ARTICLE |
Correspondence to: Bradley A. Schulte, Dept. of Pathology and Laboratory Medicine, Medical U. of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
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Summary |
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Oncomodulin (OM) is a small, acidic calcium-binding protein first discovered in a rat hepatoma and later found in placental cytotrophoblasts, the pre-implantation embryo, and in a wide variety of neoplastic tissues. OM was considered to be exclusively an oncofetal protein until its recent detection in extracts of the adult guinea pig's organ of Corti. Here we report that light and electron microscopic immunostaining of gerbil, rat, and mouse inner ears with a monoclonal antibody against recombinant rat OM localizes the protein exclusively in cochlear outer hair cells (OHCs). At the ultrastructural level, high gold labeling density was seen overlying the nucleus, cytoplasm, and the cuticular plate of gerbil OHCs. Few, if any, gold particles were present over intracellular organelles and the stereocilia. Staining of a wide range of similarly processed gerbil organs failed to detect immunoreactive OM in any other adult tissues. The mammalian genome encodes one - and one ß-isoform of parvalbumin (PV). The widely distributed
PV exhibits a very high affinity for Ca2+ and is believed to serve as a Ca2+ buffer. By contrast, OM, the mammalian ß PV, displays a highly attenuated affinity for Ca2+, consistent with a Ca2+-dependent regulatory function. The exclusive association of OM with cochlear OHCs in mature tissues is likely to have functional relevance. Teleological considerations favor its involvement in regulating some aspect of OHC electromotility. Although the fast electromotile response of OHCs does not require Ca2+, its gain and magnitude are modulated by efferent innervation. Therefore, OM may be involved in mediation of intracellular responses to cholinergic stimulation, which are known to be Ca2+ regulated. (J Histochem Cytochem 46:29-39, 1998)
Key Words: cochlea, calcium binding proteins, immunohistochemistry, parvalbumin, gerbil, rat, mouse
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Introduction |
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Parvalbumins (PVs) are small (Mr 11,500), vertebrate-specific proteins (
The PV family includes two sublineages, and ß (
PVs are less acidic (pI
5) than the ß-isoforms and have an additional amino acid residue in the C-terminal helix. At present, the mammalian genome is known to encode only one
- and one ß-isoform of PV (
PV is also expressed in a number of nonexcitatory tissues (reviewed in
The mammalian PV ß-isoform was discovered in a rat hepatoma (
Previous work had demonstrated immunoreactivity for PV in inner hair cells (IHCs) of guinea pig and gerbil cochlea and in vestibular hair cells of guinea pig, rat, and mouse (-isoform of PV, because this was the only isoform believed to be expressed in adult mammals. With the discovery of the ß PV in the mature guinea pig organ of Corti, it became necessary to re-examine this issue. The availability of immunological probes against OM has provided an opportunity to unequivocally ascertain the distribution of the PV
- and ß-isoforms in the inner ear. Using monoclonal and polyclonal probes raised against OM, we report here that the ß PV is expressed exclusively in outer hair cells of the gerbil, rat, and mouse cochlea.
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Materials and Methods |
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Tissue Collection and Processing
Inner ears were obtained from twenty Mongolian gerbils (Meriones unguiculatus) of both genders, ranging in age from 12 days after birth to 10 months, and from three each 3- to 4-month-old Sprague-Dawley rats and C57BL/6J strain mice. The gerbils were born and raised in an acoustically controlled low-noise environment. The care and use of the animals was approved by The Medical University of South Carolina's Animal Care and Use Committee under NIH grant number R01 DC00713.
All animals were anesthetized with urethane (1.5 g/kg IP) and exsanguinated by transcardial perfusion with 10 ml of near-body temperature physiological saline containing 0.1% sodium nitrite, followed by fixative. The fixatives employed for light microscopic (LM) studies included a 10% solution of formalin in 0.9% saline containing 0.5% zinc dichromate (zinc-formalin), with the pH adjusted to 5.0 with NaOH just before use, or a mixture of ethanol, chloroform, and glacial acetic acid at a volume ratio of 6~3~1 (Carnoy's solution). After coronary perfusion of fixative the bullae were opened rapidly, the stapes was removed, the round window was perforated, and fixative was gently injected into the scala vestibuli via the oval window. The length of total fixation time was approximately 30 min.
Specimens processed for LM observation were dissected from the temporal bone and decalcified in either 0.12 M EDTA (pH 7.2) for 48 hr or 8 N formic acid overnight with gentle stirring. The ears were flushed with 0.1 M PBS, pH 7.2, dehydrated in a graded series of ethanols, cleared in Histoclear (National Diagnostics; Manville, NJ), and embedded in Paraplast Plus (Curtin Matheson; Marietta, GA).
A wide range of systemic organs from two adult gerbils were fixed in Carnoy's solution and embedded in composite paraffin blocks to survey at the LM level for OM in other tissues. The specimens included brain, alimentary tract, skeletal muscle, testis, ovary, thyroid, eye, thymus, lymph node, kidney, heart, pancreas, skin, fat, tongue, adrenal, liver, and spleen. Sections from the composite blocks were stained in each protocol with the immunoperoxidase procedure described below.
For electron microscopic (EM) immunostaining, inner ears were fixed identically to those described above for LM studies except that the fixative consisted of a mixture of 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M PB, pH 7.2. After a 1-hr exposure to fixative the specimens were decalcified with EDTA as described above, sliced into half turns, dehydrated through a graded series of 50, 70, 90, and 95% ethanols, and infiltrated with Lowicryl K4M at -20C. The half turns were then oriented in Beem capsules containing fresh K4M resin and were polymerized under 360-nm wavelength UV for 24 hr at -20C and 48 hr at room temperature (RT) (
Preparation of Antibodies
A rabbit polyclonal antiserum and a mouse monoclonal antibody (MAb) were prepared against recombinant rat OM coupled to keyhole limpet hemocyanin, as described elsewhere (
For the preparation of MAbs, female BALB/c mice received two 0.2-ml injections (IP) of the antigen-adjuvant emulsion 5 weeks apart. After 15 weeks, the immunization protocol of
The polyethylene glycol-induced fusion of splenocytes with murine myeloma cells was performed using a modification of standard protocols (e.g.,
The PEG fusogen was prepared just before use by melting 30 g of PEG 1500 (Aldrich Chemicals; Milwaukee, WI) at 50-60C, mixing with 30 ml of warm RPMI-Hepes, adjusting the pH to 7.2 with NaOH, and filter-sterilizing. Two ml of this solution was added dropwise over the course of 1 min to the splenocyte-myeloma pellet. After an additional min at 37C, the PEG was gradually diluted with RPMI-Hepes, first with 2.0 ml added at a rate of 1.0 ml per min, then with an additional 8 ml added over the course of 1 min. After 2 more min at 37C, the cells were collected by centrifugation (1000 rpm, 5 min).
The cells were resuspended at 1.5 x 106 splenocytes/ml and dispensed into 96-well plates. The plating medium contained RPMI 1640 supplemented with NaHCO3, 10% heat-inactivated FBS, 2 mM L-Gln, 80 µg/ml gentamycin sulfate, and HAT components at standard concentrations. The cultures were maintained on HAT until expanded to 24-well plates. The weaning schedule consisted of 50% HAT, 25% HT, and finally standard RPMI, supplemented with 10% FBS, L-Gln, and gentamycin.
Hybridomas were assayed by ELISA and Western blot. The 1A10 antibody used for these studies belongs to the IgG1 subclass and harbors light chains, as determined with the Isotyper kit from Boehringer-Mannheim (Indianapolis, IN). Although its epitope has not been mapped, the antibody does not crossreact with rat muscle
PV or with either of the two avian thymic PVs, ATH and CPV3.
Immunoperoxidase Staining
Serial midmodiolar 5-µm-thick sections were mounted on chrome alum-subbed slides. Every twenty-fifth section was stained with hematoxylin and eosin. Selected sections were processed for immunostaining as described previously (
Control sections were processed in parallel, substituting nonimmune rabbit serum (NRS) at a dilution of 1~200 in PBS for primary antibody. As a further control, sections were exposed either to polyclonal antiserum or to MAb that had been preincubated for 24 hr at 4C with 200 µg/ml recombinant OM.
To further investigate the inconsistent immunostaining of OHC nuclei in paraffin sections, inner ears from two adult gerbils were fixed as described above with a solution of 4% paraformaldehyde and 2% glutaraldehyde, decalcified in EDTA, sliced into half turns, and embedded in Epon. One-µm-thick radial sections from these specimens were etched with a 1~1 solution of alcoholic NaOH~absolute ethanol for 15 min, followed by four rinses for 5 min each in absolute ethanol (
Immunoelectron Microscopy
Ultrathin sections of cochlear half turns were cut and picked up on formvar- and carbon-coated nickel grids (Ted Pella; Redding, CA). The grids were incubated for 1 hr with 5% NGS in PBS and then reacted overnight at 4C with either the polyclonal anti-OM at a dilution of 1~800 or the MAb at a dilution of 1~10 with NGS in PBS. Control sections were processed in parallel but substituting primary antibody with a similar dilution of NRS. The grids were rinsed in PBS and then incubated for 2 hr at RT with a 1~20 dilution of goat anti-rabbit IgG adsorbed to the surface of 15-nm colloidal gold particles (Bio Cell Research Laboratories; Cardiff, UK) for the polyclonal antiserum or goat anti-mouse IgG absorbed to the surface of 10-nm gold particles (Sigma) for the MAb. After washes with PBS and distilled water, grids were stained with uranyl acetate for 15 min and lead citrate for 3 min and were examined in a JEOL-100S electron microscope.
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Results |
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LM Immunohistochemistry
Fixation with Carnoy's solution provided the best retention of OM antigenicity with MAb 1A10 and the polyclonal antiserum. Inner ears fixed with the zinc-formalin solution showed weaker but still strong immunoreactivity with both probes. No differences in immunostaining were noted between the two decalcifying procedures.
In paraffin sections of gerbil inner ear, immunostaining for OM with MAb 1A10 was confined to OHCs in the organ of Corti (Figure 1 and Figure 2). Reaction product was distributed diffusely throughout the OHC's somata, but stereocilia were unreactive (Figure 2). The immunostaining reaction was of similar intensity in all rows of OHCs throughout all turns of the cochlea.
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As with the MAb, the polyclonal antiserum imparted strong staining to all gerbil OHCs, but differed in reacting also with cochlear IHCs (Figure 3) and some vestibular hair cells (Figure 5). The IHCs showed a longitudinal staining gradient, with those in the apex staining intensely and those in the base only weakly. The immunolabeling pattern in the mouse and rat inner ear (Figure 6) was identical to that observed in the gerbil with both immunological probes.
In paraffin sections, the nuclei of OHCs showed inconsistent staining with both the polyclonal antiserum and MAb 1A10 (Figure 2 and Figure 3), most probably owing to failure of staining reagents to penetrate uncleaved nuclei. In contrast, in etched 1-µm-thick epoxy sections, OHC nuclei were consistently strongly reactive with both the polyclonal antiserum (Figure 3, inset) and the MAb (not shown).
No staining was seen on sections in which nonimmune rabbit serum was substituted at a similar dilution for primary antiserum. Preabsorption of a 1~5 dilution of MAb with 200 µg/ml of recombinant OM eliminated all specific staining in OHCs (Figure 4). Preincubation of a 1~200 dilution of the polyclonal antiserum with 200 µg/ml of recombinant OM also blocked staining in all sites, including IHCs and vestibular hair cells (not shown). Examination of composite blocks containing a wide range of gerbil organs revealed no immunopositive sites in any of the tissues studied.
Immunoelectron Microscopy
At the EM level, immunogold labeling with MAb 1A10 again was confined exclusively to OHCs in the gerbil's organ of Corti. Colloidal gold particles were distributed diffusely over the OHC's cytoplasm and nucleus (Figure 7). Mitochondria failed to stain (Figure 7 Figure 8 Figure 9 Figure 10). The gold labeling in OHCs extended uniformly over the cuticular plate (Figure 10 and Figure 11), but stereocilia were unlabeled (Figure 11). Gold particles also were present in the region of the lateral subsurface cisternae and the postsynaptic cisternae, but poor preservation of these structures made it impossible to ascertain if immunoreactive OM was present inside or occupied cytoplasm adjacent to the cisternae.
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With MAb 1A10, no positive staining was seen in any other site in the inner ear, including Deiter's cells and nerve endings (Figure 7 and Figure 9) and IHCs (Figure 12). In agreement with the LM results, the polyclonal antiserum to OM showed weaker but consistent staining in the cytoplasm of cochlear IHCs (Figure 13). No staining was present on sections in which nonimmune rabbit serum was substituted for primary antibody.
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Discussion |
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In this study we examined the distribution of the mammalian ß PV (OM) in three different species, using both monoclonal and polyclonal immunological probes. Our results provide an interesting counterpoint to previous work on PV in the inner ear (-isoform of PV. Consistent with this hypothesis, the polyclonal anti-OM preparation likewise stains a subpopulation of vestibular hair cells, as previously reported (
PV. Moreover, the OM epitope recognized by MAb 1A10 apparently is not shared by the
-isoform.
EF-hand Ca2+ binding sites can be divided into two categories on the basis of their affinities for Ca2+ and Mg2+ (10-6 M) and negligible affinity for Mg2+ at physiological pH and ionic strength. These sites, unoccupied at resting-state intracellular Ca2+ concentrations, bind Ca2+ after an increase in cytosolic Ca2+ levels. Because the conformational change that accompanies Ca2+ binding can expose effector binding sites, Ca2+-specific sites are ideally suited for performing Ca2+-dependent regulatory roles.
The PV metal ion binding domains, known as the CD and EF sites, usually belong to the Ca2+/Mg2+ category (e.g., PV (
The literature regarding OM's putative regulatory capacity is both confusing and controversial. The preliminary reports that OM stimulates cyclic nucleotide phosphodiesterase (
The presence of the AB domain, the nonfunctional vestige of an EF-hand motif, in the PV tertiary structure prevents CaM-like interactions with helical peptide targets (
The mammalian auditory system owes its unrivaled performance to the "cochlear amplifier," a unique specialization wherein the receptor potential of the OHC does not serve to elicit a neural response, but rather, in a process of reverse transduction, is converted into a rapid motile response (
Detailed speculation regarding the role of OM in the OHC is premature. It is significant, however, that the OHC, undoubtedly the most highly differentiated hair cell in the entire acoustic-lateralis system, would be associated with such an unusual highly specialized molecule as OM.
It appears unlikely that OM is involved in the mechano-electrical transduction process or in the attendant adaptation phenomena in which the tension of the tip links of the stereocilia is being adjusted (
An involvement of OM in afferent transmission is also unlikely because of the absence of all but a minimal afferent innervation of the OHC. The small contingent of afferent fibers is unmyelinated and appears to convey proprioceptive-like rather than acoustic information (
Consequently, by elimination, we speculate that OM is involved in some way in the most specialized aspect of OHC function, the cochlear amplifier. Because electromotility continues in the absence of Ca2+, direct involvement of OM with the fast motor system can be discounted (
In response to a variety of stimuli, OHCs also display slow motility, contractions and elongations occurring on a time scale of seconds and minutes (
Existing data regarding the presence of OM in the nucleus are contradictory. Neither
The possibility that OM could function as a specialized cytosolic Ca2+ buffer within the OHC should not be dismissed. -isoform for Ca2+ buffering activity renders this hypothesis unlikely. However, until an effector protein for OM is identified, a Ca2+ buffering role for OM cannot be excluded.
The cochlear hair cell is apparently the sole site of OM expression in postnatal mammals. In the interest of optimizing its acoustic response, the organ of Corti has retained only the most minimal metabolic machinery. Viewed against this austere backdrop, the recruitment of the PV ß-isoform to the OHCs appears particularly significant. Whether it serves in a regulatory or an ion buffering capacity, it is likely that OM is vital to some unique aspect of OHC physiology or function.
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Acknowledgments |
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Supported by Research Grants R01 DC00713 (BAS), R01 DC01414 (RT), and P01 DC00422 (BAS) from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health.
We thank Ms Leslie Harrelson and Ms Nancy Smythe for editorial and technical assistance.
Received for publication February 19, 1997; accepted July 17, 1997.
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