RAPID COMMUNICATION |
Correspondence to: Bradley A. Schulte, Dept. of Pathology and Laboratory Medicine, Medical Univ. of South Carolina, 171 Ashley Avenue, Charleston, SC 29425..
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Summary |
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A smooth membrane system consisting of subsurface cisternae (SSC) underlies the lateral plasmalemma of auditory outer hair cells (OHCs). The SSC contain Ca-ATPase and are regarded as an intracellular Ca2+ reservoir like the sarcoplasmic reticulum of myocytes. Recently, it has been demonstrated that Ca-ATPase activity in sarcoplasmic reticulum is regulated by Ca2+/calmodulin-dependent protein kinases (CaM kinases). Here we investigated the presence of CaM kinases in OHCs and their possible association with the SSC. Inner ears collected from adult gerbils and from neonates at 2-day intervals between 0 and 20 days after birth were immunostained with antibodies specific for different CaM kinases. A polyclonal antiserum against CaM kinase IV yielded a strong immunostaining reaction along the lateral wall of OHCs. The staining appeared after the tenth postnatal day and continued into adulthood. No other site in the inner ear, including cochlear inner hair cells and vestibular hair cells, was reactive. The kinase's apparent association with the SSC strongly supports its involvement in intracellular Ca2+ homeostasis and suggests a role in regulating the OHCs' slow motile responses. (J Histochem Cytochem 47:712, 1999)
Key Words: cochlea, calmodulin, motility, development, immunohistochemistry, gerbil
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Introduction |
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Free cytosolic calcium (Ca2+) acts widely as an intracellular second messenger. A variety of stimuli can increase cytosolic Ca2+ levels by promoting an influx of extracellular Ca2+ through voltage- or ligand-gated Ca channels or by releasing the ion from intracellular stores sequestered in specialized elements of smooth endoplasmic reticulum. In the opposite direction, Ca-ATPases in the plasma membrane (PMCAs) and smooth endoplasmic reticulum (SERCAs) pump the ion out of the cytosol, returning its concentration to resting levels (
The signaling effect of intracellular Ca2+ can be mediated through activation of Ca2+/calmodulin-dependent protein kinases (CaM kinases). The four known isoforms of CaM kinase have a tissue- and cell type-specific distribution pattern and mediate diverse physiological activities (
In sensory hair cells, Ca2+ plays an important role in mechanoelectrical transduction (
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Materials and Methods |
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Animals and Tissue Processing
Cochleas from 42 Mongolian gerbils (Meriones unguiculatus) were examined. Specimens were taken from three gerbil pups at 2-day intervals between 0 and 20 days after birth and from young adults at 3 months of age. Inner ears from at least three different neonates were evaluated at each developmental time point. The gerbils were born and raised in a quiet room. The 3-month-old animals from this colony have been shown to have normal hearing by electrophysiological testing (
Gerbils were anesthetized by IP injection of urethane (1.5 g/kg body weight). Exsanguination was performed by transcardial perfusion with warm physiological saline containing 0.1% sodium nitrate, followed by 10 ml of a 10% formalin solution containing 0.5% zinc dichromate, pH 5.0, at room temperature (RT). The bulla was opened rapidly, the round window was perforated, and 1.0 ml of fixative was infused gently through the oval window. The inner ears were dissected free and immersed in fixative for 15 min, rinsed with saline, and decalcified by immersion in 0.12 M EDTA (pH 7.0) for 4872 hr at RT with gentle stirring. The EDTA solution was changed daily. The decalcified inner ears were dehydrated in a graded series of ethanols, cleared in Histoclear (National Diagnostics; Manville, NJ), and embedded in Paraplast Plus (Curtin Matheson; Marietta, GA). Serial midmodiolar sections were cut at 5 µm thickness and mounted on glass slides.
For surface preparations, inner ears from three young adults were fixed and processed as described above. After decalcification, the cochleas were bisected with a sharp razor blade and then sliced into half-turns. The tectorial membrane and lateral wall were removed, leaving the organ of Corti attached to the modiolus. The above procedures were performed in 0.1 M PBS, pH 7.2, at RT and the resultant specimens were held in PBS at 4C until further processing.
Immunohistochemistry
Immunostaining of tissue sections was performed as described previously (-subunit (Affinity BioReagents; Golden, CO). The sections were then rinsed in PBS and incubated with appropriate biotinylated secondary antibodies diluted 1:200. After rinsing with PBS, the sections were flooded with avidinbiotinhorseradish peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA ) for 30 min. The sections were again rinsed with PBS and reacted for 10 min in substrate medium containing 3,3'-diaminobenzidine HCl (DAB; Sigma Chemical, St Louis, MO) before dehydration and mounting. To provide accurate comparisons among cochleas at different developmental stages, sections representing all time points were stained together in the same protocol under identical conditions.
Nonimmune goat serum was substituted for the CaM kinase IV antiserum as a methods control procedure. Sections from composite blocks containing a wide range of gerbil, rat, and mouse organs processed in parallel with the cochlea sections provided positive and negative tissue controls for assessing specificity of the CaM kinase IV antiserum.
Immunostaining of Surface Preparations
The dissected specimens of cochlea were equilibrated in PBS for 20 min, immersed in 3% H2O2 for 30 min, and rinsed again with PBS for 20 min. The pieces were then immersed in 1% Triton X-100 in PBS for 1 hr and then in 0.01% Triton X-100 in PBS for 30 min. This was followed by incubation for 1 hr with 5% normal rabbit serum in 0.01% TritonPBS and immersion in a 1:100 dilution of anti-CaM kinase IV, overnight at 4C. After washing in PBS for 20 min, the pieces were incubated with biotinylated rabbit anti-goat IgG diluted 1:300 in 0.01% TritonPBS for 1 hr, rinsed in PBS, immersed in ABC reagent for 2 hr, rinsed again with PBS for 20 min, and reacted with DAB for 10 min. The stained specimens were mounted organ of Corti side up in 50% glycerol in thin wells constructed on a slide glass with cut coverglasses.
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Results |
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An immunosurvey of organs from adult rats and mice confirmed the presence of CaM kinase IV in brain, where the enzyme has previously been found (
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In sections of the adult gerbil inner ear, moderate to strong immunoreactivity for CaM kinase IV was present along the lateral borders of OHCs (Figure 3). Taller OHCs in all three rows from the apical and middle turns of the cochlea showed consistent immunopositivity, whereas the shorter OHCs in the lower basal turn and hook varied from weakly reactive to unstained. In the neonatal series, positive immunostaining first appeared in OHCs between 10 and 12 days after birth and reached adult levels by 20 days after birth (data not shown). No other site in the inner ear, including inner hair cells and vestibular hair cells, evidenced affinity for the CaM kinase IV antiserum.
In surface preparations, all three rows of OHCs showed intense staining with the CaM kinase IV antiserum (Figure 4). The strongest staining was in a rim just under the plasmalemma, although the staining appeared to extend into the cytoplasm and nuclear envelope in some cells. Such cytoplasmic and nuclear reactivity was never observed in sectioned materials and possibly represents a diffusion artifact owing to the extensive detergent permeabilization of the whole-mount preparations.
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Discussion |
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OHCs possess prominent SSC that consist of smooth endoplasmic reticulum (
Immunocytochemical demonstration of SERCA in the SSC (
The results documented the expression of CaM kinase IV in the region underlying the lateral plasmalemma of OHCs, a distribution corresponding with that of SSC. Whether the enzyme resides in membranes of SSC could not be determined with certainty at the light microscopic level of resolution. However, the appearance of CaM kinase IV immunoreactivity at 12 days after birth coincided with the development of SSC (
Immunopositivity for CaM kinase IV also correlated well with the relative abundance of SSC along the gerbil's place-frequency map. The immunostaining appeared weakest and was inconsistent in the short OHCs of the lower basal turn and hook, where the SSC are less prevalent and less well developed (
CaM kinase IV has heretofore been primarily regarded as a regulator of gene transcription (
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Acknowledgments |
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Supported by Research Grants R01 DC00713 and P01 DC00422 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health.
We thank Ms Leslie Harrelson for editorial and Ms Barbara Schmiedt for technical assistance.
Received for publication September 21, 1998; accepted September 22, 1998.
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