995) 19-29 50

30 s

30 s

A

54 48 ~" E

52

gv

E

>~

>" 5o

46

44

48

a:J

°:1

0 mM No ~" E

15

r.M

cl

I

B

12

I

Cl

v

Fig. 8. Effect of apical [Na ÷ ] steps from 150 to 0 mM on (A) the transepithelial voltage V, and (B) the transepithelial resistance R, of strial marginal cells in a preparation of stria vascularis with spiral ligament.

2 8 . 5 + 4 . 5 mV and R t from 11.6_+1.2 to 1 4 . 5 + 1 . 6 ohm-cm 2 ( N = 6; data not shown). Qualitatively similar results were obtained in the same preparation during [Na +] steps from 150 to 100 mM (solution 2). Vt increased significantly from 9.8 ___1.1 to 10.7 + 1.2 mV and R t increased from 19.9 + 1.3 to 20.8 + 1.3 ohm-cm 2 ( N = 9). A representative experiment is shown in Fig. 8.

10

Fig. 9. Effect of apical [ e l - ] steps from 153 to 15 mM on (A) the transepithelial voltage Vt and (B) the transepithelial resistance R, of strial marginal cells in a preparation of stria vascularis with spiral ligament.

16.3 -t- 3.3 to 8.0 _+ 0.5 ohm-cm 2 ( N = 2). However, the same experiment performed on stria vascularis without spiral ligament caused a significant decrease of V~ from 7.3 _+ 1.1 to - 1.5 _+ 0.4 inV. Simultaneously, R, decreased significantly from 8.8 + 0.8 to 5.0 _+ 0.3 ohmcm 2 ( N = 7). A representative experiment is shown in Fig. 10.

3.7. Effect of apical [CI-] steps on Vt and R, A decrease of the [C1-] from 153 to 15 mM (solution 3) in the apical perfusate of stria vascularis with spiral ligament caused a significant decrease of V, from 23.1 +_ 5.6 to 17.8 +__6.0 mV and a significant increase of R, from 12.8 + 1.4 to 16.0 _+ 1.8 ohm-cm 2 ( N = 6). A representative experiment is shown in Fig. 9.

10

158

~

3.8. Effect of apical [K +] steps on V, and R t An increase of the [K +] from 3.6 to 150 mM (solution 6) in the apical perfusate of stria vascularis with spiral ligament caused a significant decrease of Vt from 23.6 + 5.6 to 8.7 + 4.4 mV and R, from 12.4 + 1.4 to 8.6 + 0.7 ohm-cm 2 ( N = 6). An example is shown in the inset in Fig. 10. Qualitatively similar results were obtained from perfusion of a solution more similar to endolymph which was used in order to more-closely mimic in vivo conditions. A change from solution 1 to 7 in the apical perfusate caused a significant decrease of V, from 34.5+_1.5 to 12.6_+4.8 mV and R t from

50

M K

°'1

is,

K

I

% o

I

C1

~

10

8

Fig. 10. Effect of apical [K + ] steps from 3.6 to 151 mM (solution 7, Table 1) on (A) the transepithelial voltage Vt and (B) the transepithelial resistance R~ of strial marginal cells in a preparation of stria vascularis without spiral ligament. Insert: Effect of apical [K + ] step from 3.6 to 150 mM (solution 6) on Vt in a preparation of stria vascularis with spiral ligament. Note the difference in V~.

P. Wangemann et al. / Hearing Research 84 (1995) 19-29 60

A

30

s

55 > vE

5O

45

a:l

I

olos

I

11 o I

10 9

Fig. 11. Effect of apical DIDS (1 mM) on (A) the transepithelial voltage V~ and (B) the transepithelial resistance R t of strial marginal cells in a preparation of stria vascularis with spiral ligament.

3.9. Effect of apical D1DS on Vt, R, and Isc Addition of 1 m M D I D S to the apical perfusate of stria vascularis with spiral ligament significantly increased Vt from 22.5 _+ 5.7 to 31.5 + 6.9 m V and significantly decreased R t from 13.3_+ 1.6 to 11.4_+ 1.4 ohm-cm 2 ( N = 6). A representative experiment is shown in Fig. 11.

4. D i s c u s s i o n

The present data demonstrate for the first time that isolated strial marginal cell epithelium in vitro generates a positive transepithelial potential and secretes K + under symmetrical conditions, when both sides of the epithelium are bathed with a perilymph-like solution and transepithelial chemical driving forces are absent.

4.1. Preparation Even though the preparations under study contained more than one cell type, the observed effects on Vt and R t in response to apical and basolateral solution changes originate most likely from strial marginal cells. Effects in response to apical solution changes occurred most likely at the apical m e m b r a n e of marginal cells since the apical perfusate had access only to this membrane. Access of the apical perfusate to the basolateral m e m b r a n e of marginal cells or to any other cell within stria vascularis was ruled out by the observation that ouabain and bumetanide had no effects when added to the apical perfusate in contrast to

25

the strong effects which were observed when these drugs were added to the basolateral perfusate (Figs. 2 and 3). Considerations for basolateral solution changes are more complex since the basolateral perfusate had access to all cell types of stria vascularis and spiral ligament when present. However, a direct contribution of spiral ligament can be ruled out since spiral ligament cannot generate a potential. This tissue is comprised of a loose connective tissue which does not provide an electrical barrier across which a potential could be generated. Indeed, neither a potential nor a resistance were observed when spiral ligament without stria vascularis was placed into the micro-Ussing chamber (data not shown). Further, neither intermediate cells nor the cells surrounding the capillaries within stria vascularis comprise a sufficient electrical barrier across which a potential could be generated and measured in the micro-Ussing chamber. In contrast, basal cells could provide such a barrier due to the presence of tight junctions (Jahnke, 1975). According to the 'two cell model' (Salt et al., 1987), the large lumen-positive endocochlear potential is generated across the basal cell barrier. Such a large positive potential was not measured in the present study most likely because the basal cell barrier was at least partially compromised due to the cut edges of the preparation. Cutting of the preparation opened the intrastrial space to the basolateral perfusate and provided a low-resistance pathway across the basal cell barrier (Fig. 1B). Removal of spiral ligament most likely compromised the basal cell barrier further, which eliminated any significant contribution of the basal cells to Vt and R t. This notion is supported by the finding of a larger Vt but a slower effect of ouabain in the presence of spiral ligament as compared to in its absence (Fig. 2). The effect of ouabain can be taken as an indicator that the basolateral perfusate had access to the basolateral m e m b r a n e of strial marginal ceils. Ouabain-sensitive Na,K-ATPase has been found with high density in strial marginal cells but not in basal cells (Iwano et al., 1989; Schulte and Adams, 1989). Rapid access of the basolateral perfusate including ouabain to the basolateral membrane of marginal cells is incompatible with the presence of an electrically tight barrier consisting of basal cells. Taken together, these observations strongly support the interpretation that the measured effects on V, and R, originated from strial marginal cells.

4.2. Evidence for ion transport mechanisms in strial marginal cells." Basolateral Na, K-A TPase The involvement of the Na,K-ATPase in K + secretion and the generation of the endocochlear potential in vivo is well established (Konishi et al., 1978; Konishi

P. Wangemann et al. / Hearing Research 84 (1995) 19-29

26

and Mendelsohn, 1970). The Na,K-ATPase is thereby the major energy consuming process in stria vascularis (Kusakari et al., 1978; Marcus et al., 1978). The decline of Vt and increase of R t in response to basolateral but not apical ouabain is consistent with the presence of the Na,K-ATPase in the basolateral m e m b r a n e of strial marginal cells (Fig. 2). Similar observations have been made in vestibular dark cells from the utricle and ampulla (Marcus et al., 1987; Marcus et al., 1994). Basolateral Na + / C l - / K + cotransporter

The sensitivity of the endocochlear potential to loop-diuretics such as furosemide, piretanide and bumetanide is well established (Kusakari et al., 1978; Kusakari et al., 1978; Rybak and Whitworth, 1986). These findings in conjunction with the dependence of the endocochlear potential on the presence of Na ÷, C I - and K + in the intrastrial c o m p a r t m e n t (Shindo et al., 1992; Marcus et al., 1981; W a d a et al., 1979) suggested an involvement of the N a + / C 1 - / K + cotransporter in the generation of the endocochlear potential. Ionic profiles for Na +, K +, and C I - obtained in vivo across stria vascularis are consistent with the notion that the N a + / C I - / K + cotransporter is involved in the uptake of K ÷ (Ikeda and Morizono, 1989). Under in vivo conditions, the N a + / C 1 - / K + cotransporter is the major Na ÷ entry pathway into marginal cells since furosemide, a blocker of the N a + / C 1 - / K + cotransporter, has been shown to protect the cytosolic [ATP] and [phosphocreatine] of stria vascularis during anoxia in a fashion similar to ouabain (Kusakari et al., 1978). It is likely that this protection occurred via a similar mechanism as described for the thick ascending limb of the loop of Henle in the kidney (Wangemann and Greger, 1990). Inhibition of the N a + / C 1 - / K + cotransporter would reduced Na + entry into the cells. The Na,K-ATPase would decrease its activity after reducing the cytosolic [Na +] to its limiting level, thereby reducing the consumption of metabolic fuel such as A T P and phosphocreatine. The decline of V~ and Jx+,prob~. and the increase of R t in response to basolateral but not apical bumetanide is consistent with the presence of the N a + / C 1 - / K + cotransporter in the basolateral m e m b r a n e of strial marginal cells and its involvement in the generation of Vt and secretion of K + under symmetrical conditions in vitro (Figs. 3 and 4). Similar observations have been made in vestibular dark cells from the utricle and ampulla (Marcus et al., 1987; Marcus et al., 1994; Marcus and Shipley, 1994). Basolateral C l - conductance

The observation that [C1-] caused a transient increase in R, suggests tain a C1 conductance

a basolateral decrease of the increase in Vt and a sustained that strial marginal cells conin their basolateral m e m b r a n e

(Fig. 6). Similar observations have been made in vestibular dark cells (Marcus and Marcus, 1989). In vestibular dark cells the basolateral C I - conductance was found to be comprised of a 95 pS C1- channel (Marcus et al., 1993). Preliminary data confirm the presence of this channel in the basolateral m e m b r a n e of strial marginal cells (Takeuchi et al., 1994). Apical K + conductance

The observation that an increase in the apical [K +] caused a decline of Vt and R t suggests the presence of a major K + conductance in the apical m e m b r a n e (Fig. 10). Similar observations have been made in vestibular dark cells (Marcus et al., 1994). In both tissues, the apical K + conductance has been found to be comprised of a high density of I~K K + channels (Sunose et al., 1994; Marcus and Shen, 1994; Marcus and Shen, 1994; Sakagami et al., 1991). The observation that apical D I D S increased Vt and decreased R t suggests that D I D S increased the apical K + conductance (Fig. 11). Similar observations have been made in vestibular dark cells from the utricle and the ampulla (Marcus and Marcus, 1989; Marcus et al., 1994; Shen et al., 1994). Further, the observation that Vt increased and R, decreased during an elevation of the basolateral [K +] suggests an increase in the apical K + conductance. Similar observations have been made in vestibular dark cells where it is likely that K+-induced cell swelling mediates an increase in the apical K + conductance and K + secretion with a concurrent increase in Vt and decrease in R t (Marcus and Shen, 1994; Marcus et al., 1994; W a n g e m a n n and Marcus, 1990). It is therefore likely that cell volume in strial marginal cells is involved in the cross-talk between the basolateral m e m b r a n e taking up K ÷ and the apical m e m b r a n e secreting K ÷. Apical non-selective cation or Na + conductance

The observation that an apical decrease in the [Na +] caused a sustained increase in Vt and R t is consistent with the presence of a non-selective cation a n d / o r a Na ÷ conductance in the apical m e m b r a n e or of the paracellular pathway (Fig. 8). Similar observations have been made in vestibular dark cells (Marcus and Marcus, 1990). Recently, a 27-28 pS non-selective cation channel has been observed in the apical m e m b r a n e of strial marginal cells and vestibular dark cells (Takeuchi et al., 1992; Marcus et al., 1992; Sunose et al., 1993). However, physiologic relevance of this channel might be limited since its fractional open time was near zero at physiologic cytosolic [Ca 2÷] and [ATP]. Pharmacologic evidence suggested the presence of Na + channels in the vestibular labyrinth (Ferrary et al., 1989). The present data do not distinguish between these two types of conductances.

P. Wangemann et al./Hearing Research 84 (1995) 19-29 MC

IC

BC

FC

i i:i:;! No

:

:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::ii::::i:iiii!i:i:illlliliiii:i:. ili ::i~ii::::i!! !

Endolymph ~ii~iiiiii~ii!!#i~i~iiiiiiiiii!#i~:i:i:i::::::::l CI bl

I~ij:t Intrastrial

iiiiiiii!:.i

'-

~"

IPerilymph in |~:::::lout

Fig. 12. Cell model for ion transport in stria vascularis. MC, marginal cells; IC, intermediate cell; BC, basal cells; FC, fibrocytes; ap, apical membrane of marginal cells; bl, basolateral membrane of marginal cell; in, inner membrane of basal cells; out, outer membrane of basal cells. Note, that strial marginal cells contain the same constellation of ion transport mechanisms as found in vestibular dark cells.

Paracellular anion conductance The observation that an apical decrease in the [Cl-] caused a sustained decrease in V~ and a sustained increase in R t is consistent with the presence of an anion conductivity of the paraceUular pathway (Fig. 9). A contribution of the apical m e m b r a n e is unlikely since it is well known that a [C1-] step at a m e m b r a n e with both K + and C1- conductance produces a biphasic voltage response whereas at the apical m e m b r a n e of stria vascularis the response was monophasic (Hodgkin and Horowicz, 1959). Similar observations have been made in vestibular dark cells from the utricle and ampulla (Marcus et al., 1994; Marcus and Marcus, 1989).

Cell model for stria vascular& The present observations are consistent with the model shown in Fig. 12. Marginal cells take up K ÷ into and extrude Na ÷ from the cytosol by the Na,K-ATPase located in the basolateral m e m b r a n e . Additional uptake of K ÷ occurs via the basolateral N a + / C 1 - / K + cotransporter which is driven by the Na ÷ gradient generated by the Na,K-ATPase. Theoretically, this arrangement is very energy efficient. For each hydrolyzed ATP, 5 K + are taken up across the basolateral m e m brane under the assumption that the stoichiometries of the Na,K-ATPase and the N a + / C 1 - / K + cotransporter are the usual 3 Na + to 2 K ÷ and 1 Na + to 2 C1- to 1 K +, respectively. CI-, which has been taken up via the N a + / C I - / K + cotransporter, recycles across the basolateral m e m b r a n e via C1- channels and K + leaves the cytosol across the apical m e m b r a n e via K ÷ selective and possibly nonselective cation channels. The transepithelial potential across marginal cells is generated by the electromotive force associated with the C I conductance in the basolateral m e m b r a n e in series

27

with the electromotive force associated with a K + and possibly a Na + or non-selective cation conductance in the apical membrane. The apical m e m b r a n e potential of marginal cells, which has been found in vivo to be up to 10 m V positive with respect to scala media, provides the driving force for K + secretion across the apical m e m b r a n e channels (Offner et al., 1987; Salt et al., 1987; Ikeda and Morizono, 1989). The present data provide indirect support for the 'two cell model' which suggests that the source for the large positive endocochlear potential is a K + conductance localized in the inner m e m b r a n e of the basal ceils and that strial marginal cells do not contribute more than a few m V to the endocochlear potential (Salt et al., 1987). Support for the hypothesis that a K + conductance in a m e m b r a n e bordering the intrastrial space is involved in the generation of the endocochlear potential comes from the finding that the endolymphatic potential is more sensitive to the K + channel blocker Ba 2+ when applied via perfusion of the inner ear vasculature (Marcus et al., 1985) than when applied via perfusion of either perilymphatic scala (Marcus, 1984). However, the results of those experiments could not determine in which m e m b r a n e the Ba2+-sensitive K + conductance was located. Further evidence suggested that the endocochlear potential is generated across the basal cells rather than the marginal cells. A double barreled microelectrode, one barrel for the m e a s u r e m e n t of voltage and the other for the measurement of K +, was advanced from the spiral ligament through stria vascularis into scala media and a space was found which had a low K + concentration but a high voltage in the range of the endocochlear potential with respect to the remote reference electrode (Salt et al., 1987). It was assumed that this space was the extraceUular intrastrial c o m p a r t m e n t and that marginal cells contributed not more than a few m V to the endocochlear potential. Evidence for the presence of ion transport pathways in strial basal and intermediate cells and fibrocytes of the spiral ligament is presently limited to histological localizations of Na,K-ATPase, carbonic anhydrase and gap junctions (Iwano et al., 1989; Hsu and Nomura, 1985; Schulte and Adams, 1989; Forge, 1984; Spicer and Schulte, 1991; Schulte and Steel, 1994). The lack of evidence for conductive properties makes these observations insufficient to fully support the 'two cell model'. Further studies are therefore needed in order to determine the remaining ion transport mechanisms in strial basal and intermediate cells and in fibrocytes of spiral ligament. The present data are inconsistent with the 'single cell model' (see Introduction) which suggested that the endocochlear potential is generated by a Na + conductance in the basolateral m e m b r a n e of marginal cells. No evidence for a significant Na + conductance was

28

P. Wangemann et al. /Hearing Research 84 (1995) 19-29

f o u n d in t h e b a s o l a t e r a l m e m b r a n e of strial m a r g i n a l cells; b a s o l a t e r a l [Na +] s t e p s r e s u l t e d in only a small t r a n s i e n t i n c r e a s e in Vt, o p p o s i t e in d i r e c t i o n to that e x p e c t e d for a b a s o l a t e r a l N a + c o n d u c t a n c e . T h e absence of a significant N a + c o n d u c t a n c e is f u r t h e r supp o r t e d by t h e o b s e r v a t i o n t h a t v a s c u l a r p e r f u s i o n with Li +, which is an i n d i s t i n g u i s h a b l e r e p l a c e m e n t for N a + in m a n y e p i t h e l i a l N a + c h a n n e l s (Smith a n d Benos, 1991), f a i l e d to m a i n t a i n t h e e n d o c o c h l e a r p o t e n t i a l ( S h i n d o et al., 1992) a n d v a s c u l a r p e r f u s i o n with 10 - 4 M a m i l o r i d e , a b l o c k e r o f m a n y e p i t h e l i a l N a + channels (Smith a n d Benos, 1991), failed to inhibit t h e e n d o c o c h l e a r p o t e n t i a l ( S h i n d o et al., 1992). Homology between strial marginal cells and vestibular dark cells F u n c t i o n a l similarity b e t w e e n v e s t i b u l a r d a r k cells a n d strial m a r g i n a l cells was previously d e e m e d unlikely as long as strial m a r g i n a l cells w e r e t h o u g h t to be t h e d i r e c t s o u r c e of t h e large positive e n d o c o c h l e a r p o t e n t i a l in a c c o r d a n c e with the 'single cell m o d e l ' . I n d e e d , the e n d o c o c h l e a r p o t e n t i a l of a b o u t 80 m V has no e q u i v a l e n t in the v e s t i b u l a r l a b y r i n t h w h e r e t h e e n d o v e s t i b u l a r p o t e n t i a l in the s e m i c i r c u l a r c a n a l s is _+ 1 m V ( M a r c u s et al., 1994). M o r p h o l o g i c similarities b e t w e e n v e s t i b u l a r d a r k cells a n d strial m a r g i n a l cells, however, have long b e e n r e c o g n i z e d ( K i m u r a , 1969). R e c e n t findings illustrate similarities b e t w e e n ion t r a n s p o r t m e c h a n i s m s on the m o l e c u l a r level. V e s t i b u lar d a r k cells a n d strial m a r g i n a l cells c o n t a i n the s a m e a) apical n o n - s e l e c t i v e c a t i o n c h a n n e l ( T a k e u c h i et al., 1992; M a r c u s et al., 1992; S u n o s e et al., 1993), b) apical Is~ K + c h a n n e l ( M a r c u s a n d Shen, 1994; S u n o s e et al., 1994; M a r c u s a n d Shen, 1994), c) b a s o l a t e r a l C1 channel ( M a r c u s et al., 1993; T a k e u c h i et al., 1994), d) b a s o l a t e r a l N a , K - A T P a s e ( S c h u l t e a n d Steel, 1994) a n d e) apical p u r i n o c e p t o r (Liu et al., 1995). T h e p r e s e n t d a t a d e m o n s t r a t e t h a t t h e s e similarities f o u n d on the m o l e c u l a r level p a r t i c i p a t e in m a c r o s c o p i c , transe p i t h e l i a l e l e c t r o p h y s i o l o g i c a l events. B o t h cell types s e c r e t e K + u n d e r s y m m e t r i c a l c o n d i t i o n s in vitro a n d K + s e c r e t i o n is sensitive to b a s o l a t e r a l l y a p p l i e d b u m e t a n i d e (Fig. 4) ( M a r c u s a n d Shipley, 1994). All r e s p o n s e s to p h a r m a c o l o g i c a g e n t s a n d ion substitutions o f Vt a n d R, across strial m a r g i n a l cells w e r e q u a l i t a t i v e l y similar to those previously o b s e r v e d in v e s t i b u l a r d a r k cells. In p a r t i c u l a r , similar values w e r e o b s e r v e d in strial m a r g i n a l cells a n d v e s t i b u l a r d a r k cells ( M a r c u s et al., 1994) for V, a n d R t a) in vitro u n d e r s y m m e t r i c a l c o n d i t i o n s with a p e r i l y m p h - l i k e s o l u t i o n on b o t h sides of t h e e p i t h e l i u m (11 versus 8 m V a n d 10 versus 12 ohm-cm2), b) in vitro u n d e r in vivo-like c o n d i t i o n s with an e n d o l y m p h - l i k e solution on t h e apical side a n d a p e r i l y m p h - l i k e solutions on the b a s o l a t e r a l side ( - 2 versus - 1 m V a n d 8 versus 6 ohm-cm2). F u r t h e r , Vt in vivo across the m a r g i n a l cell

e p i t h e l i u m is n o t m o r e t h a n a few m V (Salt et al., 1987) a n d across v e s t i b u l a r d a r k cells +_ 1 m V ( M a r c u s et al., 1994), In conclusion, the m o d e l for ion t r a n s p o r t in strial m a r g i n a l cells is i d e n t i c a l to t h e m o d e l for v e s t i b u l a r d a r k cells. F u r t h e r , t h e m o d e l is c o n s i s t e n t with t h e h y p o t h e s i s that m a r g i n a l cells c o n t r i b u t e not m o r e t h a n a few m V to the e n d o c o c h l e a r p o t e n t i a l . H o w e v e r , the n a t u r e a n d l o c a t i o n of ion t r a n s p o r t m e c h a n i s m s in b a s a l cells, i n t e r m e d i a t e cells a n d fibrocytes r e m a i n to b e d e t e r m i n e d in f u t u r e studies.

Acknowledgements T h e a u t h o r s express t h e i r t h a n k s to D r J o c h e n S c h a c h t for a critical r e a d i n g of the m a n u s c r i p t . W e also t h a n k D r P e t e r S m i t h a n d M r A l a n Shipley of T h e N a t i o n a l V i b r a t i n g P r o b e Facility ( N V P F ) at M a r i n e Biological L a b o r a t o r y at W o o d s H o l e , M A for the use of the K+-selective v i b r a t i n g p r o b e a n d assistance with t h o s e m e a s u r e m e n t s . This w o r k was s u p p o r t e d by N a tional I n s t i t u t e s of H e a l t h grants DC-01098 to PW, DC-00212 to D C M a n d P41-RR-01395 to N V P F .

References Ferrary, E., Bernard, C., Oudar, O., Sterkers, O. and Amiel, C. (1989) Sodium transfer from endolymph through a luminal amiloride-sensitive channel. Am. J. Physiol. 257, F182-F189. Forge, A. (1984) Gap junctions in the stria vascularis and effects of ethacrynic acid. Hear. Res. 13, 189-200. Hodgkin, A.L. and Horowicz, P. (1959) The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Physiol. (London) 148, 127-160. Hsu, C.J. and Nomura, Y. (1985) Carbonic anhydrase activity in the inner ear. Acta Otolaryngol. Suppl. (Stockholm) 418, 1-42. Ikeda, K. and Morizono, T. (1989) Electrochemical profiles for monovalent ions in the stria vascularis: cellular model of ion transport mechanisms. Hear. Res. 39, 279-286. Ikeda, K. and Morizono, T. (1989) Electrochemical profile for calcium ions in the stria vascularis: cellular model of calcium transport mechanism. Hear. Res. 40, 111-116. lwano, T., Yamamoto, A., Omori, K., Akayama, M., Kumazawa, T. and Tashiro, Y. (1989) Quantitative immunocytochemical localization of Na*,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct J. Histochem. Cytochem. 37, 353-363. Jahnke, K. (1975) The fine structure of freeze-fractured intercellular junctions in the guinea pig inner ear. Acta Otolaryngol. Suppl. (Stockholm) 336, 1-40. Johnstone, B.M. and Sellick, P.M. (1972) The peripheral auditory apparatus. Q. Rev. Biophys. 5, 1-57. Kerr, T.P., Ross, M.D. and Ernst, S.A. (1982) Cellular localization of Na+,K+-ATPase in the mammalian cochlear duct: significance for cochlear fluid balance. Am. J. Otolaryngol. 3, 332-338. Kimura, R.S. (1969) Distribution, structure, and function of dark cells in the vestibular labyrinth. Ann. Otol. Rhinol. Laryngol. 78, 542-561.

P. Wangemann et al. / Hearing Research 84 (1995) 19-29 Konishi, T., Hamrick, P.E. and Walsh, P.J. (1978) Ion transport in guinea pig cochlea. I. Potassium and sodium transport. Acta Otolaryngol. (Stockholm) 86, 22-34. Konishi, T. and Mendelsohn, M. (1970) Effect of ouabain on cochlear potentials and endolymph composition in guinea pigs. Acta Otolaryngol. (Stockholm) 69, 192-199. Kusakari, J., lse, I., Comegys, T.H., Thalmann, I. and Thalmann, R. (1978) Effect of ethacrynic acid, furosemide, and ouabain upon the endolymphatic potential and upon high energy phosphates of the stria vascularis. Laryngoscope 88, 12-37. Kusakari, J., Kambayashi, J., Ise, I. and Kawamoto, K. (1978) Reduction of the endocochlear potential by the new 'loop' diuretic, bumetanide. Acta Otolaryngol. (Stockholm) 86, 336-341. Kiihtreiber, W.M. and Jaffe, L.F. (1990) Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J. Cell Biol. 110, 1565-1573. Liu, J., Kozakura, K. and Marcus, D.C. (1995) Evidence for P2u and P2v purinoceptors in vestibular dark cell and strial marginal cell epithelia of the gerbil. Assoc. Res. Otolaryngol. 18, 100. Marcus, D.C. (1984) Characterization of potassium permeability of cochlear duct by perilymphatic perfusion of barium. Am. J. Physiol. 247, C240-C246. Marcus, D.C., Liu, J. and Wangemann, P. (1994) Transepithelial voltage and resistance of vestibular dark cell epithelium from the gerbil ampulla. Hear. Res. 73, 101-108. Marcus, D.C., Liu, J. and Wangemann, P. (1994) Isolated stria vascularis in vitro: responses of transepithelial voltage and resistance to apical DIDS and ion substitutions. Abstr. Assoc. Res. Otolaryngol. 17, 529. Marcus, D.C. and Marcus, N.Y. (1989) Transepithelial electrical responses to C1- of nonsensory region of gerbil utricle. Biochim. Biophys. Acta 987, 56-62. Marcus, D.C., Marcus, N.Y. and Greger, R. (1987) Sidedness of action of loop diuretics and ouabain on nonsensory cells of utricle: a-micro-Ussing chamber for inner ear tissues. Hear. Res. 30. 55-64. Marcus, D.C., Marcus, N.Y. and Thalmann, R. (1981) Changes in cation contents of stria vascularis with ouabain and potassium-free perfusion. Hear. Res. 4, 149-160. Marcus, D.C., Rokugo, M. and Thalmann, R. (1985) Effects of barium and ion substitutions in artificial blood on endocochlear potential. Hear. Res. 17, 79-86. Marcus, D.C. and Shen, Z. (1994) IsK channel is apical pathway for K + secretion by inner ear epithelia. Abstr. J. Gen. Physiol. 104, 16a. Marcus, D.C. and Shen, Z. (1994) Slowly activating, voltage-dependent K + conductance is apical pathway for K + secretion in vestibular dark cells. Am. J. Physiol. Cell Physiol. 267, C857-C864. Marcus, D.C. and Shipley, A. (1994) Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe. Biophys. J. 66, 1939-1942. Marcus, D,C., Takeuchi, S. and Wangemann, P. (1992) Ca2+-activated nonselective cation channel in apical membrane of vestibular dark cells. Am. J. Physiol. 262, C1423-C1429. Marcus, D.C., Takeuchi, S. and Wangemann, P. (1993) Two types of chloride channel in the basolateral membrane of vestibular dark cell epithelium. Hear. Res. 69, 124-132. Marcus, D.C., Thalmann, R. and Marcus, N.Y. (1978) Respiratory rate and ATP content of stria vascularis of guinea pig in vitro. Laryngoscope 88, 1825-1835. Marcus, N.Y. and Marcus, D.C. (1990) Transepithelial electrical

29

responses to sodium and potassium of nonsensory region of gerbil utricle. Hear. Res. 44, 13-23. Offner, F,F., Dallos, P. and Cheatham, M.A. (1987) Positive endocochlear potential: mechanism of production by marginal cells of stria vascularis. Hear. Res. 29, 117-124. Rybak, L.P. and Whitworth, C. (1986) Comparative ototoxicity of furosemide and piretanide. Acta Otolaryngol. (Stockholm) 101, 59-65. Sakagami, M., Fukazawa, K., Matsunaga, T., Fujita, H., Mori, N., Takumi, T., Ohkubo, H. and Nakanishi, S. (1991) Cellular localization of rat Isk protein in the stria vascularis by immunohistochemical observation. Hear. Res. 56, 168--172. Salt, A.N., Melichar, I. and Thalmann, R. (1987) Mechanisms of endocochlear potential generation by stria vascularis. Laryngoscope 97, 984-991. Schulte, B.A. and Adams, J.C. (1989) Distribution of immunoreactive Na+,K+-ATPase in gerbil cochlea. J. Histochem. Cytochem. 37, 127-134. Schulte, B.A. and Steel, K.P. (1994) Expression of alpha and beta subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the W v or SId loci. Hear. Res. 78, 65-76. Shen, Z., Liu, J. and Marcus, D.C. (1994) Direct activation of a conductive pathway in the apical membrane of vestibular dark cells by a disulfonic stilbene (DIDS). Abstr. Assoc. Res. Otolaryngol. 17, 134. Shindo, M., Miyamoto, M., Abe, N., Shida, S., Murakami, Y. and Imai, Y. (1992) Dependence of endocochlear potential on basolateral Na + and C1- concentration: a study using vascular and perilymph perfusion. Jpn. J. Physiol. 42, 617-630. Smith, P.R. and Benos, D.J. (1991) Epithelial Na + channels. Annu. Rev. Physiol. 53, 509-530. Spicer, 5.5. and Schulte, B.A. (1991) Differentiation of inner ear fibrocytes according to their ion transport related activity. Hear. Res. 56, 53-64. Sunose, H., Ikeda, K., Saito, Y., Nishiyama, A, and Takasaka, T. (1993) Nonselective cation and CI channels in luminal membrane of the marginal cell. Am. J. Physiol. Cell Physiol. 265, C72-C78. Sunose, H., Ikeda, K., Suzuki, M. and Takasaka, T. (1994) Voltagedependent K channel in luminal membrane of marginal cells of stria vascularis. Hear. Res. 80, 86-92. Takeuchi, S., Irimajiri, A. and Saito, H. (1994) Ion channels in the basolateral membrane of single isolated strial marginal cells. Abstr. Assoc. Res. Otolaryngol. 17, 133. Takeuchi, S., Marcus, D.C. and Wangemann, P. (1992) Ca2 +-activated nonselective cation, maxi K + and CI channels in apical membrane of marginal cells of stria vascularis. Hear. Res. 61, 86-96. Tasaki, I. and Spyropoulos, C.S. (1959) Stria vascularis as source of endocochlear potential. J. Neurophysiol. 22, 149-155. Wada, J., Kambayashi, J., Marcus, D.C. and Thalmann, R. (1979) Vascular perfusion of the cochlea: effect of potassium-free and rubidium-substituted media. Arch. Otorhinolaryngol. 225, 79-81. Wangemann, P. and Greger, R. (1990) Piretanide inhibits the N a + 2 C I - K + carrier in the thick ascending limb of the loop of Henle and reduces the metabolic fuel requirement of this nephron segment. In: Puschett, J.B. and Greenberg, A., (Eds.), Diuretics III: Chemistry, Pharmacology, and Clinical Applications, Esevier, New York, pp. 220-224. Wangemann, P. and Marcus, D.C. (1990) K+-induced swelling of vestibular dark cells is dependent on Na + and CI- and inhibited by piretanide. Pflugers Arch. 416, 262-269.