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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].
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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
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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
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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.
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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.
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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
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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
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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
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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
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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
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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.
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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
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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). The observation that
a sustained stimulation of the vasopressin-AQP2 system leads to endolymphatic
hydrops in guinea pigs is intriguing and might be of use for a rational therapeutic
approach of Menière’s disease.
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