PRODUCTION

AND ROLE

OF INNER

EAR FLUID

Contents
1. Introduction
2. Ionic composition of endolymph
2.1, Experimental methods
2.2 Transport of K+
2.3 Sodium transport
2.4 Effect of ouabain on Na+ transport from Scala media
3. The mode of action of ethacrynic
acid
3.1 Differential effects of ouabain and ethacrynic acid
4. Model of the ion transport
mechanisms
of the secretory

cells of the stria vascularis

References

337
339
340
341
345
346
349
352
356
360

1. Introduction
1959, 1965) proposed the so-called Battery theory of acoustical transthe organ of Corti of mammalian cochlea. This theory assumes that two
biological
batteries in serial arrangement create a steady current flow across the reticular
laminar of the organ of Corti. These batteries are the electrochemical gradients produced
across the reticular laminar by the endo-cochlear potential (EP), the ionic concentration
gradients between endolymph and cell cytoplasm and the hair cell resting potential.
The theory assumes that the hairs of the hair cells are bent by shearing forces between
the tectorial membrane and the reticular laminar when the basilar membrane vibrates
in response to sound input. It is thought that the resistance across the reticular laminar
alters when the hairs are bent and thus modulates the resting current flow to produce
the receptor potentials, i.e. cochlear microphonics (CM) and the summating potential
Davis

duction

(1957,

in

(SP).
The driving force for a particular ion across the reticular laminar is represented

by:

K = E, + RT/F log,l,,;l,
where E, is the hair cell membrane potential, R,7: and F have their usual significance,
I,. and I, are the ion concentrations in the endolymph and hair cell plasma respectively,
E, is generally assumed to be about - 80 mV since this is the potential measured when
either a small or large micropipette is inserted in the organ of Corti. The negative
potential recorded by large electrodes is most likely to be the result of injury potentials
produced when the hair cells and supporting cells of the organ of Corti are damaged
(Dallos, 1968, 1973). It is uncertain whether the potentials recorded with fine electrodes
are from supporting cells or hair cells, in either case it is assumed that the value of
-80 mV for E, is a reasonable approximation. EP is easily measured with pipette
electrodes and is very close to + 80 mV in guinea pigs. The endolymph [K],, [Na],,
and [Cl-], are represented in Table 1 and if it is assumed that the intracellular [KJ;,
[NaJ and [Cl-Ii are 150, 20 and 1Om~. respectively. then the following values of
the driving forces for each ion are; Q = 160 mV. I&, = 89 mV. and V,, = 100 mV.
Honrubia and Ward (1969) altered EP by passing current between endolymph and
perilymph and recorded CM and SP so as to measure their reversal potential. They
found a linear relationship between CM and EP that indicated that EP would have
to be made negative 1.5 to 3 times the value of normal EP, i.e. 120 to 240 mV. This
potential represents the reversal potential for CM and is close to V, indicating that
* Current address: Biology Building. Llnivcrsit! of Sussex. Falmcr. Brighton. Sussex. BNI 9Q6. U.K.
337

338
TABLE

P. M. SCI.I.ICXAhu B. M. JOHNSTO~I
I. PERILYMPH

IOY CONUNTRATIONS;

[Na+]mM

I. Smith, Lowry and Wu. 1954.

2. Johnstone et al., 1963.
3. Bosher and Warren. 196X.

[K+]mM

[Na'.] 150 ITIM',~,

[K+]S'.'. [Cl ]IIO-. E\I~~L~MI~I~Iol\
CONCENTRATlONS

[Cl-1rn.u

Resting
potential

4. Sellick and Johnstone, 1972a.

5. Sellick and Johnstone, 1972b.
6. Sellick and Johnstone. 1972~.

Equilibrium potential
NaK(I

7. Boshcl c~nd Warren. 1971.

8. Citron and Exely. 1956.
Y. Inferred.

the current is carried by K and that the change in resistance across the reticular
laminar is predominantly a change in the K conductance. It is possible that the conductance change is nonspecific, i.e. that the conductance to all ions is increased since the
small amount of Na across the reticular laminar would not make a significant contribution to the receptor potential. Cl-, however, may well carry some of the current. It
is interesting that a different reversal potential is obtained for SP from plots of SP
VS. EP from the data of Honrubia and Ward. These plots are almost linear and give
a reversal potential of SP of between -20 and 0 mV indicating that SP is generated
by a different mechanism to that of CM. However, since SP is very labile there is
a possibility that the experimental data do not accurately predict the reversal potential.
Therefore, at least for the generation of CM, the endolymph ion composition and
potential provide all but a small proportion of the driving force necessary to maintain
current flow through the hair cells. If endolymph is replaced by perilymph V, will
be reduced by 85 mV, i.e. the driving force will be roughly halved. This would explain
why when Konishi et al. (1966) perfused Scala media with Ringers solution they observed
that CM was reduced by 76% before +EP fell much below its original level, Similarly
if the bodies of the hair cells are depolarized by perfusing Scala tympani, and therefore
presumably the Cortilymph with high K solutions, V, and hence CM are reduced
to the same extent since the hair cell membrane potential is reduced (Butler, 1965;
Tasaki et al., 1952; Honrubia and Ward, 1969).
The hair cells in the lateral line of the dogfish do not have high K+ endolymph
or a positive potential at their apical surfaces (Liddicoat and Roberts, 1972) and it
is interesting to compare their function with the hair cells of the mammalian labyrinth.
Presumably the current during the generation of the receptor potential is carried by
Na+ even though it has been shown that tetrodotoxin, a specific blocker of Na+ conductance, does not affect the receptor potential (Lowenstein, 1971). This appears also to
be the case for gastropod molluscan statocysts (Alkon and Bak, 1973). It is interesting
to speculate as to what advantage the electrochemical gradient between endolymph
and perilymph bestows on the labyrinthine hair cells, The driving force for Na+ is
about 130mV in the cell bathed in extracellular fluid and this is only 30mV less than
the driving force for K+ across the reticular laminar. It is difficult to see, therefore.
how the endolymph ion composition and potentials increase the driving force to any
significant extent. All that happens, it seems, is that the generator current is carried
by K instead of Na+. This may be the clue to the function of endolymph. Since
the hair cell membrane is normally selectively permeable to Kf it is likely that there
would be a large resting K+ current into the hair cell from endolymph. On the other
hand, a hair cell that functions without endolymph could not have a large resting
Na+ current since this would require considerable metabolic effort on the part of the
cell to maintain the normal ion gradients. The labyrinthine hair cell therefore gains
sensitivity by reason of the large resting current but does not have to have the metabolic
machinery
to sustain it. This is located in the stria vascularis for the cochlea or in
secretory cells in the utricular membrane and around the ampulla of the semicircular
canals. This has the advantage that the hair cell is isolated from its energy supply
with its bulk and noise, in the form of pulsatile blood flow. Therefore the difference
between the two types of hair cells may be one of the magnitude of the resting current

PRODUCTIOYAND ROLE ok IKNEK EAR

FLUHI

330

across the apex of the cell instead of the magnitude of the driving voltage. Hence
a given conductance change will produce a larger change in current in the cell with
the higher resting current and hence will be expected to have a lower threshold for
nerve stimulation.
A similar treatment can be applied to the vestibular organs. since these sensory organs
are also bathed in endolymph although of somewhat different composition (Table 1).
The most important difference between the vestibular organs and the organ of Corti
is that the potential in the endolymph is very close to zero. This halves the driving
force for K+ in both the saccule and utricle and reduces pi., and I;.,.Again it is expected
that the current be carried by K+ since VK is the largest of the three driving forces
and because of the preponderance of K+ over Na+ in endolymph and intracellular
fluid.
2. Ionic Composition of Endolymph
The electrophysiology of the cochlea. utricle and saccule has recently been reviewed
and Sellick, 1972) and therefore it is not intended to repeat the material
presented but to discuss recent material that the authors believe throws some light
on the mode of production of endolymph. The ionic concentration of the three major
ions found in cochlear, saccular and utricular endolymph that must be regarded as
the most accurate from examination of the literature are presented in Table 1. In the
case of the saccular concentration of Cl- and K+ which have not been measured.
the values are inferred from the assumption that the ionic strength is constant throughout the endolymphatic system and that the [K+] may be approximated by simply
subtracting the [Na] from 150 mM. i.e. Na+ and K+ constitute the major cations.
The equilibrium potentials for each ion derived from the Nernst equation and the
ionic composition of perilymph and endolymph together with the resting potentials
are included to illustrate the direction of the passive fluxes in each structure. It is
evident that in all three structures Na+ will tend to flow into endolymph and therefore
must be pumped out and that K+ will tend to flow out of endolymph and therefore
must be pumped in. For the saccule and utricle Cl- is at equilibrium in the resting
state but will tend to flow into the cochlear endolymph as &, differs from the endocochlear potential (EP) by 80mV. The potentials in all three structures are reduced by
anoxia or by application of metabolic poisons to negative values that are subsequently
reduced post-mortem over a period of several hours (Morizono and Johnstone. 1968;
Konishi and Kelsey, 196X; Konishi et ul.. 1961: Fernandez. 1955; Schmidt. 1963). The
reduction of the negative potentials is coincident with the reduction of the ion gradients
between endolymph and perilymph and are therefore regarded as the result of K+dominated diffusion potentials (Johnstone, 1965; Mendelsohn and Konishi, 1969; Bosher
and Warren. 1968; Kuijpers and Bonting, 1970 a, b). Therefore the resting potentials
in the utricle. saccule and cochlear duct (Scala media) are regarded as being the sum
of a negative, K-dominated diffusion potential and a positive. anoxia-sensitive electrogenie potential.
There is both experimental and anatomical evidence that the utricle and cochlear
duct are separate entities in that they are independent of each other for the production
of the electrochemical gradients between endolymph and perilymph. The saccule and
endolymphatic duct appear to provide a substantial barrier to ionic diffusion or electrical
leakage (Sellick and Johnstone, 1972a). This is supported by the occurrence of secretory
type cells in the body of the utricle and around the ampullae of the semicircular canals
similar to those found in the stria vascularis of the cochlear duct (Smith, 1956; Echandia
and Burgos, 1965).
Unlike the utricle. the saccule has no blood supply of its own and the cells in the
saccular wall are mostly simple cuboidal epithelia. similar to those that occur in
Reissners membrane. The evidence suggests that the saccule is dependent on the cochlear
duct for its potential and ion gradients and that it is a paisive diverticulum of Scala

(Johnstone

340

P. M. Skl.l.~(.k
,ANI)
B. M. .IOHNSTONI

media (Sellick and Johnstone, 1972 b). Recent evidence suggesting that this is not entirely
the case will be discussed later.
The problem of the origin of endolymph is the problem of the nature of the ion
transport that occurs in the secretory cells balanced with the leakage of ions down
their electrochemical gradients. We will attempt to describe these transport processes
and the electrochemical gradients they produce.
2.1 EXPERIMENTAL
METHODS
The major technique used for measuring the magnitude of Na+ and K+ transport
in the cochlear duct is to alter the concentration of these ions by perfusing the duct
and observing changes in ion concentration that occur after perfusion has stopped.
This method is similar to that of loading muscle or red blood cells with Na in the
cold and observing fluxes when the cells are returned to normal temperature. Perfusion
of the cochlea is carried out by inserting a perfusion pipette through the basilar membrane and into Scala media of the first turn (Sellick and Johnstone, 1972; Sellick and
Bock, 1974). The apical turn is damaged surgically to allow the perfusate to escape.
The changes in [Na] or [K] post-perfusion are measured with ion selective electrodes
inserted into Scala media via a fenestra in the basal turn. Conventional glass pipette
electrodes are inserted through the same fenestra to record EP. Damage to the apical
turn does not affect the electrical properties of the basal turn since it occurs many
length constants away. (The length constant of Scala media is 2 mm.)
Measurement of the ionic conductances for K+ and Nat of Scala media is essential
if active transport of these ions are to be measured by observing concentration changes
in endolymph. The total change in concentration of an ion consists of the sum of
the active and passive flux, the latter being equal to:
G;[EP - 58 log(i,/i,,)]

(1)
where Gi is the conductance of species i, i, concentration in perilymph, i, concentration
in endolymph, i.e. the difference between EP and the equilibrium potential for that
ion. We have estimated the value of G, and GN,, by observing the [Na+& and [K],
with ion specific electrodes after the animals had been killed by asphyxia. The assumption is that all active transport had stopped and that the ions run down their electrochemical gradients, i.e. (EP-Ei). The value of Gel cannot be determined in this way
since it is the major anion and hence alterations in [Cl-], would necessarily produce
a change in osmotic pressure and resultant water movement thus confusing the results.
The values for GI( and G,,, obtained in this way may be found in Tables 2 and
3. The mean value of GK was found to be 0.04 x IO- 7 mM/min/mV/mm of Scala media
and that of GNa 0.02 x lo- mM/min/mV/mm of Scala media. It must be kept in mind
that this is far from the best method of determining conductance since it is uncertain
whether unspecified changes that occur post-mortem alter the values measured or indeed
whether all ion transport has ceased.
Since similar values of G, are obtained when EP is reduced to its anoxic level with
ouabain in the living animal the two aforementioned objections to the mode of measurement of G, are not likely to be serious ones.
The value of Gel is assumed to be small since perfusion of Scala media with low
Cl- sulphate Ringer produces an increase in EP of about + 10 mV, whereas EC, had
been changed from 0 to -92 mV ([Cl;] = 3 mM). It was found that the increase in
EP was due to oxygen in the perfusate and that deoxygenated, low Cl- perfusates
produced negligible change in EP.
In the absence of electrogenic potentials the following equation should describe the
post-mortem EP (EP,,):
EP

pm -

GK.& +

%a.EN,+ GaEC,

G, + &a + GCI

If it is assumed that Gel is insignificant

then the means of the measured

(2)
values of

PRODUCTIONAND

ROLEOF INNCK EAR FLUID

GK and GNa predict a value of EP,, of

Therefore the ratio of Gk and GNa are
sum of G, and GNawould be close to the
current into Scala media and measuring
R, between Scala media and perilymph is
nal resistance of Scala media r, and the

341

- 17 mV, i.e. close to the - 20 mV observed.

as expected. It would be expected that the
total ion conductance as measured by passing
the voltage produced. The access resistance,
about 5 kohms and is related to the longitudilength constant ). by the following equation:

R = @r,.

(3)

The resistance of 1 mm of Scala media can be calculated from the equation:

__ r,

3,=
J(

r, + re1

(4)

where r, is the resistance of 1 mm of the walls of Scala media. r, is the longitudinal

resistance of 1 mm of endolymphatic space calculated from the preceding equation, rr
is the longitudinal resistance of perilymphatic space calculated from the specific resistance of perilymph and the area of cross-section of the perilymphatic space (Misrahy,
1958). Using a value of i of 2 mm (Johnstone et al., 1966) the resistance of 1 mm of
Scala media is calculated to be 28 kohms. This may be converted to a conductance
expressed in mM/min/mV/mm of 0.22 x lo- 7. If this value is taken as the sum of
the conductances G,, GNa and Gc, and it is assumed as a first approximation that
GNa= 2Gc, then the following values can be calculated from equation (2) for an EP,,
of - 20 mV. G, = 0.13, GNa= 0.06, Gc, = 0.03 all x lo- 7 mM/min/mV/mm of Scala
media. Note that the values of Gk and GNa are both about 3 times their measured
values. At the moment this discrepancy remains unexplained but for reasons explained
later the measured values of G, and GNa will be used to calculate active K+ and Na
flux.
2.2

TRANSPORT

OF K

It has been suggested (Kuijpers and Bonting, 1970 b; Johnstone and Sellick, 1972)
that the positive endocochlear potential of + 80 mV is the result of a positive component
of about 1OOmV produced by electrogenic transport of K+ and a negative component
of about 20mV which is mainly a K+ diffusion potential. There is ample evidence
that the negative component is the result of K+ diffusion but the assumption that
the positive component is produced by electrogenic K+ transport has hitherto not been
justified experimentally. From a consideration of the equilibrium potentials for the major
ions in endolymph of the cochlea it is evident that a positive electrogenic potential
could be produced by transport of K+ into endolymph or Cl- out of endolymph.
The latter origin can probably be disregarded as unlikely since Robinson and Sellick
(1973) have reduced the endolymph [Cl-] by perfusion to as low as 3 mM without
reducing the magnitude of +EP. Furthermore, a positive electrogenic potential occurs
in the utricle where Cl- is in electrochemical equilibrium. Therefore it is very unlikely
that electrogenic Cl- pumps contribute to the potentials of the membranous labyrinth.
In order to obtain evidence for the role of electrogenic Kf transport in the production
of + EP, the following experiments were performed (Sellick and Bock, 1974). The endolymph [K], was reduced by perfusion with normal Ringers (K+ = 5 mM) and with
K+ substituted Ringers (20m~ and 50m~ K+). Potassium specific microelectrodes,
made with K+ liquid ion exchanger similar to the method described by Walker (1971)
were used to measure [K], during and after perfusion. The mean resting values of
cochlear [K], (Table 2) of 140 + 8.6 mM were close to those of Johnstone et al. (1963)
in the guinea pig of 150 _t 5.9 mM and those of Bosher and Warren (1968) in the rat
of 157 + 5.4 mM. The events that occurred after perfusion are illustrated for three animals in Fig. 1. The following events were identical in all nine animals perfused. During
the start of perfusion various sudden changes occurred in the value of EP usually consisting of a reduction of up to 10mV during perfusion and return to initial values either
during perfusion or after perfusion had stopped. It is interesting to note that the initial

342

P. M.

SELLICK

ANI)

B.

M.

JOHNSIO~I

60

*Or

Perfuse
n

12

24

Time,

min

FIG. 1. Endocochlear potential and endolymph [Kz] measured from the first turn of the cochlea
during and after perfusion of scala media with K+-substituted Ringer. 0, 5 mM K+. 0, 20 mM
K, H, 50m~ K+. Perfusion was stopped at A.

negative movement of EP is in the opposite direction to that expected from a movement

of the basilar membrane towards Scala tympani (Butler and Honrubia, 1963) i.e. the
direction of movement in response to an increase in pressure in scala media. We later
found that the initial negative going response did not occur if Na+ was replaced with
Choline and the [K] varied between 32 mM and 150m~. Instead the initial response
to perfusion was positive going of the order of 2@30mV followed by a slow decline.
During perfusion the [K], fell to the values illustrated in Table 2. Failure to reduce
the [K], to the level of the perfusate in several animals is more likely to be the
result of inadequate perfusion rather than error in the measurement of [K], considering
the accuracy of the measurements at the resting endolymph levels. When perfusion
was stopped by withdrawing the perfusion pipette the [K], began to rise immediately
and reached stable values about 15 min later (Plateaus, Table 2). EP slowly declined
to stable levels during the K influx. Total K+ flux rates were obtained graphically
and the active component calculated from the estimated passive flux using the measured
value of GK for that particular animal. Since the measured value of EP consisted of
an electrogenic component plus a negative diffusion potential component, it was necessary to calculate the latter so that the total electrogenic component could be obtained.
The diffusion potential was calculated using the Goldman equation assuming a P,: PN,,
of 1:0.35 (giving a normal value for the K+ diffusion potential of - 26 mV), a perilymph
[K], of 5rn~ and the measured value of [K],. It was assumed that the [Na](>
TABLE

2.
___.__-

Ammal

lntt~al [K-J
W.0

ln~tral EP
CmV)

Prrfwitc
[K]
@W

LK] immediately
alter perfusion
CmM)

[K]

Plateaus
rn~ EP mV

155

x0

20

3x

I?O 20

3
4
5
6
7
8
9

I68
I15
130
130
140
160
92
170

76
x7
74

20
Xl
:::

47
??
49

::
86
80
92

SO
57
57
5.7

::
IO
6
33

I34
107
I IO
I10
76
:z,

9
21
31
35
24
ki

113

26

(I& rnM.nli mV Ill,

* IO

0 011
0.02
0 05
0 o(w
0
0 04
0 04

343

ctlve

K+Transport

x 166mmoles/min/mm

Scala Medlo

(b)

0,

-20

Active

K+ Transport

x 10M6mmoles/min/mm

Scala Media

-40 t

Kt Perfusate

FIG. 2. (a. b. c) Electrogenic

potential

vs active

transport

values of G, for each individual

calculated

animal.

from

the measured

P.

344

M.

SELLICK

ANIl

B.

M.

JOHNSTONf.

60
50

rmal Ringer perfusote

40

30

G,=0~04x10~7mMoles/min/mm/mV

20

Actwe

Active

K+Tronsport

K+Transport

x 10~7mMoles/min/mm

x 10~7mMoles/min/mm

Scala Media

Scala Media

OmM K+perfusate

G,=0~04x16~o&/min/mm/f~W

Active
FIG.

3. (a, b, c) Electrogenic
measured

K*Transpon

x167mMoks/min/mm

Scala Media

calculated
potential
vs active K+ transport
value of G, of 0.04 x lo- mM/min/mV/mm.

from

the average

PRODUCTION

AND

ROLF

OF INNI:R

EAR

FLI In

345

TABLE 3.

equal to 150 - [K],~ since it is expected that the ionic strength of endolymph
would not change and that the error in calculation from this factor would be small. Calculation of ,& using the measured values of G, produced electrogenic potential vs f;,
curves that are linear but with widely varying slopes and intercepts (Fig. 2). This variation was obviously the result of the variations in the value of G, since when the
mean value was used for all nine animals the curves had very similar slopes and intercepts (Fig. 3). This data is taken as very strong evidence that the electrogenic component
of EP is produced by an electrogenic K pump. In a previous publication (Selli&
and Bock, 1974) a calculated value of Gk was also used to calculate the active K
flux. This produces curves that intercept the abscissa at about 1 x 10m6mM/min/mm,
however. this result must be regarded very tentatively.
One of the most puzzling aspects of the results is the cause of the fall of EP following
perfusion. It is not expected that a reduction of the [KJ on the endolymph side of
the stria would inhibit K+ transport, rather it would be expected that the pump would
rely on K+ being supplied to the serosal side of the stria. Replacement of Na+ with
choline in the perfusate produced a similar fall and we must therefore conclude that
the fall of EP is due to the reduction of [K], and not to an increase in [Na],.
was

2.3 SODIUM TRANSPORT

Since Naf is transported out of endolymph, it should be possible to measure the
Na activation of the transport system by increasing the [Na], and observing Na+
efflux after perfusion had stopped. Scala media was perfused with 20, 50 and 82 mM

stop
perfusion
4

IO

14

I8

Time,
FIG.

4.EP and Nat measured

during

22

26

30

34

min

and after perfusion

with 82. 50 and 20m~

Nat

Ringers.

346

P. M. Stuxx

1-r
0

IO

H. M. JOHNSTONI-

AW

20

30

1
40

50

60

70

60

[Na+] mM
FIG. 5. Activation

of active

Na-

transport

by endolymph

[Na],

Na+ Ringer. [Na],, was measured with Na+ selective glass microelectrodes (Sellick
and Johnstone, 1972 c) and all other procedures were as for the low K+ perfusions
described in Section 2.2. Details of the high Na+ perfusions are listed in Table 3. Representative data for the three [Na+] concentrations perfused are illustrated in Fig. 4.
Perfusion of 20 mM Na. Ringers produced an initial increase of EP of about 10 mV
and a subsequent slow fall to near its original value in the following 30min. Therefore,
unlike the low K+ perfusions, EP remained near its normal value during Na+ efflux.
The [Na+lC, was reduced to 3-4 mM during this time. Perfusion with 82 mM Na, however. reduced EP and some of the Na+ efflux data was therefore taken with EP lower
than normal. The active Na efflux was calculated from the total flux and the calculated
passive flux using the measured value of GNa for each individual animal or if this was
not available the average of 0.02 x lo- mM/min/mV/mm. The active Na efflux was
plotted against the [Na],, (Fig. 5) for three 20m~, one 50m~ and two 82 mM Naf
perfusions. These data are plotted as a double reciprocal plot in Fig. 6 giving a L,,,
of 12.5 x lo- mM/min and a K,, of 100 mM.

0I

002

004006

008

IO

12

14

I.6

16

2-o

22

I/[Na+]

FIG. 6. Double

reciprocal

activation
plot of the Na+ transport
12.5 x lo- mM/min/mm.

data.

K, = 100 mM and

L,,,,, =

PRODLTTIONAND

ROLE OF INNER EAK

347

FLI tt>

Kuijpers and Bonting (1969) have characterized the Na+ activation of the Na-K+
ATPase from guinea-pig cochlea. They found that maximal activity was reached at
10 mM Na followed by a decrease between 10 and 20 mM. and that the activity then
remained unchanged up to 150 mM. K, was 4.5 mM Naf . Post et al. (1960) demonstrated
in red blood cells that the concentration at which Na+, Kt and ouabain showed maximal effects was the same for the activation of NaC-K ATPase and for the Na+ pump.
Na+ activation of the Naf-K
exchange pump was measured as a function of intracellular Na+, thus demonstrating that the activation of Na+-K+ ATPase and the activation
of the Na+-K+ pump by intracellular Na+ are comparable.
It is likely that Na transport from Scala media is carried out by the Na+ pump
associated with the Nat-K+ ATPase characterized by Kuijpers and Bonting and that
the intracellular activation of the enzyme by Na+ would bear some relationship to
the extracellular activation of the pump by Na. However. the data does not permit
a definite conclusion about the association of the Na+-K ATPase and the Na+ pump
to be drawn.
2.4 EFFECTOF OUABAINON Na+ TRANSPORT
FROMSCALA

MEDIA

Kuijpers and Bonting (1970a) have shown that 45 min after application of ouabain
to Scala vestibuli the inhibition curves for EP and the Na-K+ ATPase are very similar
and thus concluded that the Na+-K+ ATPase occupies a primary role in the production
of + EP. If the Na+-K
exchange pump directly generates +EP, its K arm would
have to be electrogenic since it must transport K+ into and Na+ out of Scala media
and produce a positive potential. In other tissues studied the Na arm is electrogenic
and the pump produces a negative potential when stimulated with high intracellular
Na+ (Thomas, 1972). It would be expected that such a pump would depend upon
endolymph Na+ for its activity and that it could be stimulated by an increase in [Nafle.
This has been shown not to be the case in the preceding section. There is the possibility
that the coupling of such a pump changes with the [Na],, i.e. becomes almost purely
electrogenic with low [Na], and loses its positive electrogenicity when [Na], is increased. This may explain why +EP falls after Scala media is perfused with high Nat
Ringer.
exchange pump is directly responsible
In an attempt to test whether the Na-K+
for the production of +EP, Naf efflux and +EP were observed during ouabain poisoning. 2 mM ouabain was included in the Scala media perfusate (20 mM Na+) and the
active Na transport calculated as before. The results are illustrated for two animals
in Fig. 7. The active Na+ efflux values for ouabain-poisoned animals were normalized
by plotting them as a ratio of the expected normal efflux for particular Na, i.e. f,/f,
where f, is the active flux for a particular [Na] from the ouabain-poisoned animal
and f, is the normal expected flux for that [Na+] as determined from the data in
Fig. 5. f,/f, increased initially to 5.5 and 14.5 and subsequently declined, i.e. there was
an initial increase in pumping rate above normal. The fact that a monotonic relationship
between ,f,/f, and the magnitude of +EP was not found during ouabain poisoning
suggests that Naf transport is independent of the generation of +EP. Indeed it was
found that the transport of Na+ increased threefold above that normally expected while
+EP was reduced by 20mV. The Na-K+
ATPase and + EP inhibition curves of
Kuijpers and Bonting are very similar to the Na- transport and +EP inhibition
obtained after perfusion of Scala media with 20 mM Na and 2 mM ouabain. The NafK ATPase is stimulated at low concentrations of ouabain while +EP is depressed
and the inhibition curves coincide only at the 30/, inhibition level. Since only one
concentration of ouabain was used in the Scala media perfusates the data cannot be
compared directly with Kuijpers Na+-K+ ATPase inhibition data, however, a similar
pattern emerges in time, with a steady decline of EP accompanied by an initial stimulation of Naf transport and subsequent decline. In other words, the Naf transport
data is qualitatively similar to the enzyme inhibition data if it is assumed that the

348

P. M.

I:

SIILIKK AND

B. M.

Time,

JOHUSI-ONI

min

FIG. 7. Effect of 2 I~IMouabain applied to scala media on EP and Na+ transport after perfusion
of 20 mM N Ringer.

concentration of ouabain at its site of action increases over the 60min following its
initial application. This could be produced if ouabain had to diffuse to its site of action
from the point of application.
There is evidence that the Nat-K+
exchange pump is located on the serosal side
of the strial border cell. Firstly, this would satisfy the condition of having to transport
Na out of and K+ into the cell and Scala media. Secondly, ouabain is effective at
lower concentrations when applied to the serosal side of the stria, i.e. when applied
to the perilymph or blood (Morizono and Johnstone, 1969; Kuijpers and Bonting, 1970a;
Konishi and Mendelsohn, 1970; Tanaka and Brown, 1970). The results confirm this
hypothesis since 2 x 10m3M ouabain applied to Scala media produced about the same
rate of reduction of EP as 1 x 10m6M ouabain applied to Scala vestibuli and hence
to the serosal side of stria vascularis. Evidence from red blood cells (Hoffman, 1966)
squid axon (Caldwell and Keynes, 1959) and turtle bladder (Solinger et u1., 1968) indicates that ouabain inhibits the Na+-Kf
pump on the side of the membrane from
which K+ is being transported. thus locating the Na-Kf
pump on the serosal side
of the strial border cell.
Therefore ouabain applied in Scala media would have to diffuse to the other side
of the strial cells to have its effect and may be expected to reach this site initially
at low concentrations. Therefore it is possible that the increase in Nat transport above
normal, observed with the application of ouabain to the Scala media, is the result of
stimulation of the Naf-K
ATPase by the initially low concentrations of ouabain
on the serosal side of the stria vascularis. It is interesting to note that a similar stimulation of Na+ efflux from squid axons was observed by Baker and Willis (1972) at
low ouabain concentrations.
The findings support the view that Na transport from scala media after perfusion
with 20mM Na+ solution is primarily carried out by the Na+-K
exchange pump
which relies on NaC-K+ ATPase for its energy supply. However, the idea that +EP
is directly generated by the Naf-K+ exchange pump is untenable unless the coupling
between Na+ and K+ transport changes during the application of ouabain.

349

PRODUCTION AND ROLI:OF INNER EAR FLLW

Time, min

FIG. 8. The effect of an intravenous

dose of 40mg/kg EthA on EP and the cochlea1
Na:. Injection was started at A.

endolymph

3. The Mode of Action of Ethacrynic Acid

The ototoxicity of ethacrynic acid (EthA) was first noticed by Maher and Schreiner
(1965) who reported that one patient experienced acute deafness that persisted for
an hour and another experienced vertigo after an oral preparation of ethacrynic acid.
This observation was followed by many reports of transient acute hearing loss in patients
after doses of EthA to relieve congestive heart failure or renal impairment (Schneider
and Becker, 1966; Pillay et al., 1969; Hanzelik and Peppercorn, 1969; and Matz et
al., 1969). Transient acute hearing loss was observed after doses of EthA between 5@
300 mg/kg orally and 50-800 mg intravenously.
Mathog et al. (1970) measured round window cochlear microphonics (CM) in cats
after intravenous injection of EthA and found that doses greater than 10 mg/kg produced
alterations in this response and depression of the action potential (AP) followed by
complete recovery in 1 hr. Silverstein and Yules (1971) observed similar changes in CM
in cats with intravenous doses of 30mg/kg of EthA. Depression of CM lasted for 5 hr
(the duration of the experiment) and endolymph [Na] increased from 7 to 20 mM
after 60 min, followed by a decrease to 1OmM after 3 hr, at which time CM and AP
were severely depressed. The [K+],. was reduced by about 10 mM 2 hr after injection.
We have confirmed these results using Na+ and K+ specific electrodes (Sellick and
Johnstone, 1974); Fig. 8. Similar changes were observed in the rat by Bosher et aI.
(1973). The dramatic changes in endolymph composition observed by Cohn et al. (1971)
in the dog remain to be confirmed. Prazma et ul. (1972) reported that intravenous
doses of l&50 mg/kg EthA in guinea pigs reversibly reduced EP, whereas CM did
not recover and the summation potential (SP) was initially increased but returned to
near normal.
Mathog et al. (1970) failed to see histopathological changes in the cochlear partition
45 min after an intravenous dose of 1@30 mg/kg EthA. However, when the animals
were killed 5 days after a dose of 30mg/kg EthA, severe vacuolization and loss of
nuclei were seen in outer hair cells and Deiters cells. Quick and Duvall (1970) observed
guinea-pig cochleas with the electron microscope after intravenous doses of 10-86 mg/kg
EthA and confirmed the changes in outer hair cells observed by Mathog. The most
striking changes were found in the stria vascularis after high doses and consisted of
an increase in the thickness of the stria due to intracellular and extracellular edema.
The marginal cells were normal but the intermediate cells were completely destroyed
or were seen in an advanced stage of atrophy, lying within the spaces created by their

350

P.

M. !%LLI(.K

ASD

H.

M. JwmSToVf:

atrophy and the extracellular effusion. A preliminary survey of the endoiymphatic sac
and the vestibular labyrinth failed to reveal any ultrastructural changes as a result
of EthA therapy. The above observations were confirmed by Silverstein and Yules ( 1970).
EthA is a powerful saluretic diuretic. Beyer rt nl. (1965). using the stop-flow technique.
showed that EthA depressed the reabsorption of sodium in both the proximal and
more distal portions of the kidney. Laragh rt al. (1966) found in renal clearance studies
that EthA caused Cl excretion to increase to a greater extent than Na excretion,
suggesting that a primary action of the drug is that it blocks Cl reabsorption; however.
data also indicated that the amount of Nat reabsorbed by ionic exchange mechanisms
actually increased after the diuretic was given and that the diuretic acts primarily to
block a NaCl reabsorptive process and to leave the Na-Kexchange untouched.
The site of action of EthA in epithelial ion-transporting systems is still largely unknown. A large amount of work has been done on its site of action in single cells
systems (red blood cell and Ehrlich ascites tumour cells) in cell free systems and on
kidney slices. The overwhelming difficulty is to use data obtained in these systems to
predict the effect of EthA on epithelial ion transport such as the stria vascularis. ill
situ.

The earliest work on the site of action of EthA in the kidney was centered around
its effect on the membrane Na+-K
ATPase. Duggan and No11 (1965) observed the
inhibitory effect of EthA and ouabain on ATPase isolated from guinea-pig kidney. Ouabain produced 50% inhibition at a concentration of 2 x 10m4~. However, inhibition
produced by EthA increased over a period of 1 hr and 5 x low4 M EthA produced
59% inhibition without preincubation and 75% with 1 hr preincubation. This phenomenon of increased potency of EthA with preincubation has been observed in most
preparations in which the effect of preincubation has been studied. Because of this
effect it is difficult to compare the degree of inhibition produced by ouabain and EthA.
Hook and Williamson (1965) gave rats oral doses of 25 mg/kg EthA, collected the
urine and subsequently removed their kidneys for Na-K+ ATPase assay. They found
no diuresis in these animals but a depression of the Na+-K+ ATPase activity thus
questioning the practice of assaying Na+-K ATPase in tlitro and extrapolating to the
effect of the drug in vivo.
Nechay et al. (1967) observed that EthA inhibited the isolated Na-K * ATPase
in dogs and that it was a relatively weak inhibitor when compared to ouabain. In
dogs pretreated with EthA the binding of the drug to the membrane fraction is lOOO2000-fold less than the binding required for 50% inhibition of the enzyme. These results
together with Hooks (1965) challenge the idea that inhibition of renal reabsorption
of Na is produced by inhibition of the Na+-K+ ATPase.
Davis (1970) observed that EthA decreased the activity of both Na+-K
ATPase
and Mgf ATPase nonselectively. Inhibition by EthA was nearly two orders of magnitude less than that obtained with ouabain. Cysteine or the sulfhydryl protecting agent
POMB added to the incubation media partially blocked ATPase inhibition of EthA
indicating that EthA is specific for sulfhydryl groups.
There is a large body of evidence that EthA is not a specific inhibitor of ATPase
but inhibits mitochondrial respiration and glycolysis. Determining whether EthA inhibits
glycolysis or mitochondrial respiration is complicated by the fact that energy-dependent
transport of monovalent cations across the cells membrane influences the rate of energy
generation in intact cells. In erythrocytes glycolysis is stimulated (Whittam and Ager.
1965; Parker and Hoffman, 1967); in other tissues mitochondrial respiration is stimulated as well (Whittam, 1964; Gordon et al.. 1967). Therefore to obtain evidence that
EthA inhibits glycolysis or mitochondrial respiration it must be demonstrated that the
effects are separate from inhibition of ion transport.
Gordon (1968) has demonstrated that EthA interfered with mitochondrial respiratory
control in the intact cells and confirmed this observation in isolated mitochondria
from Ehrlich ascites tumour cells. Gordon and Hartog (1969) demonstrated that EthA,
in contrast to ouabain, inhibits glycolysis in Ehrlich ascites tumour cells in the absence

PRODUCTIONAND ROLEOF IVNER EAR FLUID

351

of cell membrane K+ transport. In studies with ghost free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumour cells, EthA significantly
blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory
effect of this agent on one or more enzymes of the Embden Meyerhof pathway.
Klahr et 01. (1971 confirmed Gordons results by showing that EthA decreased lactate
formation by approximately 50%. Inhibition by EthA was abolished in hemolysates
by addition of dithiothreitol, a sulfhydryl protector agent and the results indicated that
EthA inhibits lactate formation from fructose-1,6-diphosphate and glucose-6-phosphate
directly and independently of their effects on cation transport in a variety of tissues.
Preincubation for 30min markedly increased inhibition of glycolysis by EthA.
It seems from the above evidence from single cell and cell-free preparations that
EthA is a rather blunt tool for the investigation of ion transport processes. There are,
however, examples of its action in some preparations that involve more the whole organ
than cell-free preparation in which EthA does seem specific for a particular ion transport
system. The work of Hook and Williamson (1965) Nechay et al. (1967) and Ebel et
al. (1972) suggest that there are large differences in the activity of EthA in citro and
irl do
in the kidney.
Whittembury (1968) Whittembury and Fishman (1969) Whittembury and Proverbio
(1970) and Proverbio et al. (1970) observed two modes of sodium extrusion from sodiumloaded kidney cortex slices; one that is accompanied by net chloride efIlux and that
is inhibited by 2 mM EthA but not by 1 or 10 mM ouabain, and the second in which
one K+ is taken up for each Naf extruded and which is inhibited by ouabain and
not by EthA. Both modes of sodium extrusion are inhibited by DNP and anoxia and
seem to be of equal magnitude. The level of EthA required to inhibit the Na+-K+
ATPase half maximally is 100 times greater than that of ouabain. The residual ATPase
activity in the absence of Na and K but in the presence of Mg+ is completely
insensitive to ouabain, but is nevertheless inhibited by high doses of EthA. Such nonspecificity has been confirmed by Davis (1970). Two pumps involved in Nat
extrusion from the kidney cortex cell have been proposed; one involves exchange for
external Kf and derives its energy from the Na+-Kf
ATPase, the other, which
should be most effective in cell volume regulation expels Na+ accompanied by Clwithout the involvement of Na+-Kf
ATPase. It is not certain if this second EthA
sensitive pump is a neutral NaCl pump or an electrogenic sodium pump with
accompanied passive Cl- movement. A decision between these two alternatives
would be possible if data on the effect of the pump on the cell membrane potential
were available.
These claims must be tempered with the observation in kidney slices of Epstein
(1972 a, b) that EthA inhibited active transport of Na+ -dependent and Na+ -independent
sugars, and reduced 0, uptake in both Na+ containing and Na+ free media indicating
that the decline was not merely reflective of diminished energy requirements of the
Nat pump. He also observed a rapid diminution of tissue ATP content during incubation of the slices with EthA and concluded that EthA exerted inhibitory effects on
several levels of cell metabolism and thus was not a specific inhibitor of individual
active transport processes. MacKnight (1969) also concluded that EthA inhibited metabolism in kidney cortex slices.
Hoffman and Kregnow (1966) showed that a large fraction of the remaining Na
efflux after maximal inhibitory doses of ouabain could be inhibited by EthA. Their
findings led them to conclude that it was an active Na transport and that it was
sensitive to external [Na+]. Lubowitz and Whittam (1968) confirmed Hoffman and
Kregnows findings that a large fraction of ouabain-insensitive Na+ efflux was Naf
dependent, but they regarded it as passive Nat exchange diffusion rather than active
transport. The same interpretation was proposed by Dunn (1970) on the basis of the
furosemide effect. Rettori and Lenoir (1972) found that the ouabain-insensitive Na+
efflux in red blood cells was inhibited by replacing Na+ with magnesium, K+ or Li
and not when replaced by choline. They concluded that the pump was inhibited by

352

P. M.

SELLKX

AYD

B.

M.

.IOHNSTONI

high external Mg+ + or K+ and not by the absence of external Na as was concluded
by Hoffman and Kregnow and hence cannot be explained as exchange diffusion.
The most compelling evidence to date that EthA selectively inhibits a Na pump
other than the Na+-K+ exchange pump iri cite is provided by Petersens (1970) work
on perfused cat submandibular gland. He measured salivary secretion during acetylcholine infusion and Kt uptake from the perfusion fluid after acetylcholine induced K
loss. He found that ouabain abolished K uptake. whereas salivary secretion was unaffected. EthA hardly affected K + uptake whereas salivary secretion was severely inhibited.
He suggests that two modes of Nat transport occur in the acinar cells of the salivary
glands; a Nat extrusion coupled with K uptake, responsible for the maintenance
of the concentration gradient across the cell membrane; and a Na transport coupled
with Cl- transport into the acinar lumen. responsible for the formation of the primary
secretion. This work could not, however, show whether it is an electrogenic Na pump
or an electroneutral NaCl pump.
In summary EthA has been shown to have effects on cellular metabolism, Na K
ATPase and Mg +-ATPase. There is evidence, however, that the drug has specific
action on NaCl transport irl rice although it is not known whether this pump is an
electrogenic Na pump with accompanying passive Cl- movement or an electroneutral
NaCl pump. There is no evidence as to its mode of action on the strial cells of the
cochlea.
3. I. DIFFERENTIAL
EFFECTSOF OUABAIN AND

ETHACRYNIC

Ac~v

Matz et al. (1969) reported a case of acute bilateral hearing loss in a patient after
an intravenous dose of 50 mg EthA. while vestibular tests consisting of 10 cm3 irrigation
with ice water in both ears gave a normal response bilaterally, suggesting that intravenous
doses of EthA affect the cochlea but not the vestibular system. This clinical finding
can be investigated by measuring EP, representing cochlear function, and UP (the utricle
potential), representing vestibular function. after an intravenous dose of EthA. During
this work it was discovered that EthA produced an anoxia-sensitive negative potential
in the cochlea and the utricle. Further investigation of this phenomenon revealed the
differences between the action of EthA and ouabain on the endolymphatic potentials
(Sellick and Johnstone, 1974).
Intravenous injection of 40-50 mg/kg EthA severely reduced EP leaving UP unaffected
(Fig. 9). This may explain Matz observation that the vestibular system was unaffected

60-

Time,

min

FIG. 9. The effect of intravenous

injection of EthA on the utricular potential and endocochlear
potential (EP). The respirator
was turned off and on as indicated at 10 min. 43 mg/kg EthA
was injected between A and B. At C the respirator
was turned off permanently
and the animal
died. Zero indicates removal of the electrodes from the utricle and Scala media and the final
zero point.

60-

;
2

40-

g
&
2
AZ
::
::
-z
w

zo-

$_

- Zero

O-20 -40
0

20

40

60
Time,

80
mln

I
100

I
120

FIG. IO. The effect of perfusion of EthA into the perilymphatic space on utricular potential
and cndocochlear potential. At (A) I mM EthA was perfused through the oval window. At (B)
the respirator was turned off permanently.

by intravenous doses of EthA which severely depressed cochlear sensitivity. The difference between the response of UP and EP to intravenous EthA could be due to the
fact that the stria vascularis is highly vascular while the utricular membrane is less
so (Smith, 1956, 1970) and intravenous EthA affects the more vascular structure. This
was confirmed by the observation that 1 x 10e3 M EthA perfused through the oval
window into the perilymphatic space reduced both EP and UP. Unlike the animals
that received an intravenous dose of EthA, animals in which EthA was perfused into
the perilymphatic space did not show recovery of EP to positive values up to X0min
after the perfusion, probably because EthA is not removed from the perilymph as it
is from plasma. However. both potentials reached maximum negative values and then
returned to lower negative values 90min after the start of the perfusion. The potentials
were not affected by anoxia at this stage (Fig. 10).
It was noticed that if animals were made anoxic when either UP (Fig. 10) or EP
(Fig. 9) were at their most negative values after application of EthA the potentials
were reduced rapidly towards zero. This phenomenon has also been reported by Thalmann et al. (1973) and represents a unique response to anoxia for these potentials.
It was further observed that the [Na],. in utricular endolymph did not change rapidly
during the positive going response indicating that the response was not due to very
rapid changes in the ion gradients and hence diffusion potentials (Fig. 11). It seems,
therefore, that a negative. anoxia-sensitive potential occurs in the cochlea and utricle
for a short time after the application of EthA. It is possible that this potential is produced
by the Na+-Kf
exchange pump which is revealed by the abolition of the positive
electrogenic potential for a short time after which it is itself abolished. If this is the
case then it would be expected that the potential would be abolished by ouabain. 2 x
10e3 M ouabain in Ringers solution perfused into the perilymphatic space reduced UP
and EP to negative values but in contrast to the EthA poisoned animals the maximum
negative potentials did not change during anoxia.
Since EthA may have produced anoxia-sensitive negative potentials in a way that
ouabain did not. both EthA and ouabain were perfused through the round window
in concentrations
of 2 x 10m3 M each. UP fell to maximum negative values of
- 39 ? 6 MV (mean and SE of three animals) and permanent anoxia produced a reduction
of these potentials in the positive direction by 4 i 1 mV. EP reached negative values
of - 18 + 9 mV and was reduced by 2 _
+ 1 mV during permanent anoxia. Thus the

354

P. M. SELLICKAND B. M.

JOHNSTONE

Zero

h
:
L

IO0

I
40
Time,

60

60

I
loo

min

FIG. 11. The effect of perfusion of 2 x low3 M EthA into the perilymph on the utricular endolymph Na+ and UP. Perfusion was started at (A) and the respirator was turned off permanently
at (B).

anoxia sensitive -EP and -UP produced by EthA were reduced by 82% and 55%, respectively, in the presence of ouabain indicating that the Na+-K+ pump either produces
the potential or is indirectly necessary for its production. If this is the case then the
magnitude of the potential may be used to predict the magnitude of Na+ transport
from Scala media. Since the average ion conductance of 1 mm of Scala media determined
from the relationship between K+ transport and the electrogenic potential is 0.14 x
lo-m~/min/mV/mm
then 13mV represents a current of 0.14 x lo- x 13 = 1.8 x
lo- mM/min. This coincides with a normal ma+], of about 25 mM from Fig. 5. If
the pump has a coupling ratio of 3Na: 2K then the actual Na flux would be 3 times
this since only one-third of the Na transported would be electrogenic. This would make
the total Na transported 5.4 x lo- mM/min/mm, still within the range of Na+ transport,
but coinciding with about 10Om~ [Na],. [Na], would be nowhere near this value
when the 13 mV anoxia sensitive potential was observed and this is an argument against
the coupling ratio of 3Na:2K. There is the possibility that EthA changes the ratio
between ma+]@ and intracellular Na+ thus making the relationship previously determined between [Na], and Na+ transport invalid. To test whether the source of the
negative anoxia sensitive potential could be stimulated by an increase in [Nalr, Scala
media was perfused with 50 mu Na+ Ringers in two animals immediately after EP
began to fall following an intravenous injection of 40 mu EthA. The animals were made
anoxic after EP had reached its maximal negative value in order to measure the anoxiasensitive component of the potential. The magnitude of the anoxia-sensitive component
did not differ significantly from that found in unperfused animals and it was concluded
that the source of the potential could not be activated further with high [Na+le.
A similar negative, anoxia-sensitive potential of about 4 mV has been observed in
the saccule after destruction of the first turn of the cochlea (Sellick and Johnstone,
1972b). Attempts to investigate the saccular potential further were unsuccessful because
observation of the potential required removal of the positive potential due to leakage
from the first turn of the cochlea. This entailed destruction of the first turn by running
a needle along the basilar membrane, a procedure that was difficult to reproduce. The
positive potential from the cochlea could be abolished with EthA, however, this destroyed the aim of investigating the potential without using EthA. There is some evidence
that the negative anoxia sensitive potential in the saccule is activated by [Na+], Similar
results were obtained in three animals but one especially could be analysed in detail.
The saccular potential became more negative as the ma+], increased after the first

355

PRODUCTION AND ROLE OF INNER EAR FLLTD

20No+
cont.
mEq/l

IO-

IO

15

20

25

30

35

40

45

50

Time,
FIG. 12. Upper:
turned on at 7
reduction of the
ment

I
55

I
60

min

the effect of transient

anoxia on the saccular potential.
The respirator
was
min. Damage to the first turn of the cochlea at 16 min caused a sudden
potential to - 17mV. Permanent
anoxia at 35min produced
a positive moveof the potential of 5 mV. Lo~rr: the [Na]
in saccular endolymph.

turn was damaged, and the initial potential after damage was equal to the potential
after asphyxia, indicating that the anoxia-sensitive potential was near zero when the
[Na], was at its normal value and increases as the [Na], increases @ig. 12). Therefore
assuming that the K+ diffusion potential does not change to a significant extent, the
line joining the initial potential after first turn damage and the potential after anoxia
can be taken as the baseline indicating zero anoxia-sensitive potential. The plot of
the anoxia-sensitive potential and the [Na+], is linear and intercepts the x-axis at
4.2 mM Na+ with a slope of 0.8 mV/mM Na (Fig. 13). It would be expected that there
would be agreement between the activation of Na+ transport from cochlear endolymph
and activation of the saccule potential by sodium if the Na transport mechanisms
are the same in the stria vascularis and the saccular membrane.

Saccular

endolymnh

[Na+],

mEq/r

FIG. 13. Plot of the saccular endolymph

Na and the saccular anoxia sensitive potential after
the first turn of the cochlea was damaged, taken from the data represented
in Fig. 12.

356

P. M.

SFLLICK

ANI)

B.

M.

JOHNSTONI

The access resistance of the saccule has been measured to be between 16 and 40 kohms
(Sellick and Johnstone, 1972 b); therefore, 0.8 mV represents a current of between 0.05
and 0.02 PA or 0.3 to 0.12 x lo- mM/minimm Na+. Activation of Na transport from
Scala media by endolymph Na + is 0.09 x 10~ mM?imin/mM Na If Na transport is
coupled to K + transport with a ratio of 3:2 then 0.09 x 10 _ must be divided
by 3 sinceonly one-third of the Na+ extruded is extruded electrogenically thus reducing
the agreement between the slopes of the activation curves and indicating that it is
more likely that Na+ is transported purely electrogenically. i.e. that it is not coupled
to K+ transport. It is difficult to assess the error in these calculations and therefore
they are not to be taken to be conclusive but rather as a rough assessment of the
agreement between the data.

4. Model of the Ion Transport Mechanisms of the

Secretory Cells of the Stria Vascularis
It is possible to propose a model of the ion-secretory processes in the strial cells
from the available experimental evidence. Since the properties of this system are not
as well characterized as other epithelial transport systems, proposed models are bound
to be highly speculative. Therefore, as with all models, the one proposed here is intended
not as a confirmed reality but rather as a catalyst for new experimental work. There
is one important simplification that must be made, and that is that ion transport between
endolymph and extracellular fluid or blood plasma be carried out by a single layer
of cells, i.e. the border cells of the stria that have the endolymph at their mucosal
borders. This simplification may nqt be warranted but it is forced upon us by the
complexity of the stria vascularis. It would be meaningless to propose a model that
includes the function of the basal, intermediate and border cells as described by Echandia
and Burgos (1965) with the current experimental data.
The model will attempt to take into account the following facts:
1. Electrochemical
gradients between endolymph and perilymph
(a) K+, Na+ and Cl- not found at their equilibrium concentrations in cochlear endolymph.
(b) EP of + 80 mV made up of approximately an electrogenic component of + 100 mV
and a K+ dominated diffusion potential of about - 20 mV.
(c) Active transport of K proportional to the magnitude of the electrogenic component of EP (Fig. 2).
(d) Na+ activation of Na transport (Fig. 5).
2. Eflects

of ouabain

(a) Reduces EP while stimulating Nat transport.

(b) More effective in reducing EP when applied to perilymph
applied to Scala media.
3. Effects

or blood than when

c?fEthA

(a) Reduces EP when applied intravenously or in perilymph.

(b) Produces transient negative anoxia-sensitive potential in utricle, cochlea and saccule that is reduced by ouabain. The magnitude of the potential cannot be increased by an increase in endolymph Na' .
4. Anoxia-sensitive negative potential has been observed in the saccule which appears
to be related to the saccular endolymph Na+, after destruction of the first turn
of the cochlea.
The existence of the following pumps are proposed.

PKOIX

CTION

AND

ROLI

OF

INNt

K EAK

FI.III)

157

Endolymph
[No] < ImEq/l
[K+ ] = MOmEq/l
[Cl-]
z 140mEqA
K+

EP=+

8OmV

I?

[Na+]<

ImEq /

[K+]=

I4OmEq/l?

[Cl-]

=50mEq/l

NaCl

3Na+

Perilym h or extracellular
[Nat! = IMOmEq/l
[K+] = 5mEqll
[Cl-] = 140mEq/l

FIG. 14. Proposed

model

for the transport

system

of the

fluid

border cells of the stria vascularis.

I. An electrogenic
K+ pump located on the mucosal side of the strial border cell
and transporting K into endolymph (Fig. 14). It seems that the mucosal border of
the secretory cells would provide most of the resistance to current flow between endolymph and extracellular fluid because it is unconvoluted, unlike the serosal barrier.
This therefore is the most efficient site for the pump as far as developing the maximal
potential in Scala media. If the pump was located on the serosal side a potential fall
would occur across the mucosal surface and potential produced by the pump would
be shunted by the low resistance of the convoluted serosal surface. There is evidence
that such a pump exists in silkworm midgut since a positive potential of about 100 mV
is produced in the lumen of the midgut, the potential being dependent on serosal K+.
independent of Na+ and inhibited by metabolic poisons and anoxia (Harvey et ul..
1968; Nedergaard and Harvey, 1968).
It would be expected that the K+ pump would produce an anoxia-sensitive hyperpolarization of the border cell since it would transport positive charge out of the cell.
Electrode penetrations of the stria from the serosal side never show a negative potential,
only vague positive steps until EP is recorded. Attempts to penetrate the cells from
the mucosal side have been largely unsuccessful; however, some negative potentials
of about - 30 mV were recorded but were not stable enough to observe their behavior
during anoxia (Johnstone, unpublished). If the border cell was hyperpolarized below
E, this would produce a gradient for K into the cell from the extracellular fluid.
thus supplying the K+ pump with K+.
Konishi and Kelsey (1973) have demonstrated that EP can be reversibly reduced
by perfusing the perilymph with K+-free solution. the inference being that the electrogenie K+ pump is deprived of K on the serosal side of the border cell. We have
performed a similar experiment observing UP instead of EP since unlike the serosal
side of the stria the serosal side of the secretory cells in the wall of the utricle are
exposed to perilymph. We found no reproducible change in UP when the [K] was
reduced by perfusion. This is not necessarily in conflict with Konishi and Kelseys finding
since the strial pumps are far more active than the utricular pumps and would therefore
be more sensitive to a reduction of K+.
It is envisaged that the K+ pump would be directly inhibited by ouabain and not
indirectly via the Na+-K ATPase. Since ouabain is known to act on the K+ receptor
site of the Na+-K+ pump its action on the K+ pump would have to be intracellular,

358

P. M. SELLICK AND B. M. JOHVSIDNI~

the larger sensitivity to serosal ouabain being explained by the easier access to the
site across the large area of the serosal surface of the border cell. The electrogenic
K+ pump in silkworm midgut has been shown not to be inhibited by ouabain (Haskell
et al., 1965).
The effect of EthA on the K pump could be via metabolic inhibition or specific
inhibition of the ion pump. Recently Kusakeri and Thalmann (1973) and Thalmann (J[
al. (1973) have demonstrated that the levels of high-energy phosphates in the stria are
not reduced by EthA poisoning whereas they are reduced by ischemia and metabolic
poisons. They observed that if EthA intoxicated ears are subjected to ischemia the
levels of ATP and P-creatine are maintained much longer than in untreated controls.
They suggest that the early effects of EthA are not caused by an impairment of energy
production or by changes in permeability, but rather are due to an interference with
energy utilization, most likely by inhibition of the energy-consuming processes mediated
by the Na+-Kf ATPase system. If the Kf pump and the Na+-K- pump are indeed
separate entities then this would not be expected to suffice for an explanation as to
why EthA inhibits the production of EP. It is more likely that EthA inhibits the K
pump directly.
2. A Naf-K+
exchange pump relying on Na+-K+ ATPase for its energy supply
and located on the serosal side of the strial border cell. This pump is initially stimulated
and then inhibited by ouabain and is inhibited by EthA after preincubation. It would
transport K + into and Na + out of the border cell and hence endolymph since there
would be a large gradient for Na+ into the border cell from endolymph especially
when [Na+],, is raised by perfusion. It is postulated that its Na arm is electrogenic
and that this electrogenicity is revealed for a short time after the application of EthA.
It is interesting to note that the potency of EthA in inhibiting Naf-Kf
ATPase increases
with incubation (Duggan and Noll. 1965). The abolition of this potential in time may
be associated with the inhibition of the Na+-K ATPase in time by EthA. The pump
would have the same disadvantages as regards producing potential in Scala media as
has been mentioned for the KC pump; however, the large membrane area of the serosal
side may allow a large number of active sites so that this may not be such a disadvantage.
In a previous publication Sellick and Bock (1974) proposed that an electroneutral
K+ component occurs when Scala media is perfused with high Na solutions and that
this could be the result of the Na-K
pump. This conclusion was based on active
K+ transport vs electrogenic potential curves using the value of G, calculated from
the assumption that active K + influx was equal to passive efflux. However, if the measured value of GK is used the curves come very close to intersecting the origin (Fig.
2) thus indicating that there is no electroneutral component. The measured value of
G, was used in this presentation because the calculated value of CC forces Gy., to
be 3 times its measured value. When this is applied to the Na+ efflux data, the Na
transport vs [Na], has a negative slope at low [Na],,. It was felt that this was
a very unlikely circumstance and that the measured values of GK and G&,,were probably
more accurate even though it was impossible to reconcile them with the calculated
resistance of 1 mm of Scala media. The fact that an electroneutral Kt influx is not
observed during the low K high Na+ perfusions does not necessarily count against
the postulate that Na is removed from endolymph by a Nat-K+
pump since, as was
proposed by Ussing in his original model for frog skin, the K exchange for Nat
at the serosal border of the cell need not reach endolymph but may diffuse back into
intracellular fluid.
It is interesting to note that a similar negative electrogenic pump exists in the saccule
in the absence and presence of EthA. Since the saccular membrane is composed of
mostly simple cuboidal epithelium, similar to that found in Reissners membrane, it
may be that these cells produce the negative potential. Similar cells are also found
in the utricular membrane along with secretory type cells similar to those found in
the stria vascularis.

PRODICTIONAND ROLL OF INMR EAK FLC ID

359

3. NaCl pump. As has been mentioned in Section 2, Cl- will tend to diffuse down
its electrochemical gradient into Scala media. In other words. it will tend to follow
K+ into endolymph. It is expected that the Cl- conductance is small since passive
Cl- movement into cochlear endolymph would short circuit the electrogenic K pump
and this would be observed as an electroneutral component in the K transport data
in Fig. 3. Unfortunately we are not sure enough of the value of G, to say whether
such a component exists. However, the Cl- conductance is expected to be finite and
hence there will be passive movement of Cl- into cochlear endolymph. It will be remembered that this is so only for cochlear endolymph since Cl- is at equilibrium in the
saccule and utricle.
There are two alternative explanations as to why Cl- is not at equilibrium in cochlear
endolymph. The first is that the hydrolic conductivity of the walls of Scala media are
low and passive Cl- influx is immediately balanced by water influx. The problem with
this explanation is that endolymph would be produced in volume and since the membraneous labyrinth is a closed system and is also incapable of sustaining large pressure
differences it is difficult to see where the extra cndolymph volume would escape. It
is conceivable that volume flow could occur across the endolymphatic sac; however.
surgical blockage produces endolymphatic hydrops only over a period of 3-4 weeks
in guinea pigs (Shea. 1974). Nevertheless this alternative cannot be completely abandoned.
The second alternative is that an electroneutral NaCl pump removes NaCl from
endolymph. If Gc, were half Gh.] then since the driving force for Cl- is twice that
of Na+ the passive fluxes of Cl- and Na would be equal and could be dealt with
by a NaCl pump with a coupling ratio of 1: I.
It is not envisaged that this pump would be responsible for the bulk of Na+ transport
out of Scala media after perfusion with high Naf Ringers since we found no detectable
change in the [Cl-],, after perfusion with normal Ringers (Na, 1.50 mM) in two animals.
However, this is not entirely conclusive since movement of water out of endolymph
during NaCl transport could have kept the [Cl-], constant. Further experiments are
needed in this area to determine the hydrolic conductivity, the Cl- conductance and
to look for the presence of active Cl- transport.
There is the possibility that a NaCl pump is the site of action of EthA since specificity
for NaCl transport over Na+-K transport has been demonstrated in kidney (Whittembury, 1968; Whittembury and Fishman. 1969; Whittembury and Proverbio. 1970: Giebisch et ul., 1971).
Mullins and Noda (1963) have derived a simple equation describing the membrane
potential to which there is a diffusion and an electrogenic component. The equation
takes the form of the constant field equation and the assumptions are that active Naand Kf fluxes are equal to passive fluxes, i.e. the equation applies only to the steady
state condition. The derivation of the equation is given by Mullens and No& (1963)
and also by Thomas (1972)

where R, T and F have their usual meaning. Py, and P, are the permeabilities of
Na+ and K+. respectively. and the subscript i and o denote inside and outside concentrations respectively; I is the coupling ratio or number of Na+ ions pumped out
per K+ pumped in.
The equation was originally intended to describe the membrane potential of a single
cell which pumped more Na+ out than K + in and thus I was larger than one and
the membrane potential was hyperpolarized in the negative direction. The electrogenic
potential produced tends toward the value of the Kt diffusion potential in the steady
state and can only be larger than this when nonequilibrium conditions occur. i.e. when
Na is injected into the cell.

360

P. M.

SELLICK

ANI)

B.

M. JOHNSTOUt.

The equation should apply to the electrogenic potential found in Scala media
except that r will be very much less than one since more K + is pumped into endolymph
than Na+ out. In the steady state the potential will not exceed the Na equilibrium
potential, EN.,. A rough calculation of the value of r can be made as follows. A ratio
of P,: P,, of 1: 0.35 predicts a K dominated diffusion potential of -26 mV in the
absence of electrogenic potentials and is approximately the value of EP soon after
the death of the animal. Using a value for EP of +XOmV a value for I of 0.015
can be calculated, i.e. in order to produce a potential in Scala media of + 80 mV superimposed on the K diffusion potential of -26 mV the stria has to transport K into
Scala media at a rate 66 times the rate that Na is transported out of Scala media.
The above considerations assume that a passive Cl- diffusion potential or an electrogcnic Cl- pump do not contribute to the potential. Since the resting K current. when
EP is + 80 mV (electrogenic potential of 106 mV). is 1.4 I 0.36 x IO mM/min, mm.
from Fig. 3 then the resting Na efflux would be expected to be 1.4 x 0.015 x IO ( =
0.02 x 10ph mM/min/mm. From Fig. 5 it can be seen, if the curve is extrapolated
through zero, that the expected Na transport when [Na+],, = 1 mM is 0.015 x IO -.
i.e. in fairly close agreement with the calculated rate. These calculations serve as a
useful check on the measured fluxes of Na+ and K~.
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