P2 purinoceptor of the globular substance in the otoconial
membrane of the guinea pig inner ear
Hideaki
Suzuki,
Katsuhisa
Ikeda,
Masayuki
Furukawa, and
Tomonori
Takasaka
Department of Otolaryngology, Tohoku University School
of Medicine, Sendai 980-77, Japan
 |
ABSTRACT |
The biological characteristics of the globular substance, a
precursor of otoconia, are unclear. In the present study, the ATP-induced internal free Ca2+ concentration
([Ca2+]i) changes of the globular substance
and the ATP distribution in the vestibular organ were investigated
using a Ca2+ indicator, fluo 3, and an adenine
nucleotide-specific fluorochrome, quinacrine, by means of confocal
laser scanning microscopy. [Ca2+]i showed a
rapid and dose-dependent increase in response to ATP with a 50%
effective concentration (EC50) of 16.7 µM. This
reaction was independent of external Ca2+, indicating the
presence of an internal Ca2+ reservoir. Neither adenosine,
,
-methylene-ATP, 3'-O-(4-benzoylbenzoyl)-ATP, ADP, nor
UTP evoked this reaction, whereas 2-methylthio-ATP induced an increase
of [Ca2+]i with an EC50 of 14.4 µM. Moreover, P2 antagonists, reactive blue 2 and suramin, and a
phospholipase C inhibitor, U-73122, inhibited the ATP-induced
[Ca2+]i increase. These findings indicate the
presence of a P2Y purinoceptor on the globular substance. In addition,
granular fluorescence was observed in the quinacrine-stained macular
sensory epithelium, indicating the presence of ATP-containing granules
in this tissue. These results suggest that a paracrine mechanism
involving ATP may exist in the macula and that this mechanism regulates
the biological behavior of the globular substance.
macula; vestibule; otoconia; adenosine 5'-triphosphate; quinacrine
 |
INTRODUCTION |
THE MACULA IS A vestibular organ that is responsible
for the perception of gravity and linear acceleration. It is composed of the sensory epithelium, which includes sensory hair cells, and the
otoconial membrane. The otoconial membrane is situated above the
sensory epithelium and consists of otoconia, the gelatinous layer, and
subcupular meshwork, a fine fibrous structure that connects the
gelatinous layer with sensory cilia of the hair cells. Otoconia are
biomineral bodies sitting on the gelatinous layer that add weight to
the otoconial membrane, resulting in its deflection and, eventually,
cilial deflection of hair cells according to the changes of direction
in gravity.
Otoconia contain calcium carbonate as the mineral component and
proteins with carbohydrates as the organic component. The mechanism of
otoconial formation, a biomineralization process, is only partially
understood, but it is thought to be different from the purely inorganic
precipitation of crystal. The globular substance is a spherical
structure floating in the gelatinous layer of the otoconial membrane in
the macula. This material is secreted from the macular sensory
epithelium and is presumed to be a precursor of otoconia (12, 33). The
morphological features of this substance have been studied by several
authors (12, 33), and Harada (12) has detected its high
Ca2+ content with an X-ray microanalyzer. More recently, we
observed the globular substance in a physiological buffer by means of
confocal laser scanning microscopy and demonstrated that this substance is a membrane-enclosed structure and has a higher internal free Ca2+ concentration ([Ca2+]i)
than the resting level of intracellular Ca2+ observed in a
variety of cells (33). In the bone formation process, which is another
biomineralization system, matrix vesicles occur as a precursor. It is
intriguing that this material manifests characteristics similar to
those of the globular substance, i.e., it is produced by microapocrine
secretion from osteogenic cells and is a membrane-enclosed structure
(11).
Despite the significance of the globular substance in the process of
otoconial formation, information about its biological reactions has so
far been very limited. In the present study, we report the
[Ca2+]i response of the globular substance
induced by ATP and its analogs, providing new insight into the
mechanism of otoconial formation. In addition, we also clarify the ATP
distribution in the macula using an adenine nucleotide-specific
fluorescent compound.
 |
MATERIALS AND METHODS |
Chemicals.
Fluo 3-pentaacetoxymethyl ester (fluo 3-AM) was purchased from
Molecular Probes (Eugene, OR). Adenosine, ADP, ATP,
,
-methyleneadenosine 5'-triphosphate (
-MeATP),
3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), UTP,
quinacrine, and ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) were obtained
from Sigma Chemical (St. Louis, MO). 2-Methylthioadenosine
5'-triphosphate (2-MeS-ATP) and reactive blue 2 (RB2) were bought from
Research Biochemicals International (Natick, MA). Suramin, U-73122, and U-73343 were purchased from Wako Pure Chemical (Osaka, Japan). PC-12
cells, derived from rat pheochromocytoma, were provided by Riken Cell
Bank (Tsukuba, Japan). Dulbecco's modified Eagle's medium (DMEM),
RPMI 1640, and fetal bovine serum (FBS) were obtained from GIBCO BRL
Life Technologies (Palo Alto, CA).
Sample preparation.
Adult albino guinea pigs, 6-10 wk old, were anesthetized by
diethylether inhalation and decapitated. Temporal bones were collected, and the utricular maculae were dissected under a dissection microscope in O2-gassed artificial perilymph (APL) composed of (in mM)
150 NaCl, 3.5 KCl, 1 CaCl2, 1 MgCl2, 2.3 tris(hydroxymethyl)aminomethane, 2.8 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid,
and 3 D-glucose (pH 7.4). The samples were then transferred
onto a glass coverslip or into a thin-bottomed petri dish, and the
otoconial membrane was gently detached from the sensory epithelium by
forceps. For the superfusion experiments to study
[Ca2+]i changes, the membrane on the glass
coverslip was incubated with 3 µM fluo 3-AM as a free
Ca2+ indicator in APL for 1 h at room temperature. The care
and use of the animals were in accordance with the Guidelines of the
Declaration of Helsinki.
Cell culture.
PC-12 cells were cultured in DMEM supplemented with 10% FBS. For
fibroblast culture, the dermis and subcutaneous tissue were excised
from the abdominal skin of the guinea pig and cultured in RPMI 1640 containing 10% FBS. The samples were placed in thin-bottomed petri
dishes and incubated in humidified 5% CO2-95% air at
37°C until the cells grew nearly confluent.
Quinacrine staining.
For the observation of the ATP distribution, the samples in the petri
dish were incubated with 5 µM quinacrine in APL (for utricle) or in
the culture medium (for cultured cells) for 30 min at room temperature
as previously described (37) and then washed before confocal
microscopic examination.
Superfusion experiment.
Coverslips with fluo 3-loaded samples were placed in a superfusion
chamber. Solutions were continuously saturated with O2 and
pumped into the chamber at a flow rate of 0.8 ml/min by a peristaltic
pump and removed by a siphon. Reagents were applied by changing the
superfusion solutions.
Confocal laser scanning microscopy.
The samples were directly observed under a laser scanning confocal
imaging system (Bio-Rad MRC-600; Bio-Rad Microscience Division, Watford, UK) with an argon ion laser as the light source coupled to an
inverted microscope (Olympus IMT-2; Olympus, Tokyo, Japan). The
excitation wavelength was 488 nm. The laser power was 10 mW at the
source and was reduced by a neutral density filter of ND-2 (1% transmission) for fluo 3 and ND-1 (10% transmission) for
quinacrine. The objective was ×40 with a numerical aperture of 0.95 (SPlan Apo 40; Olympus). Emitted fluorescence, which passed through a long-pass filter (515-nm cutoff), was collected by a photomultiplier tube and displayed as a 768 × 512 pixel resolution image through a
host computer (standard IBM PC-AT).
Dynamic changes in the intensity of fluo 3 fluorescence were observed
every 1 min and displayed in a 256-step arbitrary fluorescence scale of
0 (no fluorescence) to 255 (most intense fluorescence). The obtained
data were not converted to the absolute value of the internal
Ca2+ concentration because of the error inherent in
single-wavelength detection of fluorometric intensities and are
therefore expressed as relative values. A value of relative
fluorescence was calculated as
follows
|
(1)
|
{smtxt}where F is the intensity of the observed fluorescence,
Fo is the intensity of the fluorescence at time
zero, and BG is the background fluorescence.
Statistics.
Data values are expressed as means ± SE. Statistical significance was
analyzed using a two-tailed Student's t-test, and a P
value of <0.05 was considered significant.
 |
RESULTS |
ATP-induced [Ca2+]i response of the globular
substance.
Figure 1 represents
pseudocolor images of typical ATP-induced
[Ca2+]i increases in the globular substance.
Fluo 3 fluorescence rapidly increased 2 min after the application of
100 µM ATP and then fell gradually (Fig. 1). Not all globular
substances responded to ATP. Thirty-two of fifty globular substances
(64%) showed a relative fluorescence value of 2.0 or more at the peak
in response to 100 µM ATP, whereas the others did not. To
quantitatively evaluate the [Ca2+]i
responses, we calculated the arithmetic mean of relative fluorescence values of all globular substances that appeared in the confocal microscopic field. Mean relative fluorescence at the peak reached five-
to sixfold the value of the resting level. The reaction was independent
of external Ca2+, i.e., superfusion with
Ca2+-free APL supplemented with 1 mM EGTA (corresponding to
10
8 M Ca2+) did not suppress this
ATP-induced response (Fig. 2), indicating the release of Ca2+ from internal stores of the globular
substance. There was no significant difference in the proportion of the
responding population in the presence or absence of external
Ca2+.

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Fig. 1.
ATP-induced internal free Ca2+ concentration
([Ca2+]i) changes of the globular
substance. Otoconial membrane of the utricular macula was dissected
under a dissection microscope and incubated with 3 µM fluo
3-pentaacetoxymethylester (AM) in artificial perilymph (APL) for 1 h at
room temperature. Sample was then placed in a superfusion chamber, and
dynamic changes of the fluorescence were observed every 1 min under a
confocal laser scanning imaging system (Bio-Rad MRC-600) as described
in MATERIALS AND METHODS. APL was continuously saturated
with O2 and pumped into the superfusion chamber at a flow
rate of 0.8 ml/min; 100 µM ATP was applied by changing the
superfusion solution at time 0. Each digit represents time (in
min) from the application of ATP. Intensities of fluo 3 fluorescence in
arbitrary scale are displayed in pseudocolor, as indicated in color
calibration bar. Scale bar (top left) = 10 µm.
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Fig. 2.
ATP-induced [Ca2+]i changes of the globular
substance. Otoconial membranes were treated as in Fig. 1. Samples were
superfused with APL ( ; n = 36) or with
Ca2+-free APL supplemented with 1 mM EGTA ( ;
n = 14); 100 µM ATP was applied by changing the superfusion
solution as indicated in the graph. NS, not significant.
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Neither adenosine, 
-MeATP, BzATP, ADP, nor UTP evoked this
reaction, whereas 2-MeS-ATP induced a significant increase in fluorescence (Fig. 3). The
[Ca2+]i response was elicited by 10-300
µM ATP and 3-300 µM 2-MeS-ATP in a dose-dependent manner (Fig.
4). The concentrations that yielded the
half-maximal response (EC50) were 16.7 µM for ATP and
14.4 µM for 2-MeS-ATP.

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Fig. 3.
Effects of purinergic agonists on [Ca2+]i
response of the globular substance. Otoconial membranes were superfused
with APL, and 100 µM of a purinergic agonist was applied by changing
the superfusion solution. Each value represents mean ± SE of maximum relative fluorescence within 5 min after application of the agonist.  -MeATP, , -methylene-ATP; 2-MeS-ATP, 2-methylthio-ATP;
BzATP, 3'-O-(4-benzoylbenzoyl)-ATP. ** P < 0.01, *** P < 0.001.
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Fig. 4.
Dose-response relationships for ATP and 2-MeS-ATP. Otoconial membranes
were superfused with APL, and various concentrations of ATP ( ) or
2-MeS-ATP ( ) were applied by changing the superfusion solution. Each
value represents mean ± SE of maximum relative fluorescence within 5 min after application of the agonist. The 50% effective concentrations
(EC50) are 16.7 and 14.5 µM for ATP and 2-MeS-ATP,
respectively.
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P2 antagonists, RB2 and suramin, completely inhibited the ATP-induced
[Ca2+]i increase (Fig.
5). Moreover, preincubation with 10 µM
U-73122, a phospholipase C inhibitor (14), significantly inhibited the ATP-induced [Ca2+]i increase, whereas the
same concentration of U-73343, an inactive analog of U-73122 (14), had
no effect (Fig. 6).

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Fig. 5.
Effects of P2 antagonists on ATP-induced
[Ca2+]i response of the globular substance.
Otoconial membranes were initially superfused with an antagonist
dissolved in APL for 5 min, followed by exposure to both the antagonist
and 100 µM ATP. Each value represents mean ± SE of maximum relative
fluorescence within 5 min after application of ATP. RB2, reactive blue
2. ** P < 0.01.
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Fig. 6.
Effect of a phospholipase C inhibitor on ATP-induced
[Ca2+]i response of the globular substance.
Otoconial membranes were initially superfused with either 10 µM
U-73122 (phospholipase C inhibitor) or 10 µM U-73343 (inactive
analog) in APL for 10 min, followed by exposure to both the agent and
100 µM ATP. As a control, sample was exposed to 100 µM ATP without
pretreatment. Each value represents mean ± SE of maximum relative
fluorescence within 5 min after application of ATP. *** P < 0.001.
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These results indicate the presence of a P2 purinoceptor on the
globular substance. The pharmacological characteristics of the
reactions strongly suggest that the receptor belongs to the P2Y family.
ATP distribution in the macula.
Many fluorescent spots were observed in the sensory epithelium of the
quinacrine-stained specimen (Fig.
7A). The fluorescence was most
intense at a depth of 2-5 µm from the apical surface. The spots
were 2-5 µm in diameter and 4-8 µm apart from one
another. At a higher magnification, each fluorescent spot consisted of several smaller granules 0.4-0.9 µm in diameter (Fig.
7B). These findings indicate the presence of ATP-containing
granules in the sensory epithelial cells. Meanwhile, the otoconial
membrane showed little or no fluorescence of quinacrine.

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Fig. 7.
Confocal photomicrographs showing ATP distribution in the utricular
macula and cultured cells. Samples were incubated with 5 µM
quinacrine in APL (for utricle) or in the culture medium (for cultured
cells) at room temperature for 30 min, washed, and then directly
observed under a confocal laser scanning microscope as described in
MATERIALS AND METHODS. A: confocal fluorescent image of the utricular sensory epithelium. Optical section image at a 3 µm depth from the apical surface shows many fluorescent spots. Scale
bar = 25 µm. B: higher magnification of A. Each
fluorescent spot is composed of several smaller granules, 0.4-0.9
µm in diameter. Scale bar = 5 µm. C: ordinary
light microscopic image of PC-12 cells. Scale bar = 25 µm. D:
simultaneous confocal fluorescent image of C. Fluorescent
granules are seen in the cytoplasm of the cells. Scale bar = 25 µm.
E: higher magnification of D. Scale bar = 5 µm.
F: ordinary light microscopic image of cultured fibroblasts derived from guinea pig skin. Scale bar = 25 µm. G:
simultaneous confocal fluorescent image of F. Fluorescence is
much less than for the utricular sensory epithelium and PC-12 cells.
Scale bar = 25 µm.
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Fluorescent granules were also seen in the cytoplasm of PC-12 cells
(Fig. 7, D and E), which are known to have
ATP-containing secretory vesicles (35), whereas fibroblasts from the
abdominal skin of the guinea pig showed much less fluorescence (Fig.
7G). No fluorescence was detected in the specimens without
quinacrine treatment that served as controls.
 |
DISCUSSION |
The present study is the first report demonstrating that extracellular
ATP induces a [Ca2+]i increase in the
globular substance. It is unusual that the dynamic change of
[Ca2+]i remained elevated throughout the
exposure to ATP and even after the removal of ATP (Fig. 2). This would
indicate that the globular substance cytoplasts lack a significant
capacity for Ca2+ homeostasis and may suggest that this
process is a vestigial response of the globular substance, which
becomes nonliving otoconial bodies.
A number of authors have documented the biological actions of ATP in
the inner ear. In vivo studies have shown that cochlear perilymphatic
and endolymphatic perfusion with ATP alters cochlear electrical
activity (15, 21) and that this effect is inhibited by purinergic
antagonists (16, 21). In vitro experiments have shown that isolated
inner ear component cells exhibit various physiological and biochemical
reactions in response to ATP and its analogs. In terms of ATP
responses, outer hair cells in the organ of Corti are one of the
best-documented types of cells in the inner ear. Isolated outer hair
cells contract in length (5, 29, 38), depolarize (2, 23), and show
membrane currents (6, 18) and an intracellular Ca2+
increase (2, 25) in response to ATP. These reactions are also observed
in the inner hair cells (8), supporting cells (2, 7), strial marginal
cells (31, 34), and lateral wall epithelial cells (13) of the cochlea.
At the morphological level, the ATP-binding sites of cochlear hair
cells are the stereocilia, cuticular plate, and basolateral margins
(20). Moreover, ATP and its analogs have been shown to reduce the
K+ secretion of strial marginal cells (19) and induce
inositol phosphate release from the lateral wall and sensory epithelium of the cochlea (24, 27).
For vestibular organs, there are fewer studies on the effects of ATP.
ATP-induced membrane currents, intracellular Ca2+ increase,
and cell motility have been reported in vestibular hair cells (28). ATP
and its analogs have also been shown to elicit inositol phosphate
release from the vestibular sensory epithelium (26), reduce the
K+ secretion of vestibular dark cells (34), and modulate
the vestibular transepithelial potential (3).
These lines of evidence suggest that purine-mediated humoral mechanisms
influence the function of the inner ear. The receptors that respond
specifically to ATP but not to adenosine are referred to as P2
purinoceptors. P2 purinoceptors used to be pharmacologically classified
into multiple subtypes, that is, P2X, P2Y,
P2Z, P2U, P2T, and P2D.
Studies with purinergic agonists revealed the presence of various types
of P2 receptors in the cochlea and in the vestibule. P2X,
P2Y, P2Z, and P2U receptors are
present in the cochlear lateral wall (13, 19, 27), P2X and
P2Y in the organ of Corti (24, 26), P2Y and
P2Z in outer hair cells (25), P2Y and
P2U in vestibular dark cells (19), and P2Y in
the vestibular sensory epithelium (26) and semicircular canal (3). More
recently, a new framework of P2 receptors has been established, based
not only on the agonist potency order but also on the transduction mechanism and molecular structure. According to this nomenclature, P2
receptors are divided into P2X (ligand-gated ion channels) and P2Y (G
protein-coupled receptors) families. Seven subtypes of the P2X family
(P2X1-7) and seven subtypes of the P2Y family
(P2Y1-7) have been identified to date (1, 4). The
former P2Y and P2U receptors correspond to
P2Y1 and P2Y2 in the new classification,
respectively (1).
The present experiments with purinergic agonists and antagonists showed
that a P2 receptor exists in the globular substance. The ATP-induced
[Ca2+]i increase was independent of external
Ca2+ (Fig. 2), indicating that Ca2+ was
mobilized from an internal reservoir in the globular substance. The
inhibition of the response by U-73122 (Fig. 6) indicates that the
Ca2+ mobilization is linked to a phospholipase C-dependent
signaling mechanism, which is probably coupled to G protein. These
results indicate that the purinoceptor of the globular substance
belongs to the P2Y family and not to the P2X family. The rank order of agonist potency is similar to that of a cloned chick P2Y1
receptor expressed in Xenopus oocytes (36) but different from
that of a cloned turkey P2Y1 receptor expressed in a human
astrocytoma cell line (9) with respect to the inability of ADP to
elicit the response in the present study. Therefore, the purinoceptor of the globular substance may be a variant of P2Y1 or a new
subtype. The subtype of the receptor could be identified by molecular
cloning techniques. However, it is very difficult to cleanly isolate
the globular substance from adjacent structures such as otoconia, the
gelatinous layer, sensory epithelial cells, nonsensory epithelial lining cells including dark cells, and transitional cells. The sample
in the present study is, therefore, too crude for biochemical, Northern
blot, or polymerase chain reaction analyses.
The globular substance is a noncellular structure and does not possess
a nucleus (33), suggesting that it is incapable of synthesizing
proteins. Why, then, does this material have a purinoceptor? Electron
microscopic studies have demonstrated that the globular substance is
generated on the surface of the macular sensory epithelium by an
apocrine-secretion-like mechanism (12). Moreover, it has been shown
that a P2Y (comparable to P2Y1) receptor is
probably present in the vestibular sensory epithelium and that the ATP response of this tissue is accompanied by the release of inositol phosphate (26). When these observations are taken into account, the P2
receptor of the globular substance may have originated from that of
macular sensory epithelial cells.
Although the role of intracellular ATP is well known, it has been
thought that extracellular release of ATP rarely occurs because of its
impermeability across the cell membrane. Nonetheless, it is also known
that extracellular ATP influences many biological processes, such as
platelet aggregation, vasodilation and constriction, neurotransmission,
cardiac function, and smooth muscle contraction (10).
The source of ATP in the inner ear fluids is controversial. Because of
a tight blood-labyrinth barrier in the normal inner ear, it seems
unlikely that middle- to large-sized molecules in the systemic
circulation directly enter into the inner ear fluids under
physiological conditions. Extracellular ATP may exist in the inner ear
via three sources. First, ATP may come from tissue injury or a
pathological condition in the inner ear. ATP can be released from
endothelial cells during the sudden breakage of blood vessels, from
aggregating platelets by degranulation, and from red blood cells under
conditions of ischemia and hypoxia (10). In fact, Munoz et al. (22)
observed that ATP in the cochlear fluids is increased during hypoxia.
Second, ATP could be coreleased with the putative neurotransmitters of
the afferent (glutamate) or efferent (acetylcholine) systems that
innervate sensory hair cells (7). There is accumulating evidence that ATP is released from sympathetic and parasympathetic nerve endings and
acts as a cotransmitter of both acetylcholine and norepinephrine (17).
However, direct evidence of this phenomenon in the inner ear is not
available at present. In addition, because nerve endings are located on
the basolateral surface facing the perilymphatic space of sensory hair
cells, coreleased ATP is unlikely to reach the endolymphatic space
where the globular substance lies. Third, ATP may be secreted from
endolymphatic surface-lining cells. This type of ATP release would
directly affect the globular substance, which floats in the endolymph.
Using quinacrine, a fluorescent compound that preferentially binds to
adenine nucleotides, particularly to ATP, White et al. (37) have
demonstrated that strial marginal cells in the cochlear lateral wall
have ATP-containing granules. With the use of the same method, we
demonstrated the presence of an ATP-containing granular structure in
the macular sensory epithelium, suggesting that an autocrine or
paracrine mechanism involving ATP may exist in the macula to regulate
the secretion and maturation of the globular substance. Interestingly,
a similar purine-mediated mechanism has been shown to be involved in
the regulation of ion channels of respiratory epithelial cells (30) and
pituitary gland cells (32).
In conclusion, the ATP-induced [Ca2+]i
response of the globular substance and ATP distribution in the guinea
pig macula were investigated by means of confocal laser scanning
microscopy. ATP elicited a rapid and dose-dependent increase in
[Ca2+]i with an EC50 of 16.7 µM. This reaction was independent of external Ca2+,
indicating that Ca2+ was mobilized from an internal
reservoir in the globular substance. Experiments with purinergic
agonists and antagonists and a phospholipase C inhibitor showed that
this ATP-induced response was mediated by a purinoceptor that belongs
to the P2Y family. Furthermore, granular fluorescence was observed in
the quinacrine-stained macular sensory epithelium, indicating the
presence of ATP-containing granules in this tissue. These results
suggest that ATP may regulate the biological behavior of the globular
substance via a paracrine mechanism. The biological significance of ATP
and purinoceptors in the process of otoconial formation remains to be
investigated in future studies.
 |
FOOTNOTES |
Address for reprint requests: H. Suzuki, Dept. of Otolaryngology,
Tohoku Univ. School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai
980-77, Japan.
Received 3 October 1996; accepted in final form 18 June 1997.
 |
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