Immunocytochemical Localization of Intermediate Filaments in the Guinea Pig Vestibular Periphery with Special Reference to Their Alteration after Ototoxic Drug Administration

Usami, Shin‐ichi; Hözawa, Jirö; Shinkawa, Hideichi; Saito, Shinichi; Matsubara, Atsushi; Fujita, Sachiko

doi:10.3109/00016489309130231

Cited by 14 publications

(10 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although they provide mechanical stability to cells, cytokeratins probably serve other roles as well, including the organization of organelles and the nucleus (15)(16)(17). Cytokeratins, including cytokeratins 8, 9, and 19 and otokeratin, are known to occur in cells of the inner ear (13,(18)(19)(20)(21)(22)(23). A definitive subclassification of our inner-ear cytokeratin within the type II cytokeratin family is difficult because of the sequence conservation within this family and the lack of anuran cytokeratin sequences to use for comparison.…”

Section: Discussionmentioning

confidence: 99%

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Cyr

Bell

Hudspeth

2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Extensive biochemical characterization of cells in the inner ear has been hampered by a lack of tools with which to identify inner-ear proteins. By using a single-chain antibody fragment isolated from a bacteriophage-displayed library, we have identified a cytokeratin that is abundant in nonsensory cells of the frog inner ear. Although the progenitors of hair cells exhibit strong immunoreactivity to this cytokeratin, the signal declines in immature hair cells and vanishes as the cells mature. The correlation between diminished immunoreactivity and hair-cell differentiation indicates that the cytokeratin is down-regulated during the transition from a nonsensory to a sensory cell and suggests that the marker is an early index of hair-cell differentiation. O ur awareness of faint sounds and weak accelerations rests on the sensitivity of hair cells, which respond to subnanometer deflections of their hair bundles (1). The exquisite sensitivity of hair cells unfortunately renders them acutely susceptible to trauma from acoustical overstimulation. In addition, aminoglycoside antibiotics and some chemotherapeutic agents can damage or kill these sensory cells. Because they are postmitotic, hair cells can be replaced only by the differentiation of precursors. Although hair cells do not regenerate in the adult mammalian cochlea, replacement does occur in mammalian vestibular organs and more extensively in nonmammalian receptor organs (2, 3).Little is known about the proteins involved in hair-cell differentiation and function. Although the identification and characterization of inner-ear proteins are impeded by the paucity of cells, the isolation of specific reagents directed against inner-ear proteins makes biochemical studies feasible. To generate such reagents, we produced a bacteriophage-displayed antibodyfragment library directed against proteins of the bullfrog inner ear (4). From this library, we isolated a single-chain Fv fragment (scFv) that specifically labels a protein expressed almost exclusively in the inner ear. We describe here the further characterization and identification of this protein as a cytokeratin whose expression declines during hair-cell differentiation. Materials and MethodsThe production of a library of bacteriophage-displayed scFvs and the selection of a clone producing an scFv with a high specificity for an inner-ear antigen have been described (4). For the present study, soluble scFvs from clone scFv-278 were isolated by the procedures in that reference.Immunocytochemistry on Saccular Whole Mounts. Bullfrog sacculi were isolated and the otolithic membranes were mechanically removed. Sacculi were fixed for 1 h at 4°C in 4% (wt͞vol) formaldehyde in PBS (68 mM NaCl͞58 mM Na 2 HPO 4 ͞17 mM NaH 2 PO 4 , pH 7.4), rinsed in PBS, permeabilized for 1 h in PBS containing 1% (vol͞vol) Triton X-100, and washed in PBS. Samples were blocked for 2 h in PBS containing 2.5% (vol͞vol) Liquid Block (Amersham Pharmacia) and 3% (vol͞vol) goat serum, then incubated overnight at 4°C in a mixture of 1.8 mg͞liter aff...

show abstract

Section: Discussionmentioning

confidence: 99%

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Cyr

Bell

Hudspeth

2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…2B). Furthermore, double-labeling experiments with a monoclonal antibody staining neurofilaments exclusively in the nerve endings of type I cells (13) revealed that all KCNQ4-expressing cells were of type I (data not shown). The absence of KCNQ4 labeling of type II hair cells (which lack the calyx nerve ending) was confirmed by immunoelectron microscopy.…”

Section: Resultsmentioning

confidence: 99%

KCNQ4, a K ⁺ channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway

Kharkovets

Hardelin

Safieddine

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

382

414

View full text Add to dashboard Cite

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, an autosomal dominant form of progressive hearing loss in humans. In the mouse cochlea, the transcript has been found exclusively in the outer hair cells. By using specific antibodies, we now show that KCNQ4 is situated at the basal membrane of these sensory cells. In the vestibular organs, KCNQ4 is restricted to the type I hair cells and the afferent calyx-like nerve endings ensheathing these sensory cells. Several lines of evidence suggest that KCNQ4 underlies the I K,n and gK,L currents that have been described in the outer and type I hair cells, respectively, and that are already open at resting potentials. KCNQ4 is also expressed in neurons of many, but not all, nuclei of the central auditory pathway, and is absent from most other brain regions. It is present, e.g., in the cochlear nuclei, the nuclei of the lateral lemniscus, and the inferior colliculus. This is the first ion channel shown to be specifically expressed in a sensory pathway. Moreover, the expression pattern of KCNQ4 in the mouse auditory system raises the possibility of a central component in the DFNA2 hearing loss.

show abstract

“…The cellular levels of all of the above-mentioned enzymes, which very likely play key roles in active transport of ions in their respective cells, were found to be affected by the gentamicin treatment. Furthermore, the intermediate filament vimentin has been shown to be involved in response to tissue damage (Oesterle et al 1990;Calvo et al 1991;Gallanti et al 1992;SundarRaj et al 1992;Usami et al 1993) and has been reported to be present within the cochlea (Anniko et al 1987;Schulte and Adams 1989b;Shi et al 1993). Staining for vimentin was found to be changed in some cells in response to gentamicin treatment.…”

Section: Cytochemical Effects Of Gentamicin Treatmentmentioning

confidence: 99%

“…Calcium-binding proteins and the IP 3 R very likely that is essential for cochlear function (Kikuchi et al 1995). The intermediate filament vimentin has been shown to be involved in response to tissue damage (Oesterle et al 1990;Berggren et al 1990;Calvo et al 1991;Gallanti et al 1992;SundarRaj et al 1992;Usami et al 1993) and has been reported to be present within the cochlea (e.g., Anniko et al 1987;Schulte and Adams 1989b;Shi et al 1993). Staining for neurofilaments was used to judge nerve fiber density.…”

Section: Introductionmentioning

confidence: 99%

Changes in Cytochemistry of Sensory and Nonsensory Cells in Gentamicin-Treated Cochleas

Imamura

Adams

2003

JARO - Journal of the Association for Research in Otolaryngolog

View full text Add to dashboard Cite

Effects of a single local dose of gentamicin upon sensory and nonsensory cells throughout the cochlea were assessed by changes in immunostaining patterns for a broad array of functionally important proteins. Cytochemical changes in hair cells, spiral ganglion cells, and cells of the stria vascularis, spiral ligament, and spiral limbus were found beginning 4 days post administration. The extent of changes in immunostaining varied with survival time and with cell type and was not always commensurate with the degree to which individual cell types accumulated gentamicin. Outer hair cells, types I and II fibrocytes of the spiral ligament, and fibrocytes in the spiral limbus showed marked decreases in immunostaining for a number of constituents. In contrast, inner hair cells, type III fibrocytes and root cells of the spiral ligament, cells of the stria vascularis, and interdental cells in the spiral limbus showed less dramatic decreases, and in some cases they showed increases in immunostaining. Results indicate that, in addition to damaging sensory cells, local application of gentamicin results in widespread and disparate disruptions of a variety of cochlear cell types. Only in the case of ganglion cells was it apparent that the changes in nonsensory cells were secondary to loss or damage of hair cells. These results indicate that malfunction of the ear following gentamicin treatment is widespread and far more complex than simple loss of sensory elements. The results have implications for efforts directed toward detecting, preventing, and treating toxic effects of aminoglycosides upon the inner ear.

show abstract

Immunocytochemical Localization of Intermediate Filaments in the Guinea Pig Vestibular Periphery with Special Reference to Their Alteration after Ototoxic Drug Administration

Cited by 14 publications

References 20 publications

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

KCNQ4, a K ⁺ channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway

Changes in Cytochemistry of Sensory and Nonsensory Cells in Gentamicin-Treated Cochleas

Contact Info

Product

Resources

About

Immunocytochemical Localization of Intermediate Filaments in the Guinea Pig Vestibular Periphery with Special Reference to Their Alteration after Ototoxic Drug Administration

Cited by 14 publications

References 20 publications

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

KCNQ4, a K + channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway

Changes in Cytochemistry of Sensory and Nonsensory Cells in Gentamicin-Treated Cochleas

Contact Info

Product

Resources

About

KCNQ4, a K ⁺ channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway