ARTICLE |
Correspondence to: Srdjan M. Vlajkovic, Div. of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: s.vlajkovic@auckland.ac.nz
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Summary |
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Cellular, molecular, and physiological studies have demonstrated an important signaling role for ATP and related nucleotides acting via P2 receptors in the cochlea of the inner ear. Signal modulation is facilitated by ectonucleotidases, a heterologous family of surface-located enzymes involved in extracellular nucleotide hydrolysis. Our previous studies have implicated CD39/NTPDase1 and CD39L1/NTPDase2, members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, as major ATP-hydrolyzing enzymes in the tissues lining the cochlear endolymphatic and perilymphatic compartments. NTPDase1 hydrolyzes both nucleoside triphosphates and diphosphates. In contrast, NTPDase2 is a preferential nucleoside triphosphatase. This study characterizes expression of these E-NTPDases in the mouse cochlea by immunohistochemistry. NTPDase1 can be immunolocalized to the cochlear vasculature and neural tissues (primary auditory neurons in the spiral ganglion). In contrast, NTPDase2 immunolabeling was principally localized to synaptic regions of the sensory inner and outer hair cells, stereocilia and cuticular plates of the outer hair cells, supporting cells of the organ of Corti (Deiters' cells and inner border cells), efferent nerve fibers located in the intraganglionic spiral bundle, and in the outer sulcus and root region of the spiral ligament. This differential expression of NTPDase1 and 2 in the cochlea suggests spatial regulation of P2 receptor signaling, potentially involving different nucleotide species and hydrolysis kinetics. (J Histochem Cytochem 50:14351441, 2002)
Key Words: E-NTPDase, CD39, ectonucleotidase, ATP, P2 receptor, cochlea, mouse, immunohistochemistry
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Introduction |
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The multiple physiological and pathophysiological roles of extracellular purines and pyrimidines are now well established (
Ectonucleotidases play a major role in modulation of P2 receptor-mediated effects by hydrolyzing extracellular nucleotides. A large number of nucleotide-hydrolyzing enzymes have been cloned from mammalian tissues, including members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family that hydrolyze nucleoside 5'-tri- and -diphosphates. Three members of this family (NTPDase1-3) are expressed in the mammalian brain (
ATP-hydrolyzing activity in the cochlea is largely mediated by E-NTPDases (
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Materials and Methods |
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Animals
Experiments described in this study were approved by the University of Auckland Animal Ethics Committee. Adult Swiss white mice (23 months old) were deeply anesthetized with Nembutal (Virbac Laboratories; Auckland, NZ), and perfused transcardially with cold 4% paraformaldehyde (PFA; Scharlau Chemie, Barcelona, Spain) in 0.1 M phosphate-buffered solution (PB: Na2HPO4 77.5 mM; NaH2PO4 22.5 mM, pH 7.4). The cochleae were removed and postfixed in 4% PFA overnight at 4C.
Immunohistochemistry
NTPDase1 and NTPDase2 antibodies were raised in rabbits by direct inoculation of the encoding cDNA in pcDNA3 (
Mouse cochleae fixed in 4% PFA were decalcified in 5% EDTA/PB solution for 7 days, then cryoprotected overnight in 30% sucrose/PB solution. The sections were then rinsed in 0.1 M PB, snap-frozen in isopentane at 80C, and cryosectioned at 20 µm. Sections were placed in 48-well plates (Nalge Nunc; Naperville, IL), rinsed with sterile 0.1 M PBS (Na2HPO4 77.5 mM; NaH2PO4 22.5 mM; NaCl 154 mM, pH 7.4), and permeabilized with 1% Triton X-100 for 1 hr. Nonspecific binding sites were blocked with 2% non-fat milk powder and 1.5% normal goat serum (Vector Laboratories; Burlingame, CA). Primary antibodies to NTPDase1 or NTPDase2 were applied at a titer of 1:5000 in 0.1 M PBS containing 1.5% normal goat serum (Vector) and 0.1% Triton X-100 and incubated overnight at 4C. Vectastain Elite ABC kit (Vector) containing a secondary biotinylated goat anti-rabbit antibody and avidinbiotinperoxidase complex was used for detection of NTPDase immunoreactivity. Incubations with the secondary antibody and avidinbiotin complex were done for 40 min at room temperature (RT). The reaction was visualized using diaminobenzidine (DAB; Vector) as a chromogen. Negative control sections were incubated with the preimmune rabbit serum (titer 1:5000) overnight at 4C. Brain tissue was used as a positive control as described by
Confocal Immunofluorescence
Laser scanning confocal microscopy of whole mounts of cochlear tissues was used to provide high resolution of immunostaining. After fixation in 4% PFA, cochlear tissues were microdissected from the modiolus and rinsed in 0.1 M PBS. The blocking and permeabilization steps were the same as described above for immunohistochemistry. Primary NTPDase1 and NTPDase2 antibodies (titer 1:5000) were applied overnight at 4C. The tissues were then incubated with Alexa 488 goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR; dilution 1:200) for 2 hr at RT. The tissues were rinsed several times in PBS, mounted in fluorescence medium (DAKO; Carpinteria, CA), and screened for NTPDase1- or NTPDase2-specific immunofluorescence using a confocal microscope (TCSAD; Leica Leisertechnik, Heidelberg, Germany).
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Results |
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NTPDase1 Immunolocalization
The most prominent NTPDase1 immunoreactivity was observed in the cochlear vasculature (Fig 1A). Immunopositive blood vessels were detected in the spiral ligament, stria vascularis, spiral limbus, osseous spiral lamina, and Rosenthal's canal (Fig 1A, Fig 1C, and Fig 1D). NTPDase1-specific immunostaining appeared to be associated with endothelial cells/smooth muscle cells of the radiating arterioles and endothelial cells of the capillaries (Fig 2B). No immunoreactivity was observed in the sensory and supporting cells of the organ of Corti (Fig 1B and Fig 2A). NTPDase1-specific immunoreactivity was also detected in the perikarya of the spiral ganglion neurons (SGNs) (Fig 1D). In the spiral ligament, a narrow zone of fibrocytes bordering the otic capsule consistently showed immunolabeling (Fig 1C), whereas Reissner's membrane, which separates scala media from scala vestibuli (not shown), was unstained. Nonspecific staining was observed in the tectorial membrane and osseous spiral lamina in control sections stained with the preimmune serum (Fig 1A inset). Staining in the acellular tectorial membrane, attributable to binding to collagen fibers, is frequently observed in immunocytochemical studies in the cochlea (
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NTPDase2 Immunolocalization
NTPDase2 immunoreactivity was differentially distributed in the tissues bordering scala media. It was observed in the organ of Corti (Fig 1E) but was notably absent from Reissner's membrane (not shown) and marginal cells of the stria vascularis (Fig 1G). Strong immunostaining was observed in both inner and outer hair cell regions. Immunoreactivity in the reticular lamina corresponds to the cuticular plates and stereocilia of the outer hair cells, but not their lateral walls (Fig 1F, Fig 2C, and Fig 2D). The pronounced punctate staining beneath the outer hair cells is most likely attributable to labeling of efferent nerve endings, based on their size and number. Supporting Deiters' cells that form cups underneath the outer hair cells showed moderate immunostaining. Apart from the inner hair cells, strong immunolabeling in the inner hair cell region may also include supporting inner border cells. Intense NTPDase2 immunoreactivity was detected in the inner spiral plexus and outer spiral bundle regions, which provide afferent innervation to the inner and outer hair cells, respectively (Fig 1F). Nerve fibers projecting through the osseous spiral lamina to these regions were stained immunopositive with NTPDase2 antibody (Fig 1E). As for the lateral wall tissues, strong immunostaining was found in the spiral prominence, outer sulcus cells, and their root processes, which are intimately associated with the blood vessels of the spiral ligament (Fig 1G and Fig 2E). Spiral ganglion cell bodies were not stained (compare with NTPDase1 labeling), whereas the efferent nerve fibers of the intraganglionic spiral bundle showed intense immunoreactivity (Fig 1H). Apart from the tectorial membrane, control sections stained with the preimmune serum (Fig 1F, Fig 1G, and Fig 1H insets; Fig 2F) lacked immunolabeling.
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Discussion |
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This study demonstrates an extensive NTPDase1 and NTPDase2 distribution in mouse cochlear tissues, consistent with the postulated role for E-NTPDases in setting the level and kinetics of neurohumoral nucleotide signaling in the inner ear (
The differential expression of NTPDase1 and 2 in vascular, sensory, neural, and secretory tissues is consistent with the wide range of proposed actions of extracellular nucleostides in the cochlea (
The most prominent NTPDase1 immunostaining was observed in blood vessels of the stria vascularis and spiral ligament. Abundant NTPDase1 immunoreactivity in the cochlear vasculature was comparable to NTPDase1 signal levels in blood vessels of the mouse brain (
Both E-NTPDases were also present in neural tissues of the cochlea. NTPDase1 was strongly expressed by the spiral ganglion cell bodies, whereas NTPDase2 immunostaining was more prominent at the dendritic processes of the spiral ganglion neurons providing afferent innervation to the inner and outer hair cells. Co-localization of E-NTPDases and the P2X2 receptor at the SGN cell bodies and their afferent synapses with the inner and outer hair cells (
The present study demonstrates strong NTPDase2 expression at the outer hair cell stereocilia extending to the cuticular plates. In contrast, NTPDase1 was absent from the organ of Corti. This co-localization of NTPDase2 and P2X2 receptor (
The NTPDase2 immunolabeling of Deiters' cells shown here further demonstrates P2 receptor signal termination mechanism, because P2X and P2Y receptor expression has previously been reported for these cells (
A number of studies have indicated that extracellular ATP levels in the cochlear endolymphatic space regulate hearing sensitivity through activation of P2X receptors that reduce the electrochemical driving force for sound transduction via an electrical shunt across the cochlear partition (
In conclusion, this study provides a description of NTPDase1 and NTPDase2 distribution in mouse cochlea. The results indicate tissue-specific actions of NTPDase1 and 2, which are well placed to provide spatial regulation of P2 receptor signaling in the cochlear endolymphatic compartment.
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Acknowledgments |
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Supported by the Auckland Medical Research Foundation, Deafness Research Foundation of New Zealand, and Health Research Council of New Zealand. JS was supported by the American Liver Foundation and the Canadian Institutes of Health Research and SCR by NIH, USA.
We thank Dr Norbert Braun and Professor Herbert Zimmermann (AK Neurochemie, Biozentrum der J.W. Goethe-Universität, Frankfurt am Main, Germany) for their kind gift of the NTPDase2 plasmid used for generation of the NTPDase2 antibody.
Received for publication May 14, 2002; accepted August 1, 2002.
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