Immunohistochemical localization of fibronectin in the chinchilla cochlea.

The purpose of this study was to better understand the molecular composition of the cochlea. Fibronectin (FN), a well characterized adhesive glycoprotein, was localized by immunofluorescence microscopy in fresh and fixed cochlear tissues, and in fixed kidney tissue, using a polyclonal, affinity-purified, rabbit, anti-fibronectin antibody and a secondary antibody coupled to FITC. The FN antibody was free from cross-reactivity with other known basement membrane and cell matrix molecules. FN reactivity in the cochlea was most intense in the basilar membrane, latero-basal borders of Boettcher's cells, otic capsule, endothelial basement membranes (particularly those of the stria vascularis), and as a diffuse, fan-shaped network radiating into the spiral ligament. Little FN labelling was present in the epithelial basement membranes. Negative control tissue showed no immunoreactivity; whereas, positive kidney control tissue showed appropriate FN immunoreactivity in the mesangium of the glomerulus. The most significant finding of this study was that FN is a major component of the basilar membrane and its distribution appears to correspond to the amorphous ground substance. FN was not localized in the organ of Corti or at the tips of the hair-cell stereocilia.

Adenylate cyclase and G-proteins as a signal transfer system in the guinea pig inner ear.

In many eukaryotic cells G-proteins play a key role in signal transduction through outer cell membranes. To study this pathway in the auditory organ of mammals we examined tissue preparations from the stria vascularis and the organ of Corti from the guinea pig inner ear. The activity of adenylate cyclase was measured by stimulation at the site of the enzyme, the hormone receptors and the modulating G-proteins. In the organ of Corti we found a low enzyme activity in all cochlear turns. The stria vascularis, however, showed a constant high concentration of beta 2-adrenergic receptors and of stimulating G-proteins in all cochlear turns. In contrast, the activity of the enzyme increased from the apical to the basal turn. Adenylate cyclase could be stimulated or inhibited in a concentration-dependent manner by drugs selectively effecting the G-proteins. Our results suggest a structure of the adenylate cyclase complex in the inner ear similar to other organs. Pathophysiological correlations to hearing loss associated with pseudohypoparathyroidism are discussed.

Immunohistochemical demonstration of the co-expression of vimentin and cytokeratin(s) in the guinea pig cochlea.

Using a decalcification procedure for guinea pig petrous bones, we were able to prepare cryostat sections for immunohistochemical studies. A panel of monoclonal antibodies against intermediate filament proteins was then used to show a co-expression of cytokeratin and vimentin in the epithelial cells of the stria vascularis.

Elemental composition of individual cells and tissues in the cochlea.

Localization of elements at the cellular and sub-cellular levels was performed with the energy dispersive X-ray microanalysis technique, using shock-frozen, freeze-dried and araldite-embedded mouse (CBA/CBA) cochleae sectioned dry. Anatomical identification occurred in the STEM (scanning transmission electron microscopy) mode. In inner hair cell stereocilia the K/Na ratio was 70:1 but only 20:1 in the cytoplasm. In outer hair cell cytoplasm the K/Na ratio was 11:1 while the ratio in stereocilia was similar to that in inner hair cells. Ca was identified in stereocilia and the upper part of the cytoplasm of both outer and inner hair cells. The elemental composition in the subtectorial space is endolymph-like and that in the inner tunnel of the organ of Corti is similar to extracellular fluid. Considerable regional differences in elemental composition occur in the tectorial membrane with regard to P, K and Ca. The highest concentration of Ca occurs in the basal part of the tectorial membrane towards the sensory hairs. The highest concentration of K occurs in the basal and outer parts whereas the middle part of the tectorial membrane contains low levels of both K and Ca. The elemental composition changes in two main directions: 1) from the limbal (growing) region to the tip of the tectorial membrane, and, 2) from upper to lower surfaces. The three cell types of the stria vascularis differ considerably in elemental composition. The highest concentration of K occurs in marginal cells. The basal cells contain more K than do the intermediate cells. A significantly higher concentration of Ca, Cl and Na occurs in marginal cell cytoplasm than in any other cell type in the stria vascularis.

Several distinct receptor binding enkephalins in olivocochlear fibers and terminals in the organ of Corti.

Biochemical studies centering on the use of reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassays (RIA) demonstrate the presence in the guinea pig organ of Corti of at least 3 enkephalin-related peptides, two of which are identified as Met- and Leu-enkephalin, respectively. Enkephalins were identified and quantitated by HPLC-RIA in the isolated second turn of the organ of Corti, but were not found in stria vascularis or auditory nerve dissected from the cochlea. Three enkephalin-immunoreactive HPLC fractions inhibited the binding of labeled naloxone to rat brain membranes. All enkephalins identified by the combined HPLC-RIA procedure had an apparent molecular weight similar to that of Met- and leu-enkephalin peptide standards. Immunocytochemistry, performed with the best-characterized Met-enkephalin antiserum used in the RIAs, localized the enkephalin-like immunoreactivity to lateral efferent fibers and terminals under inner hair cells of the organ of Corti. Other antisera raised against Met-enkephalin, not used for RIA, visualized enkephalin-like immunoreactivity in medial efferent fibers under outer hair cells as well. This enkephalin-like immunoreactivity may reflect the presence in the medial efferent system of other structurally similar peptides in addition to those detected biochemically. Efferent fiber lesion, by evulsion of the vestibular nerve close to the vestibulocochlear anastomosis in which the olivocochlear fibers run, eliminated enkephalin-like immunoreactivity and the enkephalin-related peptides identified by HPLC-RIA.

Basophilic deposits in the stria vascularis. A clinicopathological update.

Basophilic deposits in the stria vascularis have been found in association with a multitude of disease entities. Their true significance and nature remain obscure. In the temporal bone collection at the Massachusetts Eye and Ear Infirmary are 22 cases with basophilic deposits in the stria vascularis. The ages of the subjects studied ranged from two days to 83 years. Atrophy of the stria vascularis was the main pathology seen in their temporal bones. The deposits were found in both ears in 82% of cases. They tend to show a striking polymorphism. Special histochemical stains suggest they contain glycogen. Hypertension and uremia were two health problems most of the subjects were suffering from during life. A theory is advanced as to the immune complex nature of these deposits. A review of the literature concerning this subject is also offered.

Adenine nucleotides of the organ of Corti under metabolic stress.

The purpose of the reported experiments was to measure the concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in the organ of Corti in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions the concentrations of ATP, ADP, and AMP amounted to 15.8, 3.9, and 0.53 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since ischemia of 65 seconds leads to an increase of 155%. Under normal conditions the adenylate energy charge, the energy status, and the phosphorylation state amounted to 0.83, 4.1, and 2.5 gram wet weight/mumole, respectively. Within 10 minutes of ischemia the energy charge had declined by 26%, the energy status by 50%, and the phosphorylation state by 76%. The apparent equilibrium constant of adenylate kinase of the organ of Corti was found to be 0.55. The potential significance of these data and their relationship to the situation in the stria vascularis are discussed.

Changes in cation contents of stria vascularis with ouabain and potassium-free perfusion.

Perfusion of the perilymphatic space of guinea pig cochleae with K-free medium leads to a gradual decline of the endocochlear potential (EP) over 30-50 min to a negative value (mean: -12 mV). The input resistance of scala media does not decrease during this time. The ATP and K content of the stria vascularis are reduced by similar amounts (26 and 34%, respectively) during this period. Perfusion of 1 mM ouabain produces a different pattern of response: strial ATP remains normal while strial K content is strongly reduced (by 77%). Strial Na rises in a complementary way to the K loss. These results demonstrate that a reduction of the K concentration of the perilymph leads to an inhibition of the generator of the positive component of the EP rather than to a general increase of cochlear duct membrane conductance. In addition, they suggest, in concert with other considerations (such as the slower rate of decline of the EP during K-free vascular perfusion (Wada, J., Kambayashi, J., Marcus, D.C. and Thalmann, R (1979): Arch. Otorhinolaryngol. 225, 79-81)), that the mode of action may be different from that of ouabain. In spite of the lack of teleological support, we offer the hypothesis that the strial generator of the EP may primarily utilize K from perilymph and that vascular K may not have access to the generator.

Autoradiographic localization of vitamin A in the stria vascularis of the rat cochlea.

Vitamin A is present in the membranous cochlea at a higher concentration than in most other mammalian tissues. In order to localize this substance in the cochlea, 11(3)[H] retinyl acetate was administered to vitamin A-deficient rats. An autoradiographic study of these cochleas revealed labelling of the stria vascularis; the sensory cells and ganglion cells were not labelled.

Analysis of proteins of the stria vascularis of the normal and the waltzing guinea pig.

Proteins of the stria vascularis of the normal and the genetically deaf waltzing guinea pig were analysed by one and two-dimensional acrylamide gel electrophoresis. Cochlear proteins were labeled in vivo by replacing the perilymph with a solution containing radioactive precursors. With the two-dimensional analysis, more than 200 polypeptides were resolved. Proteins that are exposed on the endolymphatic surface of the stria vascularis were identified by lactoperoxidase-catalysed iodination. Seven polypeptides were identified with this technique. No consistent changes in protein patterns of the stria vascularis from the waltzing guinea pig were detected.

The morphologic effects of histamine on the lateral cochlear wall.

The chinchilla lateral cochlear wall (stria vascularis, spiral ligament, and spiral prominence) was examined by morphologic and histochemical techniques following various doses of intravenous histamine. The three main findings were as follows: (1) the basic ultrastructure was not altered by histamine; (2) there is a time- and dose-dependent change in the rate of stria vascularis vessel permeability to a small protein tracer (horseradish peroxidase), but the mode of transport (large pore system) is unchanged; and (3) glycogen depletion in stria marginal cells occurs with its apparent mobilization into stria intercellular spaces.

Element content of intracochlear fluids, outer hair cells, and stria vascularis as determined by energy-dispersive roentgen ray analysis.

Roentgen ray spectrometry was used to obtain element spectra from isolated samples of perilymph, endolymph, outer hair cells, and stria vascularis obtained by microdissection from the freeze-dried inner ears of chinchillas. The spectra of perilymph and endolymph residues indicated that no cross-contamination of the two cochlear fluids occurs during freeze-drying or sampling. The spectra of outer hair cells suggested that the extracellular fluid in the organ of Corti spaces is similar to perilymph in its ionic content. The spectra of stria vascularis samples indicated low sodium and high phosphorus contents. Energy-dispersive roentgen ray analysis of freeze-dried inner ears appears to be a promising method for low-contamination measurement of ion distribution in the cochlea.

Adenylate energy charge, energy status, and phosphorylation state of stria vascularis under metabolic stress.

The purpose of the reported experiments was to measure the strial concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions, the concentrations of ATP, ADP, and AMP were found to be 11.4, 3.7, and 0.6 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since its concentration doubles within 6 sec. of ischemia and reaches a peak level of about 1500% of the control following 65 sec. of ischemia. Under normal conditions, the "adenylate energy charge," the "energy status," and the "phosphorylation state" amount to 0.84, 3.0, and 1.52 gram wet weight/mumole, respectively. In ischemia of 10 min. duration, the adenylate energy charge decreases 3 fold, the energy status 7 fold and the phosphorylation state 14 fold. The size of the adenylate pool shows a slight increase in the earliest stage of ischemia, but declines progressively thereafter. The apparent equilibrium constant of strial adenylate kinase was found to be 0.48. The advantages and limitations of the different adenylate ratios, as indicators of metabolic health and as regulatory parameters, are discussed.