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Cellular localization of Na+, K+-ATPase in the mammalian cochlear duct: Significance for cochlear fluid balance
Am J Otolaryngol 3:332--338, 1982
Cellular Localization of Na+,K+-ATPase in the Mammalian Cochlear Duct Significance for Cochlear Fluid Balance THOMASP. KERR, B.A.,* MURIELD. Ross, PH.D.t AND STEPHENA. ERNST, PH.DA" Cytochemical and autoradiographic procedures were employed to determine the cellular distribution of Na+,K+-ATPase in the guinea pig cochlea. The highest activity was associated with the stria vascularis and was restricted almost entirely to the contraluminal extensions of the marginal cells. Elevated levels of activity were also observed in stromal cells of the spiral prominence, external sulcus, and spiral limbus. The pattern of activity in the latter tissues was unusual in being symmetrically distributed along the plasma membranes of the reactive cells. In the organ of Corti, only neural elements showed appreciable activity. Possible functions of the enzyme are discussed in relation to cochlear fluid balance. (Key words: Cochlea; Na+,K+-ATPase; Ouabain.)
The e x p a n s i o n of e n d o l y m p h v o l u m e associated with M~ni~re's disease m a y represent the net effect of several pathogenetic factors. These could include defects in the permeability of m e m b r a n e s b o u n d i n g the e n d o l y m p h a t i c spaces, together w i t h excessive formation or insufficient resorption of e n d o l y m p h . Of the various processes relating specifically to the f o r m a t i o n of e n d o l y m p h , none are of greater significance t h a n those responsible for its u n i q u e ionic composition. The h i g h potassium and low s o d i u m concentrations of m a m m a l i a n e n d o l y m p h , first reported by Smith et al., 1 are k n o w n to be supported by active transport with expenditure of metabolic energy. ~ The enzyme Na+,K+-ATPase has received consideration as a possible transport m e c h a n i s m for maintenance of cochlear cation gradients since the first conclusive b i o c h e m i c a l studies by Kuijpers and Bonting 3 a n d by Matschinsky and Thalmann 4 demonstrated its intense activity in the stria vascularis. In addition, the well-established func-
tion of the transport ATPase in sustaining low i n t r a c e l l u l a r s o d i u m c o n c e n t r a t i o n s bears a corollary linking the enzyme to maintenance of an appropriate water balance between intracellular a n d e x t r a c e l l u l a r f l u i d c o m p a r t m e n t s . Thus, homeostatic regulation of cellular volume is accomplished in part by removal of water, which passively follows sodium ion extruded from the cell by the enzyme. ~,6 A final aspect pertaining to the process of endolymph formation concerns the involvement of the enzyme in transport of water between extracellular fluid compartments. In tissues noted for salt-coupled transepithe]{al water transport, Na+,K+-ATPase is g e n e r a l l y r e g a r d e d as the p r i m a r y active t r a n s p o r t m e c h a n i s m t h a t p o w e r s the fluid flux.7-1o METHODS FOR LOCALIZATION OF ION PUMP SITES
From the University of Michigan Medical School, Medical Sciences II, Ann Arbor, Michigan 48109. Received May 14, 1982. Accepted for publication May 24, 1982. Supported by N A S A NSG 9047 and NIH AM 27559. Presented at the Midwinter Research Meeting of the Association for Research in Otolaryngology,January 18-21, 1982, St. Petersburg Beach, Florida. * Doctoral Candidate, Program in PhysiologicalAcoustics. ~rDepartment of Anatomy and Cell Biology. Address correspondence and reprint requests to Dr. Ernst. 0196-07091821090010332
Progress in the area of cochlear fluid balance will depend increasingly u p o n observations at the cellular level. The information still needed includes measurements of intracellular resting potentials and ionic activities, determinations of ionic permeabilities in various cell membranes, and cellular localization of the ion pump sites. The exact distribution of Na+,K+-ATPase among cochlear cells and tissues w o u l d therefore seem to h o l d c o n s i d e r a b l e i n t e r e s t , n o t o n l y for
$01.40 Q W. B. Saunders Co. 332
KERR ET AL.
Figure 1. A, light photomicrographic survey showing the distribution of autoradiographic silver grains in the outer wall of the cochlea after incubation with '~H-ouabain. Dense clusters of silver grains cover the basolateral compartments of marginal cells in the stria vaseularis (SV), while the apical cytoplasm is relatively free of label (see Fig. 2). Ouabain binding sites are also associated with cells in the spiral prominence (SP) and external sulcus (see Fig. 3). Labeled cells extend below the level of the basilar membrane. R: Reissner's membrane. Brightfield, methylene blue. Second cochlear turn. B, this photomicrograph shows, at higher magnification, the distribution of auabain-binding sites in the region of the spiral prominence. Na+-K + pump sites are associated with cells of the prominence, as well as with the strial marginal cells at the upper left. Phase-contrast, second cochlear turn. C, this photomicrograph shows labeling of stromal cells in the body of the spiral limbus. Phase-contrast, second cochlear turn.
Volume 3 Number 5 September 1982 333
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American Journal of Otolaryngology 334
KERRET AL. Figure 2. Electron photomicrograph showing the distribution of K+-dependent phosphatase activity iu the stria vascularis. Electron-dense deposits of reaction product outlinethe extensions of the marginal cells near the center of the field. The luminal surface of the marginal cells, bordering the endolymph (EL),is devoid of reaction product. Little or no reaction can be seen in basal cells (B). intermediate cells (*), ar capillaries (C), or in the spiral ligament {SL). Second cochlear turn. -~[
clarifying the role of the enzyme in normal cochlear function, but also for its possible relevance to the alteration of fluid balance occurring in M~ni6re's disease. R e c e n t l y d e v e l o p e d cytochemical methods can provide information on this subject, and have been used both to indicate which cell types in various ion-transporting epithelia contain the highest density of Na+-K+ pump sites and to show that in certain types of cells the p u m p s may be asymmetrically distributed to a particular region of the plasma membrane.1', '2 O n e of t h e s e t e c h n i q u e s , originated by Ernst, '3''4 detects inorganic phosphate released from an artificial substrate (nitrophenyI phosphate) by the catalytic activity of transport ATPase. In the presence of strontium ion, phosphate is precipitated at regions of high activity, then converted to an electron-dense product, which may finally be visualized in the electron m i c r o s c o p e . A s e c o n d m e t h o d , d e v i s e d by Stirling, is depends upon the well-documented ability of a specific sodium-pump inhibitor, ouabain, to bind selectively to Na+,K+-ATPase, thereby blocking catalytic activity. In this procedure, tissues are incubated with ~H-ouabain, and regions with high concentrations of Na+-K+ pump sites are subsequently identified by light microscope autoradiography. Since these techniques localize the same enzyme by different principles, we employed both methods concurrently for cross-validation of results. The distribution of activity described below was further substantiated by control experiments appropriate to the respective techniques. To assure the specificity of phosphatase reaction product observed b y electron microscopy, we verified that the reaction was K+-dependent and ouabain-inhibitable, and that the product was deposited on the cytoplasmic surface of cell plasma membranes. These criteria are compatible with the biochemical characterization established for the phosphatase component of the Na*,K+-ATPase enzyme complex, u,16 To demonstrate the specificity of the autoradiographic method, we carried out quantitative assays of 3H-ouabain u s i n g liquid scintillation spectrometry. In segments of outer wall incubated with 5 ~M aH-ouabain, dilution with 5 mM unlabeled ouabain produced a decrement of 100 per cent in tissue-bound label, suggesting the
,,m
saturable nature of the binding. Omission of Mg ++, a cofactor necessary for binding of ouabain to Na+,K+-ATPase in vitro, led to an 85 per cent reduction in tissue labeling. Moreover, the number of binding sites, normalized to tissue dry weight, increased from the apical to the basal turn, consistent with the earlier biochemical assays of enzymatic activity by Kuijpers and Bonting2 Scatchard analysis of ouabain/enzyme interaction at ouabain concentrations as high as 5/,M was consistent with a single population of receptors having a dissociation constant of approximately 2/,M. The following account summarizes our observations on the cellular localization of the enzyme in cochlear structures, particularly those with p o s s i b l e functions in e n d o l y m p h a t i c homeostasis. CELLULAR DISTRIBUTION OF COCHLEAR CATION PUMP SITES
In the outer wall of the guinea pig cochlea, both methods show highest Na+,K+-ATPase activity in the stria vascularis (Figs. 1 and 2), with intermediate levels in cells of the external sulcus and spiral prominence (Figs. 1 and 3). While these observations are consistent with the biochemical data of Kuijpers and Bonting, a the present study also extends their findings by the unexpected demonstration of activity in the spiral lirnbus (Fig, 1), and by showing that activity in the organ of Corti appears to be associated primarily with nerve fibers. Thus, elevated levels of the enzyme were localized to the synaptic regions below the inner and outer hair cells, to the vicinity of the inner and outer spiral bundles, to unmyelinated fibers crossing the tunnel, to the terminal node of Ranvier in the habenula perforata, and to the myelinated segments of fibers coursing through the osseous spiral lamina. Very little activity was found in sensory or supporting cells of the organ of Corti or in Reissner's membrane. There was, however, some suggestion of increased activity in the membrane near its attachment to the limbus. At the ultrastructural level, almost all reaction product in the stria vascularis was restricted to the contraluminal extensions of the marginal cells (Fig. 2), with little reaction on the luminal surface. The distribution of activity in the spiral
Volume 3 Number 5 September 1982
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KERRET AL. Figure 3. Electronphotomicrographshowing the distributionof K+-dependentphosphataseactivityin the spiral prominence. At the center of the field and at lower right, reaction product outlines the extensions of a stromal cell (S). Notice that the reaction product is distributed to the cytoplasmicmembrane surfaces. Little or no activitycan be seen in the prominence epithelial cell (PE). in the root cell (R},or in the capillary (C). EL: endolymph.Second cochlearturn. prominence, external sulcus, and spiral limbus was remarkable in being limited ahnost wholly to the stromal cells (Fig. 3). Although the reactive cells display an extensive network of tendrils with high mitochondrial density, they have no apparent communication with the lumen of the cochlear duct. Instead, the extensions of these cells seem c o n f i n e d to the connective tissue matrix and pericapillary spaces beneath the epithelial surface. The reaction product was distributed quite evenly along their plasma membranes, rather than being polarized to one particular surface. This pattern of activity stands in contradistinction to that observed in the stria and in other tissues implicated in transport of cations from one extracellular fluid compartment to another. POSSIBLE SIGNIFICANCE FOR COCHLEAR FLUID BALANCE
Stria Vascularis The cytoarchitecture of the strial marginal cells closely resembles that seen in many other ion-transporting epithelia; '7 moreover, the ultrastructural d i s t r i b u t i o n of Na+,K+-ATPase along the basolateral infoldings of these cells is virtually identical to the molecular organization found in several other tissues--both secretory and resorptive in function--where the enzyme is the only mechanism yet demonstrated to energize transepithelial ion transport2 In keeping with a major role i n cochlear ion transport, in vitro measurements by Marcus et al. is indicate that Na:~,K+-ATPase activity accounts for nearly half the oxygen consumption of the stria vascularis, a tissue long known for its extremely high respiratory rate. 19 Localization of most strial transport ATPase to the contraluminal aspect of the marginal cells is consistent with the view that the enzyme participates in the transport of potassium ion toward the endolymph. The pump sites are favorably situated to mediate uptake of potassium from the strial interstitial spaces, concentrating K+ within the marginal cell for eventual extrusion across the luminal membrane. Similar proposals were advanced by Johnstone and Sellick 29and by Kusakari and Thalmann2~; various data compatible with such a mechanism are cited by Ross et al. in this issue. 22 With the demonstration that most Na+-K + pump sites are distributed along
the marginal cell contraluminal membrane, the possibility arises that sodium ion from the adjacent strial interstitial spaces may contribute to marginal cell intracellular sodium, required at the cytoplasmic membrane surface for optimal enzymatic activity2~,24 and K+ uptake. It seems possible that the same mechanisms responsible for maintenance of the endolymphatic ion profile in the course of normal physiologic function may also play a part in regulating endolymph volume. The available evidence indicates that the osmotic pressures of endolymph and perilymph are normally equal (in young rats, at least25), and that the osmotic pressure of endolymph can be attributed almost entirely to its ionic constituents. 25'26 In addition, Bosher and Warren 24 provided evidence that certain structures bounding the endolymphatic compartment may be freely permeable to water. These results suggest that endolymph may gain or lose water to maintain osmotic equilibrium with surrounding extracellular fluids. Johnstone and Robertson27 pointed out that an inappropriate elevation in the rate of entry of ions into endolymph might favor an expansion of volume. Their calculations suggest that an increase in endolymphatic NaC1 or KC1 amounting to only 1 mM per hour could produce an osmotic pressure gradient sufficient to expand endolymphatic volume at rates comparable to those observed in guinea pigs with surgically induced hydrops. It should, however, be noted that an osmotic increase in endolymph formation may be of little consequence unless accompanied by other defects as, for example, in compensatory radial or longitudinal resorption (cf. Kimura, 2s Wilbrand and Stahle29).
Spiral Prominence, External Sulcus, and Spiral Limbus The distribution of transport ATPase observed in stromal cells of the spiral prominence and limbus represents, in our knowledge, a specialization unique to the cochlea. Since these cells do not appear to form a boundary between fluid compartments, they cannot be regarded as directly involved in transepithelial ion transport. Stromal tissues of the prominence and liraVolume 3 bus share certain structural features, including a Number 5 rich vascularization and extensive extracellular September 1 9 8 2 spaces, which may suggest some limited simi337 larity in function. Many investigators have be-
COCHLEAR TRANSPORT OF ATPase
lieved these structures to be involved in transport of ions and/or fluids (see discussions by Duvall s~ and Ishiyama et al.3'). Experiments with vital dyes "~ and electron-dense tracer particles "s,"4 indicated that the extracellular spaces of both tissues communicate with perilymph. The investigations of von Ilberg and colleagues ~s,34 demonstrated that the stromal cells, found in the present study to be possessed of significant transport ATPase activity, also show a vigorous pinocytotic uptake of tracer particles from the extracellular spaces. While further study will be necessary to determine the role of the reactive stromal cells, it is evident that they represent much more than simple connective tissue components.
American Journal of Otolaryngology 338
14.
15. 16. 17. 18. 19.
Acknowledgments. The authors thank Dr. Seth R. Hootman for many useful suggestions in the course of this research and in the preparation of the manuscript.
21.
References
22.
1. Smith CA, Lowry OH, Wu ML: The electrolytes of the labyrinthine fluids, Laryngoscope 64:141-153, 1954 2. Bosher SK, Warren RL: Observations on the electrochemistry of the cochlear endolymph of the rat: a quantitative study of its electrical potential and ionic composition as determined by means of flame spectrometry. Proc R Soc Lend B 171:227-247, 1988 3. Kuijpers W, Bonting SL: Studies on (Na+-K+)-actlvated ATPase XXIV. Localization and properties of ATPase in the inner ear of the guinea pig. Biochim Biophys Acta 173:477-485, 1969 4. Matschinsky FM, Thalmaun R: Energy metabolism of the cochlear duct, in: Paparella M (add: Biochemical Mechanisms in Hearing and Deafness. Springfield, II1., Charles C. Thomas, 1970, pp 265-288 5. MacKnight ADC, Leaf A: Regulation of cellular volume. Physiol Roy 57:510-573, 1977 6. Hoffman EK: Control of cell volume, in" Gupta BL, Moreton RB, Oschman JL, et al (eds.): Transport of Ions and Water in Animals. New York, Academic Press, 1977, pp 285-332 7. van Os CH, Slegers JFG: Correlation between (Na+,K+)activated ATPase activities and the rate of isotonic fluid transport of gallbladder epithelium. Biochim Biophys Acta 241:89-96, 1971 8. Fromter E: Solute and water transport across epithelia, in: Vosteen K-H, Schuknecht H, Pfaltz CR, et al (eds.): Meniere's Disease. New York, Thieme-Stratton, 1981, pp 23-31 9. Ernst SA, Riddle CV, Karnaky KJ Jr: Relationship between localization of Na+,K+-ATPase, cellular fine structure, and reabsorptive and secretory electrolyte transport. Curr Top Membranes Transport 13:355385, 1979 10. DiamondJW: Standing-gradientmodel of fluid transport in epithelia. Fed Proc 30:6-13, 1971 11. Ernst SA, Hootman SR: Microscopical methods for the localization of Na+,K+-ATPase. Histochem J 13:397418, 1981 12. Ernst SA, Mills JW: Autoradiographic localization of tritiated ouabaimsensitive sodium pump sites in ion transporting epithelia. J Histochem Cytochem 28:72-77, 1980 13. Ernst SA: Transport adenosine triphosphatase cytochemistry. I, Biochemical characterization of a cyto-
20.
23. 24. 25.
26. 27.
28. 29.
30. 31.
32. 33.
34.
chemical medium for the ultrastructural localization of ouabain-sensitive, potassium-dependent phosphatase activity in the avian salt gland. J Histochem Cytochem 20:13-22, 1972 Ernst SA: Transport adenosine cytochemistry, If. Cytochemical localization of ouabain-sensitive, potassium-dependent phosphatase activity in the secretory epithelium of the avian salt gland. J Histochem Cytochem 20:23-38, 1972 Stirling CE: Radieautographic localization of sodium pump sites in rabbit intestine. J Cell Biol 53:704-714, 1972 Robinson JD, Flashner MS: The (Na§ Enzymatic and transport properties. Biochim Biophys Acta 549:145-176, 1979 Keynes RE): From frog skin to sheep rumen: a survey of transport of salts and water across multicellular structures. Q Rev Biophysics 2:177-281, 1969 Marcus OC, Thalmann R, Marcus NY: Respiratory rate and ATP content of stria vascularis of guinea pig in vitro. Laryngoscope 8B:1825-1835, 1978 Chou JTY, Rodgers K: Respiration of tissue lining the mammalian membranous labyrinth. J Laryngo] Otol 76:341--351, 1962 Johnstone BM, Sellick PM: The peripheral auditory apparatus. Q Rev Biophysics 5:1-57, 1972 Kusakari J, Thalmann R: Effects of anoxia and ethacrynic acid upon ampullar endolymphatic potential and upon high energy phosphates in ampullar wall. Laryngoscope 86:132-147, 1976 Ross MD, Ernst SA, Kerr TP: Possible functional roles of Na+,K+-ATPase and their relevance to M6niere's disease. Am J Otolaryngol 3:353-360, 1982 Sellick PM, Johnstone BM: Differential effects of ouabain and ethacrynic acid on the labyrinthine potentials. Pfluegers Arch 352:339-350, 1974 Schuurmans Stekhoven F, Bonting SL: Transport adenosine triphosphatases: properties and functions, Physiol Rev 61:1-76, 1981 Bosher SK, Warren RL: A study of the electrochemistry and osmotic relationships of the cochlear fluids in the neonatal rat at the time of the development of the endocochlear potential. J Physiol 212:739-761, 1971 Makimoto K, Takeda T, Silverstein H: Chemical composition in various compartments of inner ear fluid. Arch Oto-R_hino-Laryngol220:259-264, 1978 Johnstone BM, Robertson D: The physiology and biophysics of hydrops, in: Vosteen K-H, Schuknecht H, Pfaltz CR, et al (eds.]~ Maniare's Disease. New York, Thiome-Stratton, Inc., 1981, pp. 44-46. Kimura RS: Animal models of endelymphatic hydrops. Am J Otolaryngol Nov/Dec 1982 (in press) Wilbrand HF, Stahle J: Temporal bone in patients with Maniere's disease, In: Vosteen K-H, Schuknecht H, Pfaltz CR, Wersall J, Kimura RS, Morgonstern C, Juhn SK (eds.): Meniere's Disease. New York, ThiemeStratton, 1981, pp ~41-147 Duvall AJ: The ultrastructure of the external sulcus in the guinea pig cochlear duct. Laryngoscope 79:1-29, 1969 Ishiyama E, Keels EW, Weibel J: New anatomical aspects of the vasculo-epithelial zone of the spiral limbus in mammals. Acta Otolaryngol (Stockh) 70:319-326, 1970 Tonndorf J, Duvall AJ, Reneau JP: Permeability of intracochlear membranes to various vital stains. Ann Otol Rhenol Laryngol 71:801-841, 1962 llberg C yon: Elektronenmikroskopische untersuchungen uber diffusion u n d resorption yon thoriumdioxyd an der meerschweinchenschnecke. 3. Mitteilung: limhus spiralis. Arch Klin Exp OhrenNasen-Kehlkopfheilk 192:163-175, 1968 llberg C von, Spoendlin H, Vosteen KH: Die ultrastruktur und funktion des sulcus externus und der prominentia spiralis der meerschweinchenschnecke. Darstellung mittels thoretrast. Arch Kiln Exp Ohren-NasenKehlkopfheilk 192:124-136, 1968
Cellular Localization of Na+,K+-ATPase in the Mammalian Cochlear Duct Significance for Cochlear Fluid Balance THOMASP. KERR, B.A.,* MURIELD. Ross, PH.D.t AND STEPHENA. ERNST, PH.DA" Cytochemical and autoradiographic procedures were employed to determine the cellular distribution of Na+,K+-ATPase in the guinea pig cochlea. The highest activity was associated with the stria vascularis and was restricted almost entirely to the contraluminal extensions of the marginal cells. Elevated levels of activity were also observed in stromal cells of the spiral prominence, external sulcus, and spiral limbus. The pattern of activity in the latter tissues was unusual in being symmetrically distributed along the plasma membranes of the reactive cells. In the organ of Corti, only neural elements showed appreciable activity. Possible functions of the enzyme are discussed in relation to cochlear fluid balance. (Key words: Cochlea; Na+,K+-ATPase; Ouabain.)
The e x p a n s i o n of e n d o l y m p h v o l u m e associated with M~ni~re's disease m a y represent the net effect of several pathogenetic factors. These could include defects in the permeability of m e m b r a n e s b o u n d i n g the e n d o l y m p h a t i c spaces, together w i t h excessive formation or insufficient resorption of e n d o l y m p h . Of the various processes relating specifically to the f o r m a t i o n of e n d o l y m p h , none are of greater significance t h a n those responsible for its u n i q u e ionic composition. The h i g h potassium and low s o d i u m concentrations of m a m m a l i a n e n d o l y m p h , first reported by Smith et al., 1 are k n o w n to be supported by active transport with expenditure of metabolic energy. ~ The enzyme Na+,K+-ATPase has received consideration as a possible transport m e c h a n i s m for maintenance of cochlear cation gradients since the first conclusive b i o c h e m i c a l studies by Kuijpers and Bonting 3 a n d by Matschinsky and Thalmann 4 demonstrated its intense activity in the stria vascularis. In addition, the well-established func-
tion of the transport ATPase in sustaining low i n t r a c e l l u l a r s o d i u m c o n c e n t r a t i o n s bears a corollary linking the enzyme to maintenance of an appropriate water balance between intracellular a n d e x t r a c e l l u l a r f l u i d c o m p a r t m e n t s . Thus, homeostatic regulation of cellular volume is accomplished in part by removal of water, which passively follows sodium ion extruded from the cell by the enzyme. ~,6 A final aspect pertaining to the process of endolymph formation concerns the involvement of the enzyme in transport of water between extracellular fluid compartments. In tissues noted for salt-coupled transepithe]{al water transport, Na+,K+-ATPase is g e n e r a l l y r e g a r d e d as the p r i m a r y active t r a n s p o r t m e c h a n i s m t h a t p o w e r s the fluid flux.7-1o METHODS FOR LOCALIZATION OF ION PUMP SITES
From the University of Michigan Medical School, Medical Sciences II, Ann Arbor, Michigan 48109. Received May 14, 1982. Accepted for publication May 24, 1982. Supported by N A S A NSG 9047 and NIH AM 27559. Presented at the Midwinter Research Meeting of the Association for Research in Otolaryngology,January 18-21, 1982, St. Petersburg Beach, Florida. * Doctoral Candidate, Program in PhysiologicalAcoustics. ~rDepartment of Anatomy and Cell Biology. Address correspondence and reprint requests to Dr. Ernst. 0196-07091821090010332
Progress in the area of cochlear fluid balance will depend increasingly u p o n observations at the cellular level. The information still needed includes measurements of intracellular resting potentials and ionic activities, determinations of ionic permeabilities in various cell membranes, and cellular localization of the ion pump sites. The exact distribution of Na+,K+-ATPase among cochlear cells and tissues w o u l d therefore seem to h o l d c o n s i d e r a b l e i n t e r e s t , n o t o n l y for
$01.40 Q W. B. Saunders Co. 332
KERR ET AL.
Figure 1. A, light photomicrographic survey showing the distribution of autoradiographic silver grains in the outer wall of the cochlea after incubation with '~H-ouabain. Dense clusters of silver grains cover the basolateral compartments of marginal cells in the stria vaseularis (SV), while the apical cytoplasm is relatively free of label (see Fig. 2). Ouabain binding sites are also associated with cells in the spiral prominence (SP) and external sulcus (see Fig. 3). Labeled cells extend below the level of the basilar membrane. R: Reissner's membrane. Brightfield, methylene blue. Second cochlear turn. B, this photomicrograph shows, at higher magnification, the distribution of auabain-binding sites in the region of the spiral prominence. Na+-K + pump sites are associated with cells of the prominence, as well as with the strial marginal cells at the upper left. Phase-contrast, second cochlear turn. C, this photomicrograph shows labeling of stromal cells in the body of the spiral limbus. Phase-contrast, second cochlear turn.
Volume 3 Number 5 September 1982 333
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American Journal of Otolaryngology 334
KERRET AL. Figure 2. Electron photomicrograph showing the distribution of K+-dependent phosphatase activity iu the stria vascularis. Electron-dense deposits of reaction product outlinethe extensions of the marginal cells near the center of the field. The luminal surface of the marginal cells, bordering the endolymph (EL),is devoid of reaction product. Little or no reaction can be seen in basal cells (B). intermediate cells (*), ar capillaries (C), or in the spiral ligament {SL). Second cochlear turn. -~[
clarifying the role of the enzyme in normal cochlear function, but also for its possible relevance to the alteration of fluid balance occurring in M~ni6re's disease. R e c e n t l y d e v e l o p e d cytochemical methods can provide information on this subject, and have been used both to indicate which cell types in various ion-transporting epithelia contain the highest density of Na+-K+ pump sites and to show that in certain types of cells the p u m p s may be asymmetrically distributed to a particular region of the plasma membrane.1', '2 O n e of t h e s e t e c h n i q u e s , originated by Ernst, '3''4 detects inorganic phosphate released from an artificial substrate (nitrophenyI phosphate) by the catalytic activity of transport ATPase. In the presence of strontium ion, phosphate is precipitated at regions of high activity, then converted to an electron-dense product, which may finally be visualized in the electron m i c r o s c o p e . A s e c o n d m e t h o d , d e v i s e d by Stirling, is depends upon the well-documented ability of a specific sodium-pump inhibitor, ouabain, to bind selectively to Na+,K+-ATPase, thereby blocking catalytic activity. In this procedure, tissues are incubated with ~H-ouabain, and regions with high concentrations of Na+-K+ pump sites are subsequently identified by light microscope autoradiography. Since these techniques localize the same enzyme by different principles, we employed both methods concurrently for cross-validation of results. The distribution of activity described below was further substantiated by control experiments appropriate to the respective techniques. To assure the specificity of phosphatase reaction product observed b y electron microscopy, we verified that the reaction was K+-dependent and ouabain-inhibitable, and that the product was deposited on the cytoplasmic surface of cell plasma membranes. These criteria are compatible with the biochemical characterization established for the phosphatase component of the Na*,K+-ATPase enzyme complex, u,16 To demonstrate the specificity of the autoradiographic method, we carried out quantitative assays of 3H-ouabain u s i n g liquid scintillation spectrometry. In segments of outer wall incubated with 5 ~M aH-ouabain, dilution with 5 mM unlabeled ouabain produced a decrement of 100 per cent in tissue-bound label, suggesting the
,,m
saturable nature of the binding. Omission of Mg ++, a cofactor necessary for binding of ouabain to Na+,K+-ATPase in vitro, led to an 85 per cent reduction in tissue labeling. Moreover, the number of binding sites, normalized to tissue dry weight, increased from the apical to the basal turn, consistent with the earlier biochemical assays of enzymatic activity by Kuijpers and Bonting2 Scatchard analysis of ouabain/enzyme interaction at ouabain concentrations as high as 5/,M was consistent with a single population of receptors having a dissociation constant of approximately 2/,M. The following account summarizes our observations on the cellular localization of the enzyme in cochlear structures, particularly those with p o s s i b l e functions in e n d o l y m p h a t i c homeostasis. CELLULAR DISTRIBUTION OF COCHLEAR CATION PUMP SITES
In the outer wall of the guinea pig cochlea, both methods show highest Na+,K+-ATPase activity in the stria vascularis (Figs. 1 and 2), with intermediate levels in cells of the external sulcus and spiral prominence (Figs. 1 and 3). While these observations are consistent with the biochemical data of Kuijpers and Bonting, a the present study also extends their findings by the unexpected demonstration of activity in the spiral lirnbus (Fig, 1), and by showing that activity in the organ of Corti appears to be associated primarily with nerve fibers. Thus, elevated levels of the enzyme were localized to the synaptic regions below the inner and outer hair cells, to the vicinity of the inner and outer spiral bundles, to unmyelinated fibers crossing the tunnel, to the terminal node of Ranvier in the habenula perforata, and to the myelinated segments of fibers coursing through the osseous spiral lamina. Very little activity was found in sensory or supporting cells of the organ of Corti or in Reissner's membrane. There was, however, some suggestion of increased activity in the membrane near its attachment to the limbus. At the ultrastructural level, almost all reaction product in the stria vascularis was restricted to the contraluminal extensions of the marginal cells (Fig. 2), with little reaction on the luminal surface. The distribution of activity in the spiral
Volume 3 Number 5 September 1982
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336
KERRET AL. Figure 3. Electronphotomicrographshowing the distributionof K+-dependentphosphataseactivityin the spiral prominence. At the center of the field and at lower right, reaction product outlines the extensions of a stromal cell (S). Notice that the reaction product is distributed to the cytoplasmicmembrane surfaces. Little or no activitycan be seen in the prominence epithelial cell (PE). in the root cell (R},or in the capillary (C). EL: endolymph.Second cochlearturn. prominence, external sulcus, and spiral limbus was remarkable in being limited ahnost wholly to the stromal cells (Fig. 3). Although the reactive cells display an extensive network of tendrils with high mitochondrial density, they have no apparent communication with the lumen of the cochlear duct. Instead, the extensions of these cells seem c o n f i n e d to the connective tissue matrix and pericapillary spaces beneath the epithelial surface. The reaction product was distributed quite evenly along their plasma membranes, rather than being polarized to one particular surface. This pattern of activity stands in contradistinction to that observed in the stria and in other tissues implicated in transport of cations from one extracellular fluid compartment to another. POSSIBLE SIGNIFICANCE FOR COCHLEAR FLUID BALANCE
Stria Vascularis The cytoarchitecture of the strial marginal cells closely resembles that seen in many other ion-transporting epithelia; '7 moreover, the ultrastructural d i s t r i b u t i o n of Na+,K+-ATPase along the basolateral infoldings of these cells is virtually identical to the molecular organization found in several other tissues--both secretory and resorptive in function--where the enzyme is the only mechanism yet demonstrated to energize transepithelial ion transport2 In keeping with a major role i n cochlear ion transport, in vitro measurements by Marcus et al. is indicate that Na:~,K+-ATPase activity accounts for nearly half the oxygen consumption of the stria vascularis, a tissue long known for its extremely high respiratory rate. 19 Localization of most strial transport ATPase to the contraluminal aspect of the marginal cells is consistent with the view that the enzyme participates in the transport of potassium ion toward the endolymph. The pump sites are favorably situated to mediate uptake of potassium from the strial interstitial spaces, concentrating K+ within the marginal cell for eventual extrusion across the luminal membrane. Similar proposals were advanced by Johnstone and Sellick 29and by Kusakari and Thalmann2~; various data compatible with such a mechanism are cited by Ross et al. in this issue. 22 With the demonstration that most Na+-K + pump sites are distributed along
the marginal cell contraluminal membrane, the possibility arises that sodium ion from the adjacent strial interstitial spaces may contribute to marginal cell intracellular sodium, required at the cytoplasmic membrane surface for optimal enzymatic activity2~,24 and K+ uptake. It seems possible that the same mechanisms responsible for maintenance of the endolymphatic ion profile in the course of normal physiologic function may also play a part in regulating endolymph volume. The available evidence indicates that the osmotic pressures of endolymph and perilymph are normally equal (in young rats, at least25), and that the osmotic pressure of endolymph can be attributed almost entirely to its ionic constituents. 25'26 In addition, Bosher and Warren 24 provided evidence that certain structures bounding the endolymphatic compartment may be freely permeable to water. These results suggest that endolymph may gain or lose water to maintain osmotic equilibrium with surrounding extracellular fluids. Johnstone and Robertson27 pointed out that an inappropriate elevation in the rate of entry of ions into endolymph might favor an expansion of volume. Their calculations suggest that an increase in endolymphatic NaC1 or KC1 amounting to only 1 mM per hour could produce an osmotic pressure gradient sufficient to expand endolymphatic volume at rates comparable to those observed in guinea pigs with surgically induced hydrops. It should, however, be noted that an osmotic increase in endolymph formation may be of little consequence unless accompanied by other defects as, for example, in compensatory radial or longitudinal resorption (cf. Kimura, 2s Wilbrand and Stahle29).
Spiral Prominence, External Sulcus, and Spiral Limbus The distribution of transport ATPase observed in stromal cells of the spiral prominence and limbus represents, in our knowledge, a specialization unique to the cochlea. Since these cells do not appear to form a boundary between fluid compartments, they cannot be regarded as directly involved in transepithelial ion transport. Stromal tissues of the prominence and liraVolume 3 bus share certain structural features, including a Number 5 rich vascularization and extensive extracellular September 1 9 8 2 spaces, which may suggest some limited simi337 larity in function. Many investigators have be-
COCHLEAR TRANSPORT OF ATPase
lieved these structures to be involved in transport of ions and/or fluids (see discussions by Duvall s~ and Ishiyama et al.3'). Experiments with vital dyes "~ and electron-dense tracer particles "s,"4 indicated that the extracellular spaces of both tissues communicate with perilymph. The investigations of von Ilberg and colleagues ~s,34 demonstrated that the stromal cells, found in the present study to be possessed of significant transport ATPase activity, also show a vigorous pinocytotic uptake of tracer particles from the extracellular spaces. While further study will be necessary to determine the role of the reactive stromal cells, it is evident that they represent much more than simple connective tissue components.
American Journal of Otolaryngology 338
14.
15. 16. 17. 18. 19.
Acknowledgments. The authors thank Dr. Seth R. Hootman for many useful suggestions in the course of this research and in the preparation of the manuscript.
21.
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