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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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, alpha ,beta -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. Fluo 3-pentaacetoxymethyl ester (fluo 3-AM) was purchased from Molecular Probes (Eugene, OR). Adenosine, ADP, ATP, alpha ,beta -methyleneadenosine 5'-triphosphate (alpha beta -MeATP), 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), UTP, quinacrine, and ethylene glycol-bis(beta -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
Relative fluorescence = (F − BG)/(F<SUB>o</SUB> − BG) (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
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Materials & Methods
Results
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References

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 (bullet ; n = 36) or with Ca2+-free APL supplemented with 1 mM EGTA (black-square; n = 14); 100 µM ATP was applied by changing the superfusion solution as indicated in the graph. NS, not significant.

Neither adenosine, alpha beta -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. alpha beta -MeATP, alpha ,beta -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 (bullet ) or 2-MeS-ATP (black-square) 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.

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.

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.

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
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Abstract
Introduction
Materials & Methods
Results
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References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abbracchio, M. P., and G. Burnstock. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol. Ther. 64: 445-475, 1994[Medline].

2.   Ashmore, J. F., and H. Ohmori. Control of intracellular calcium by ATP in isolated outer hair cells of the guinea-pig cochlea. J. Physiol. (Lond.) 428: 109-131, 1990[Abstract].

3.   Aubert, A., C. H. Norris, and P. S. Guth. Influence of ATP and ATP agonists on the physiology of the isolated semicircular canal of the frog (Rana pipiens). Neuroscience 62: 963-974, 1994[Medline].

4.   Brake, A. J., and D. Julius. Signaling by extracellular nucleotides. Annu. Rev. Cell Dev. Biol. 12: 519-541, 1996. [Medline]

5.   Canlon, B., and D. Dulon. Dissociation between the calcium-induced and voltage-driven motility in cochlear outer hair cells from the waltzing guinea pig. J. Cell Sci. 104: 1137-1143, 1993[Abstract/Free Full Text].

6.   Chen, C., A. Nenov, and R. P. Bobbin. Noise exposure alters the response of outer hair cells to ATP. Hear. Res. 88: 215-221, 1995[Medline].

7.   Dulon, D., R. Moataz, and P. Mollard. Characterization of Ca2+ signals generated by extracellular nucleotides in supporting cells of the organ of Corti. Cell Calcium 14: 245-254, 1993[Medline].

8.   Dulon, D., M. Sugasawa, C. Blanchet, and C. Erostegui. Direct measurements of Ca2+-activated K+ currents in inner hair cells of the guinea-pig cochlea using photolabile Ca2+ chelators. Pflügers Arch. 430: 365-373, 1995[Medline].

9.   Filtz, T. M., Q. Li, J. L. Boyer, R. A. Nicholas, and T. K. Harden. Expression of a cloned P2Y purinergic receptor that couples to phospholipase C. Mol. Pharmacol. 46: 8-14, 1994[Abstract].

10.   Gordon, J. L. Extracellular ATP: effects, sources and fate. Biochem. J. 233: 309-319, 1986[Medline].

11.   Hale, J. E., and R. E. Wuthier. The mechanism of matrix vesicle formation. J. Biol. Chem. 262: 1916-1925, 1987[Abstract/Free Full Text].

12.   Harada, Y. Metabolic disorder, absorption area and formation area of the statoconia. J. Clin. Electron Microsc. 15: 1-18, 1982.

13.   Ikeda, K., M. Suzuki, M. Furukawa, and T. Takasaka. Calcium mobilization and entry induced by extracellular ATP in the non-sensory epithelial cell of the cochlear lateral wall. Cell Calcium 18: 89-99, 1995[Medline].

14.   Kimball, B. C., D. I. Yule, and M. W. Mulholland. Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G587-G593, 1996[Abstract/Free Full Text].

15.   Kujawa, S. G., C. Erostegui, M. Fallon, J. Crist, and R. P. Bobbin. Effects of adenosine 5'-triphosphate and related agonists on cochlear function. Hear. Res. 76: 87-100, 1994[Medline].

16.   Kujawa, S. G., M. Fallon, and R. P. Bobbin. ATP antagonists cibacron blue, basilen blue and suramin alter sound-evoked responses of the cochlea and auditory nerve. Hear. Res. 78: 181-188, 1994[Medline].

17.   Kupfermann, I. Functional studies of cotransmission. Physiol. Rev. 71: 683-732, 1991[Free Full Text].

18.   Lin, X., R. I. Hume, and A. L. Nuttall. Voltage-dependent block by neomycin of the ATP-induced whole cell current of guinea-pig outer hair cells. J. Neurophysiol. 70: 1593-1605, 1993[Abstract/Free Full Text].

19.   Liu, J., K. Kozakura, and D. C. Marcus. Evidence for purinergic receptors in vestibular dark cell and strial marginal cell epithelia of the gerbil. Auditory Neurosci. 1: 331-340, 1995.

20.   Mockett, B. G., G. D. Housley, and P. R. Thorne. Fluorescence imaging of extracellular purinergic receptor sites and putative ecto-ATPase sites on isolated cochlear hair cells. J. Neurosci. 14: 6992-7007, 1994[Abstract].

21.   Munoz, D. J. B., P. R. Thorne, G. D. Housley, T. E. Billett, and J. M. Battersby. Extracellular adenosine 5'-triphosphate (ATP) in the endolymphatic compartment influences cochlear function. Hear. Res. 90: 106-118, 1995[Medline].

22.   Munoz, D. J. B., P. R. Thorne, G. D. Housley, and T. E. Billett. Adenosine 5'-triphosphate (ATP) concentrations in the endolymph and perilymph of the guinea-pig cochlea. Hear. Res. 90: 119-125, 1995[Medline].

23.   Nakagawa, T., N. Akaike, T. Kimitsuki, S. Komune, and T. Arima. ATP-induced current in isolated outer hair cells of guinea pig cochlea. J. Neurophysiol. 63: 1068-1074, 1990[Abstract/Free Full Text].

24.   Niedzielski, A. S., and J. Schacht. P2 purinoceptors stimulate inositol phosphate release in the organ of Corti. Neuroreport 3: 273-275, 1992[Medline].

25.   Nilles, R., L. Jarlebark, H. P. Zenner, and E. Heilbronn. ATP-induced cytoplasmic [Ca2+] increase in isolated cochlear outer hair cells. Involved receptor and channel mechanisms. Hear. Res. 73: 27-34, 1994[Medline].

26.   Ogawa, K., and J. Schacht. Receptor-mediated release of inositol phosphates in the cochlear and vestibular sensory epithelia of the rat. Hear. Res. 69: 207-214, 1993[Medline].

27.   Ogawa, K., and J. Schacht. P2Y purinergic receptors coupled to phosphoinositide hydrolysis in tissues of the cochlear lateral wall. Neuroreport 6: 1538-1540, 1995[Medline].

28.   Rennie, K. J., and J. F. Ashmore. Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals. Neurosci. Lett. 160: 185-189, 1993[Medline].

29.   Schacht, J., and H. P. Zenner. Evidence that phosphoinositides mediate motility in cochlear outer hair cells. Hear. Res. 31: 155-160, 1987[Medline].

30.   Schwiebert, E. M., M. E. Egan, T.-H. Hwang, S. B. Fulmer, S. S. Allen, G. R. Cutting, and W. B. Guggino. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 81: 1063-1073, 1995[Medline].

31.   Sunose, H., K. Ikeda, Y. Saito, A. Nishiyama, and T. Takasaka. Nonselective cation and Cl channels in luminal membrane of the marginal cell. Am. J. Physiol. 265 (Cell Physiol. 34): C72-C78, 1993[Abstract/Free Full Text].

32.   Surprenant, A., G. Buell, and R. A. North. P2X receptors bring new structure to ligand-gated ion channels. Trends Neurosci. 18: 224-229, 1995[Medline].

33.   Suzuki, H., K. Ikeda, and T. Takasaka. Biological characteristics of the globular substance in the otoconial membrane of the guinea pig. Hear. Res. 90: 212-218, 1995[Medline].

34.   Suzuki, M., K. Ikeda, H. Sunose, K. Hozawa, C. Kusakari, Y. Katori, and T. Takasaka. ATP-induced increase in intracellular Ca2+ concentration in the cultured marginal cell of the stria vascularis of guinea-pigs. Hear. Res. 86: 68-76, 1995[Medline].

35.   Wagner, J. A. Structure of catecholamine secretory vesicles from PC12 cells. J. Neurochem. 45: 1244-1253, 1985[Medline].

36.   Webb, T. E., J. Simon, B. J. Krishek, A. N. Bateson, T. G. Smart, B. F. King, G. Burnstock, and E. A. Barnard. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett. 324: 219-225, 1993[Medline].

37.   White, P. N., P. R. Thorne, G. D. Housley, B. Mockett, T. E. Billett, and G. Burnstock. Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea: a possible source of extracellular ATP? Hear. Res. 90: 97-105, 1995[Medline].

38.   Zenner, H. P., R. Zimmermann, and A. H. Gitter. Active movements of the cuticular plate induce sensory hair motion in mammalian outer hair cells. Hear. Res. 34: 233-240, 1988[Medline].


AJP Cell Physiol 273(5):C1533-C1540
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society




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