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 [K’J;, [Na’J 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,+ Ga‘EC,

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

0

Perfuse n

6

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

I

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

I

343

ctlve

K+Transport

x 166mmoles/min/mm

Scala Medlo

(b)

0,

G

-20

Active

K+ Transport

x 10M6mmoles/min/mm

Scala Media

-40 t

Kt Perfusate

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

potential

vs active

K’

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 [K’J 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

0

6

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

I

I

20

30

1 40

I

I

I

I

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’

1

002

’

1

’

1 ”

004006

008

”

IO

”

12

”

14

” I.6 ”

16

’

“1

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

6

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 Deiter’s 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 Hook’s (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 cell’s 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 Gordon’s 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 Kregnow’s 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 Petersen’s (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.

0

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

1

h : ‘L

s

IO0

I

m

I 40 Time,

I

I

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 [Na’lr, 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-

0

I

5

I

I

IO

15

I

20

I

25

I

I

I

I

I

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 since’only 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?f‘EthA

(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.I’II)

157

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

EP=+

8OmV

I?

[Na+]