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P1: FLT Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 C 2003) Cellular and Molecular Neurobiology, Vol. 23, No. 3, June 2003 (° Review Aquaporin-Mediated Fluid Regulation in the Inner Ear Eric Beitz,1,3 Hans-Peter Zenner,2 and Joachim E. Schultz1 Received September 30, 2002; accepted October 10, 2002 SUMMARY 1. The sensory functions of the inner ear (hearing and balance) critically depend on the precise regulation of two fluid compartments of highly desparate ion composition, i.e., the endolymph and the perilymph. 2. The parameters volume, ion composition, and pH need to be held at homeostasis irrespective of the hydration status of the total organism. 3. Specific cellular water channels, aquaporins, have been shown to be essential for the fluid regulation of several organs, e.g., kidney, lung, and brain. 4. Because of functional similarities of water regulation in the kidney and inner ear this review initially summarizes some aquaporin functions in the kidney and then focuses on 6 out of 11 mammalian aquaporins that are present in the inner ear (AQP1-6). 5. Their potential role in the inner ear fluid control will be discussed on the basis of the respective expression patterns and individual pore properties. 6. Further, a working model is presented of how the endolymphatic sac may contribute to inner ear fluid regulation. KEY WORDS: inner ear; endolymph; fluid regulation; aquaporin; vasopressin. INNER EAR FLUIDS, SOUND PERCEPTION, AND DISEASE The inner ear is a tiny, yet anatomically and functionally highly sophisticated organ for sensing sound and gravity. The growth and development of the inner ear usually is completed at birth or shortly thereafter to enable young animals to communicate vocally with the mother animal and to stay and walk. Its size is principally delimited by the length of the sound waves, which it can register and translate into a neuronal signal. Therefore, much to the dismay of biochemists, its size does not vary very much from the smallest to the largest animal species. For example, in man the length of 1 Department of Pharmaceutical Biochemistry, School of Pharmacy, University of Tübingen, 72076 Tübingen, Germany. 2 Department of Otorhinolaryngology, University of Tübingen, 72076 Tübingen, Germany. 3 To whom correspondence should be addressed at Department of Pharmaceutical Biochemistry, School of Pharmacy, Morgenstelle 8, 72076 Tübingen, Germany; e-mail: [email protected]. 315 C 2003 Plenum Publishing Corporation 0272-4340/03/0600-0315/0 ° P1: FLT Cellular and Molecular Neurobiology [cemn] 316 pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Beitz, Zenner, and Schultz the incapsuled cochlear spiral is about 3.5 cm. The sensory properties of the inner ear are truly astounding: sustained sound perception in the kilo-Hertz range, reliable discrimination of sound frequencies, which are only 0.2% apart, and perception of a wide dynamic range of signals with amplitude differences of more than six orders of magnitude. Without the inner ear’s degree of precision the analysis of complex auditory signals, such as speech, would be impossible. Further, irrespective of the physical situation, the posture is constantly monitored and the respective muscles are constantly fine-tuned for balance and position control purposes. The whole set-up of the middle and the inner ear from the general anatomy to terminally differentiated sensory hair cells is tailored to comply with these demands. First, an impedance matching of the sound signal by the ossicular chain takes place in the middle ear. The sound signals are then mechanically transmitted through vibrations of the oval window membrane into the entirely fluid-filled inner ear. The basic elements for the conversion of mechanical signals into electrical signals are ionic transduction channels, which are located in the stereocilia of sensory hair cells, allowing potassium ions to enter the cell. These channels are directly gated by the deflection of the stereocilia possibly through so-called tip-links and thus open and close mechanically in response to the auditory signal. The resulting potassium influx gives rise to depolarization of the sensory hair cells, which are functionally linked at their basal sides to the afferent neurons of the cochlear nerve probably via glutaminergic synapses (Dallos, 1996; Slepecky, 1996). From the above it is obvious that the inner ear fluids play important roles in the process of sound perception. Firstly, fluid is the basic medium in which the mechanical sound signal travels hydrodynamically. This requires a tight volume control in order to establish and maintain correct pressure and volume within the system. The delicate balance between stiffness and mass that may be influenced by overpressure or volume increase has to be carefully regulated. Secondly, as a specialized fluid compartment, the endolymph serves as an extracellular reservoir and buffer of K+ ions that are needed for signal transduction. In fact the inner ear as a labyrinthine tubular system contains two distinctly separated fluid compartments of defined, yet differing ionic composition. In the cochlea, the perilymph and the endolymph network, are divided only by the two cell layer thick Reissner’s membrane (Fig. 1(B)). The perilymph is considered to be a filtrate of the cerebrospinal fluid or blood and consequently contains a high Na+ concentration (about 145 mM), low K+ (about 5 mM), and 1– 2 mM Ca2+ . The endolymph in contrast is unique as an extracellular fluid in that it contains only 1 mM Na+ , yet 155 mM K+ and Ca2+ in the nanomolar range. Thus it very much mirrors the cytosolic ion composition (Konishi et al., 1984; Salt et al., 1989; Sterkers et al., 1987). It is contained in a tube that runs throughout the gyrate cochlea (scala media), the vestibulum and which ends in a reservoir-like endolymphatic sac (Fig. 1). Between the cochlear endolymph and the endolymphatic sac, a rather static ion gradient exists. Actually, in the endolymphatic sac the fluid composition is more akin to an extracellular fluid (about 140 mM Na+ , 15 mM K+ , and 0.5 mM Ca2+ ; Mori et al., 1987; Ninoyu and Meyer zum Gottesberge, 1986). For the production, absorption, and regulation of the endolymph volume and composition predominantly two anatomically distant sites are discussed. The stria vascularis in the scala media is thought to be the tissue, which extrudes K+ into the endolymph. Probably the stria P1: FLT Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Inner Ear Aquaporins 317 Fig. 1. Schematic drawings of the endolymph compartment of the inner ear with indication of aquaporin expression sites. (A): The endolymph compartment is shown as a solid black line running through the cochlea and the vestibulum ending up in the endolymphatic sac. (B): Shown here is a section through one turn of the cochlea. The separation of the endolymph (scala media, SM) from the perilymph compartments (scala tympani and vestibuli, ST and SV) through the basiliar (BM) and Reissner’s membrane (RM) is clearly visible. The cell types, which express aquaporins are labelled. The inset shows a microscopic picture of a guinea pig cochlea from the same view. Abbreviations are BM – basiliar membrane, C – Claudius cells, D – Deiters cells, ESC – external sulcus cells, H – Hensen’s cells, IHC – inner hair cells, IP – inner pillar cells, ISC – inner sulcus cells, OHC – outer hair cells, OP – outer pillar cells, RM – Reissner’s membrane, SL – spiral ligament, SM – scala media, SP – spiral prominence, ST – scala tympani, StV – stria vascularis, SV – scala vestibuli, TM – tectorial membrane. vascularis also is involved in water transport. The function of the endolymphatic sac is somewhat more controversial. In the guinea pig, obliteration of the endolymphatic sac causes endolymphatic hydrops, i.e., endolymph volume increase, suggesting a role in endolymph transfer (Kimura and Schuknecht et al., 1965). Yet this would P1: FLT Cellular and Molecular Neurobiology [cemn] 318 pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Beitz, Zenner, and Schultz require a directional flow of fluid from the stria vascularis to the endolymphatic sac. So far, there was no flux detectable beyond a passive bidirectional diffusion of injected marker molecules into the scala media (Salt and Thalmann, 1988). The sensory cells of the inner ear are exposed to the endolymph at their apical sides whereas the basolateral parts are bathed in a perilymph environment (Fig. 1(B)). This contributes to a high electrochemical gradient of about 150 mV over the apical end of the hair cells, which drives the K+ ions into the hair cells when the transduction channels open. This empowers the cell to detect tiny signals with high precision. Furthermore, it allows the system to quickly reset because of a rapid K+ efflux via the basolateral membrane. K+ ions are then discussed to be swiftly recycled into the endolymph compartment through the marginal cells of the stria vascularis involving a gap junction network (Kikuchi et al., 2000). Hereditary mutations in gap junction proteins (connexin 26) unavoidably result in deafness. The close relationship between endolymph maintenance and sensory functions is stressed by the finding that disturbances in the endolymph regulation causes balance and hearing problems (Rybak, 1994). Such incidences may be precipitated by application of the loop diuretics furosemide or ethacrynic acid. The wanted therapeutic effect of these drugs is due to the inhibition of the Na+ -K+ -2Cl− cotransporter localized in the ascending limb of the loop of Henle, which results in solute and water diuresis. In the inner ear, the presence of this very carrier as part of the potassium recycling machinery in the marginal cells of the stria vascularis is the basis for the ototoxic effects of loop diuretics. An inhibition that may occur even after a single dose of furosemide, leads to an edema within the stria vascularis and probably interferes with the K+ and water transport into the endolymph compartment. Obviously, the inner ear shares a number of functional mechanisms for water and ion regulation with the kidney, which are the molecular basis for unwanted side effects of diuretics (Humes, 1999). Notable are the oto- and nephrotoxic aminoglycoside antibiotics (gentamycin, kanamycin, etc.) and platinum-containing chemotherapeutics (cisplatin) although their effects on the fluid control in the stria vascularis are secondary. As much as the water regulating system of the kidney and the hearing aparatus are concerned, this will be described in more detail later. A well-documented clinical manifestation of endolymph dysregulation is Menière’s disease (Hamann and Arnold, 1999). Here, either the underlying overproduction or reduced absorption of the endolymph may have severe pathophysiological consequences, such as endolymphatic hydrops or a permeability increase of tight junctions resulting in vertigo attacks, hearing loss, tinnitus, and a sensation of fullness in the inner ear (Zenner et al., 1994). The mechanisms that lead to endolymphatic hydrops are not known. The fact that quite often only one ear is affected and the presence of macrophages in the endolymphatic sac led to the hypothesis of an allergic autoimmune reaction. Other speculations are a neural or viral etiology. Our own finding that systemically applied vasopressin, the regulator of water permeability of the kidney collecting duct, evokes endolymphatic hydrops in guinea pigs (Kumagami et al., 1998) will be discussed later in conjunction with a model for the fluid regulation of the endolymphatic sac. Taken together, water regulation of the inner ear fluid compartments is of utmost importance for functional stability of the hearing and balancing system. P1: FLT Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Inner Ear Aquaporins 319 CELLULAR WATER CHANNELS—AQUAPORINS The presence of pore-like molecules dedicated to transmenbraneous water fluxes was suggested by the ingenius regulation of the water permeability in the collecting duct, by the unexpectedly low Arrhenius activation energy observed for the rapid water flux across certain cell membranes (<5 kcal/mol vs. >10 kcal/mol in a plain lipid bilayer), and the inhibitory effect of Hg2+ ions on water fluxes. The discovery of cellular water channels, the proteins were named aquaporins (AQP), provided the molecular basis and consequently dramatically enhanced the field (Agre et al., 2001). Aquaporins are integral membrane proteins with a high specificity for the transport of water or small uncharged solutes, such as glycerol or urea across cell membranes. Importantly, the permeation strictly follows the existing osmotic or chemical gradient as the driving force. Structurally, all aquaporins share the same overall topology with six membrane spanning helices and two short pore helices which protrude into the membrane and form a pseudo-seventh transmembrane domain (Fig. 2; Kozono et al., 2002). The latter guides passing molecules by hydrogen bonds through the pore-forming center of the protein. Independent of the monomeric operation, the water channels form a tetrameric unit in the membrane. In humans, 11 isoforms (AQP0 to AQP10) exist. They have marked differences in their water and solute permeabilities and in the mode of regulation as well as in their tissue distribution and subcellular localization. So far, the best examined mammalian water channels are those with a pronounced function in kidney physiology, i.e., aquaporins AQP1 to AQP4 (Nielsen Fig. 2. Structure of an aquaporin monomer as seen from the intracellular space. The membrane spanning helices are labeled consecutively with numerals (1–6) and the two short pore helices are marked with asterisks. The site of the actual water pore is indicated by the black circle in the center of the monomer. The diamond labels the position of the fourfold axis of the tetrameric aquaporin. P1: FLT Cellular and Molecular Neurobiology [cemn] 320 pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Beitz, Zenner, and Schultz et al., 2002). They significantly contribute to the regulation of urine volume. AQP1 is located in the descending part of the loop of Henle, and facilitates the reabsorption of 60–80% of the primary urine volume. This is a hormone-independent process, which is driven solely by the hypotonicity of the primary urine with respect to the high osmolarity in the surrounding tissue (up to 1200 mOsm/kg). The reabsorption of another 10–20% in the collecting duct is fine-tuned by the antidiuretic hormone vasopressin (ADH). This involves the activation of the vasopressin V2-receptor, an increase in intracellular cAMP and, consequently, the phosphorylation of AQP2 at Ser256, which is stored in cytosolic vesicles. The phosphorylation event triggers by an as yet unknown mechanism the directional translocation of these vesicles to the apical plasma membrane. There, AQP2 is integrated into the plasmalemma and increases the water permeability. The entering water leave the cell basolaterally via AQP3 and AQP4 into the surrounding tissue. This upregulation of the apical water permeability is fully reversible, i.e., dephosphorylation of membrane-bound AQP2 triggers its removal via targeted endocytosis and probably replenishes the cytosolic vesicle pool for another round of shuttling. Disturbances in this network (lack of vasopressin, mutations in the V2-receptor or in the AQP2 protein) can cause diabetes insipidus. Even temporary challenges, such as the downregulation of AQP2 expression during a lithium treatment may lead to polyuria. The physiological role of aquaporins in the kidney and other fluid controlling tissues seems rather straight forward. However, puzzling novel observations indicate additional functions for aquaporins apart from fluid regulation. For example, AQP0 may also be important for the structural maintenance of cell to cell interactions of fiber cells in the eye lens (Fotiadis et al., 2000). Further, in acid secreting intercalated cells of the kidney collecting duct AQP6 represents an intracellular pore protein, which may operate as an ion channel (Yasui et al., 1999). A number of observations have always indicated that distinct similarities in the physiological processes exist, which are responsible for the fluid regulation in the kidney and in the inner ear. The advance in biomolecular technologies in recent decades has enabled us to investigate the molecular basis for these obvious similarities, as outlined later. DISTRIBUTION OF AQUAPORIN ISOFORMS IN THE INNER EAR After the first aquaporin proteins had been characterized there was little doubt they would also be involved in the fluid regulation of the inner ear. This has been investigated by several groups using the reverse transcriptase-polymerase chain reaction (RT-PCR) and immunological techniques. Despite the immense difficulties in the dissection of clearly defined tissue samples and sections from the inner ear because of the tiny size and a highly complex anatomy enclosed in a hard boney shell considerable progress has been made. Below we review what is known about the expression and localization of various aquaporin isoforms in anatomically and functionally defined areas of the inner ear (Table I). Additionally, we will summarize data on signal transduction systems which may directly contribute to regulatory inputs. In Fig. 1 the standard anatomical outlay of the inner ear is depicted and the areas are indicated, which were prepared by microdissection (endolymphatic sac, vestibulum, +/− (RT-PCR) +/+ (RT-PCR, in-situ, IF, Western) AQP3 Endolymphatic sac +/− (RT-PCR) AQP6 External sulcus cells, spiral prominence M1 almost absent in cochlea, M1:M23 = 1:3 in vestibulum, mRNA for M1 in endolymphatic sac. Takumi et al., 1998, Beitz et al., 1999, Li and Verkman, 2001. apical turns only. Beitz et al., 1999, Mhatre et al., 1999. Beitz, unpublished. April 22, 2003 Note. The data were collected in rats, mice, or guinea pigs. The methods of detection are indicated (reverse transcription polymerase chain reaction – RT-PCR; immunofluorescence – IF; Western blot or in-situ hybridization); further the gros tissue and the particular cell types of expression are shown. AQP4 exists in two splice variants (M1 and M23); the ratios based on Western blotting are given. Cochlea +/− (RT-PCR, IF) Supporting cells of the cochlea and vestibulum, Hensen’s cells, inner sulcus cells Stankovic et al., 1995, Takumi et al., 1998, Beitz et al., 1999, Li and Verkman, 2001. also V2-receptor, vesicle transport proteins. Kumagami et al., 1998, Beitz et al., 1999, Merves et al., 2000, Mhatre et al., 2002. Beitz et al., 1999 Comment, references pp820-cemn-463429 AQP5 Cochlea, vestibulum endolymphatic sac Endolymphatic sac +/+ (RT-PCR, IF) AQP2 Nonepithelial cells, spiral ligament, fibrocytes Cell types Cellular and Molecular Neurobiology [cemn] AQP4 M1, M23 Cochlea, vestibulum lining cells of the endolymphatic sac Endolymphatic sac Tissue +/+ (RT-PCR, IF) mRNA/protein AQP1 Isoform Table I. Aquaporin Expression Pattern in the Postnatal Inner Ear P1: FLT 22:44 Style file version Oct 23, 2000 Inner Ear Aquaporins 321 P1: FLT Cellular and Molecular Neurobiology [cemn] 322 pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Beitz, Zenner, and Schultz organ of Corti, stria vascularis, Reissner’s membrane) or sections through the cochlea and vestibulum and investigated by RT-PCR or immunofluorescence. AQP1 was present in most areas of the inner ear at the mRNA and protein level (Beitz et al., 1999; Huang et al., 2002; Li and Verkman, 2001; Stankovic et al., 1995; Takumi et al., 1998). Immunofluorescence was predominantly detected in fibrocytes in close proximity to the bone capsule, in the spiral ligament, in cells below the basilar membrane and in mesothelial cells lining the scala tympani in the cochlea. Notably, AQP1 was not found in epithelial cells nor in cells involved in the endolymph formation. Thus, a decisive involvement of AQP1 water permeability in endolymph or perilymph regulation is rather unlikely. It may have an as yet undefined role in the maintenance of the bone and the basilar membrane. In the 4-day-old rat, AQP2 mRNA was present exclusively in the endolymphatic sac (Beitz et al., 1999). In addition, AQP3 and AQP4 mRNAs were detected in this tissue. Immunofluorescence confirmed the expression of AQP2 in neonatal rats at Day 18 after gestation (Merves et al., 2000). Recent evidence indicates that AQP2 may also be expressed in the mammalian cochlea in particular structures bordering the endolymph (Mhatre et al., 2002). The findings for the endolymphatic sac were particularly striking because the coexpression of the three AQP isoforms AQP2, AQP3, and AQP4 so far was only known to occur in the principal cells, which are the main cell type lining the collecting duct in the kidney. There, AQP2 trafficking is regulated by vasopressin-dependent vesicle shuttling as mentioned earlier. Not surprisingly then, the presence of mRNAs for the vasopressin receptor and the major vesicle transport proteins VAMP2, syntaxin-4, and rab3a in the endolymphatic sac of rats was demonstrated by RT-PCR (Beitz et al., 1999; Kumagami et al., 1998). The lining of the collecting duct of the kidney is interspersed with acid secreting cells, so-called intercalated cells, which express the intracellular AQP6 (type A intercalated cells; Yasui et al., 1999). In the inner ear, protons need to be actively secreted into the endolymph in the cochlea in order to maintain a pH of 7.4 because of the strongly positve potential of the endolymph (+80 mV) a constant passive efflux of protons occurs from this compartment. This problem is somewhat less pressing in the endolymphatic sac, where the pH of the endolymph is around 6.6 (Karet et al., 1999). Recently, by RT-PCR with mRNA from the endolymphatic sac of 9-day-old rats we obtained a weak signal, which turned out to be the expected AQP6 product (unpublished data). How the intracellular AQP6 may be involved in pH regulation of the endolymph certainly is a pressing question. The presence of AQP2 and 6 in the endolymphatic sac extends the similarities with the kidney collecting duct from morphology to the molecular level. AQP4, the aquaporin with the highest water permeability, is also expressed in the sensory parts of the inner ear (Beitz et al., 1999; Huang et al., 2002; Li and Verkman, 2001; Takumi et al., 1998). It is not identified in the hair cells themselves, i.e., inner, outer, and vestibular hair cells, but in supporting cells within the cochlea (Hensen’s cells, Claudius cells, inner sulcus cells) and the vestibular end organs (Fig. 1). Interestingly, there is a significant difference in the ratio of two splice variants of AQP4 (M1 and M23) which are present in the vestibulum and the cochlea (Takumi et al., 1998). The M23 splice variant lacks the first 22 amino acids and appears 2–3 kDa smaller on Western blots than the full length M1 AQP4 isoform. In the cochlea the P1: FLT Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Inner Ear Aquaporins 323 AQP4 M23 isoform is by far predominant whereas in the vestibulum both variants are expressed in reasonable amounts (M1:M23 = 1:3; Takumi et al., 1998). Potential functional consequences of these differences in the expression of AQP4 splice variants are enigmatic at present and have to await the elucidation of functional differences between M1 and M23 AQP4 splice variants. A highly regionalized expression pattern is also observed for AQP5 (Beitz et al., 1999). RT-PCR established the presence of AQP5 mRNA in the organ of Corti and in Reissner’s membrane. By immunofluorescence AQP5 was localized in external sulcus cells at the spiral prominence of the scala media. Curiously, only the upper turns of the cochlea appear to express AQP5 (Mhatre et al., 1999). Because of the spatial resolution of an auditory stimulus along the length of the cochlea low frequencies stimulate hair cells in the apical turns whereas high frequencies stimulate close to the base. Although highly speculative one may envision that AQP5 may play an as yet undefined role in low frequency perception which is considered the “primitive trait” in vertebrate hearing. AQUAPORIN KNOCKOUTS AND HEARING IMPAIRMENT Mouse aquaporin knockout models are available for AQP1, AQP3, AQP4, and AQP5 (Verkman, 2000). Of those AQP4 turned out to the only water pore, which is essential for normal for hearing (Li and Verkman, 2001). The fact that the deafness of AQP4 knockout mice was overlooked for 4 years indicates that only very subtle behavioral differences exist in rodents with impaired hearing. A morphological analysis of AQP4 knockout mice did not reveal any abnormalities of the inner ear. In fact, this animal has only a mild urinary concentration defect due to impaired water reabsorption in the kidney collecting duct (Li and Verkman, 2001). This raises the question about the basic function of AQP4. The AQP4 expression pattern and localization in neuronal tissues may provide a hint. Usually, AQP4 is localized to the basolateral membrane of various types of supporting cells in the brain (astroglial cells), the eye (Müller cells) and, as already mentioned earlier, the inner ear (Hensen’s and Claudius cells). These cells accompany neurons, bipolar cells or sensory hair cells, respectively, which carry considerable K+ -ion fluxes during excitation. The highly permeable AQP4 water channel would functionally fit into such a scenario when one takes the transient osmotic imbalance in the surrounding interstitium into account which may occur locally because of the charge movements. A concurrent water flux through the AQP4 pore of the supporting cells may at least partially offset local swells in K+ ion concentrations. Currently, this is considered as a general paradigm in the physiology of excitable tissues (Li and Verkman, 2001). The deaf AQP4 knockout mice strikingly demonstrate that the AQP4 water permeability of the supporting cells is functionally almost as important for excitation as is the ion flux through K+ channels itself. AQP1, AQP3, and AQP5 knockout mice appear to have normal hearing as deduced from auditory brain stem response measurements questioning the proposed involvement of AQP5 in low frequency hearing (Li and Verkman, 2001; Mhatre et al., 1999). But the immuno localization of AQP5 was done in rat cochleae leaving the possibility of interspecies differences in this case. AQP6 knockout mice are not yet P1: FLT Cellular and Molecular Neurobiology [cemn] 324 pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000 Beitz, Zenner, and Schultz available. In addition to AQP1 knockout mice there are rare human null mutants. These individuals exhibit a surprisingly mild phenotype (Preston et al., 1994). Only a slight urinary concentration defect was noted after water deprivation (King et al., 2001); hearing deficits were not reported. The lack of AQP2 knockout mice is due to a simple reason: it is lethal and an AQP2 knock-in mouse model of autosomal recessive nephrogenic diabetes insipidus does not survive beyond the first week after birth (Verkman, 2000). Clearly, this model is not suitable for hearing analysis because of the later onset of hearing in mice, which is around postnatal Day 12. On the other hand, there are humans with mutations in the AQP2 gene, which lead to nephrogenic diabetes insipidus by misfolding or an intracellular routing failure of the water channel (Deen et al., 2000). A single report describes two related patients with nephrogenic diabetes insipidus who also suffer from Menière’s disease (Comacchio et al., 1992). To conclude from this single case that a nonfunctional AQP2 leads to hearing problems would certainly be premature. Screening for AQP2 mutations in individuals with Menière’s disease has revealed no sequence alterations (Mhatre et al., 2002). Below, we propose a model for the regulation of the endolymph, which is congruent in many respects with the regulatory features established for the kidney collecting duct. MODEL OF THE FLUID REGULATION IN THE ENDOLYMPHATIC SAC Four experimental observations form the basis for our water regulation model of the endolymphatic sac (Fig. 3). (A) The endolymph is hypertonic to the perilymph and the serum (305 mOsm/kg vs. 285 mOsm/kg, Konishi et al., 1984). (B) AQP2 is present in the endolymphatic sac and key regulators of AQP2 are identical to the kidney collecting duct (Beitz et al., 1999; Kumagami et al., 1998). (C) The endolymphatic sac shows a very high endocytotic activity in the basal state and vasopressin inhibits endocytosis in vitro (Kumagami et al., 1998). (D) Vasopressin application leads to endolymphatic hydrops in vivo (Kumagami et al., 1998; Takeda et al. 2000). The sole driving force for water movement over epithelia is osmosis. This means, that the hypertonicity of the endolymph facilitates a unidirectional water flow into the endolymph compartment. Because of the rather small differences in osmolality between the endolymph and perilymph (10–20 mOsm/kg) the rate of the nonfacilitated water diffusion is very slow unless augmented by the presence of aquaporins. So far, AQP2 is the only established water pore, which has been unequivocally localized to the endolymph lining epithelium. Nevertheless, the subcellular localization in the endolymphatic sac is to be determined. The presence of the major components of the kidney AQP2 regulatory equipment strongly suggests that a similar AQP2 shuttling process will also operate in the endolymphatic sac. Vasopressin stimulation at the basolateral site should therefore increase the number of AQP2 water channels in the apical membrane via a cAMP-triggered phosphorylation thus greatly enhancing the water influx into the endolymph compartment (see Fig. 3(A) for a model). The physiological function of the vasopressin effect might be an effective short-term protection of the endolymph volume during periods of hypovolemia. However, it is known when vasopressin levels are pathologically increased over a prolonged period P1: FLT Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 22:44 Inner Ear Aquaporins Style file version Oct 23, 2000 325 Fig. 3. Model of the vasopressin-dependent water regulation in the endolymphatic sac. Shown is a cell under basal and stimulated conditions. (A): In the presence of vasopressin AQP2 water channels are inserted into the apical membrane allowing osmotic water flux into the endolymph compartment. (B): Without vasopressin pinocytosis of endolymph fluid dominates whereas AQP2 water channels are stored intracellularly leading to a reduction of the endolymph volume. See text for further explanations. of time, this will ultimately result in endolymphatic hydrops in agreement with the above proposal (Kumagami et al., 1998). In diabetes insipidus patients the vasopressin regulatory system is inoperative (because of defects in vasopressin biosynthesis or release, the vasopressin receptor or the AQP2 protein). According to the proposed model those patients cannot increase the water influx into the endolymph compartment and, thus, are usually essentially protected against the development of a hydrops. P1: FLT Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 326 22:44 Style file version Oct 23, 2000 Beitz, Zenner, and Schultz Fig. 3. (Continued.) How can the endolymph volume be reduced? The only possibility to transport water against an osmotic gradient out of the endolymph compartment is by pinocytosis of the cell layer lining the fluid compartment. Two ways are possible by which the cells could dispose of the internalized hyperosmolar fluid load: by transpinocytosis or generation of localized, transbasolateral water and ion efflux. Pinocytosis would involve a directional transport of the ingested vesicles from the apical to the basolateral site and subsequent disposal into the microvasculature. The alternative is the ion secretion over the basolateral membrane and a concurrent water flux (through AQP3 and AQP4). Which of the two possibilities actually has been realized is unclear at present and must wait until appropriately designed experiments are feasible—an arduous task if one considers the known difficulties in handling the endolymphatic sac or primary cultures of it. The excess fluid in the interstitium can finally enter the network of surrounding fenestrated capillaries. It has been demonstrated that in P1: FLT Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 Inner Ear Aquaporins 22:44 Style file version Oct 23, 2000 327 the basal state, i.e., without hormonal stimulation, endocytosis is rapid. Vasopressin inhibits pinocytosis from the endolymphatic sac and this process may potentially contribute passively to an increase of the endolymph volume in addition to the vasopressin-stimulated water transport through AQP2 (Kumagami et al., 1998). The underlying mechanism for the inhibition of endocytosis in the endolymphatic sac by vasopressin is unknown so far. In summary, this “leaky boat” model predicts a local endolymph turn-over by regulated osmotic water influx into the endolymph through AQP2 (the leaks in the boat) and the simultaneous removal of endolymph fluid by isosmotic pinocytosis (scooping out the water bucket by bucket). The overall process is hormonally balanced via vasopressin with respect to the systemic hydration status. Additional local regulatory mechanisms may exist. So far, mainly two further observations point in this direction. First, it was reported that the endolymphatic sac secretes a hormone, “saccin,” with diuretic activity (Qvortrup et al., 1996). Second, the seven transmembrane, calcium sensing receptor (CaSR), is expressed in the endolymphatic sac indicating the potential of a cellular responsiveness towards changing Ca2+ concentrations (Beitz et al., 1999). This receptor has rather prominent roles in the parathyroid and the kidney in the calcium homeostasis by triggering parathyroid hormone secretion and renal calcium reabsorbation (Hendy et al., 2000). The proposed model could account for a further important feature in endolymph regulation: it would remove “used” fluid and replace it with AQP2-“filtered” liquid and thus clean the endolymph from accumulating metabolites, cellular debris, and other unwanted material, which may accumulate therein. CONCLUSION Aquaporins have proven to be absolutely essential for the sensory functions of the inner ear (AQP4) or are very good candidates for an involvement in the regulation of the endolymph volume (AQP2) and pH control (AQP6). 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