Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI.

CONCLUSION: The data indicate important roles for phosphodiesterase (PDE) 3, 4, 5, and related cAMP and cGMP pools in the regulation of inner ear fluid homeostasis. Thus, dysfunction of these enzymes might contribute to pathologies of the inner ear. OBJECTIVE: The mechanisms underlying endolymphatic hydrops, a hallmark of inner ear dysfunction, are not known in detail; however, altered balance in cAMP and cGMP signaling systems appears to be involved. Key components of these systems are PDEs, enzymes that modulate the amplitude, duration, termination, and specificity of cAMP and cGMP signaling. METHOD: To evaluate the role of PDE3, 4, and 5 and associated cAMP and cGMP pools in inner ear function, the effect of cilostamide (PDE3 inhibitor), rolipram (PDE4 inhibitor), and sildenafil (PDE5 inhibitor), administrated via mini-osmotic pumps, on mouse inner ear fluid homeostasis was evaluated using 9.4T in vivo MRI in combination with intraperitoneally administered Gadolinium contrast. Also, using human saccule as a model, the expression of PDEs and related signaling molecules and targets was studied using immunohistochemistry. RESULTS: PDE3, PDE4, as well as PDE5 inhibitors resulted in the development of endolymphatic hydrops. Furthermore, PDE3B, PDE4D, and some related signaling components were shown to be expressed in the human saccule.

Hair bundles are specialized for ATP delivery via creatine kinase.

When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.

Biochemical properties and immunohistochemical localization of carbonic anhydrase in the sacculus of the inner ear in the salmon Oncorhynchus masou.

Carbonic anhydrase (CA) in the inner ear sacculus was examined by activity assay, Western blotting and immunohistochemistry to determine its role in otolith calcification. An immunoreactive protein with a molecular mass of approximately 28 kDa was detected by Western blotting. The CO2 hydration activity in the cytosol fraction of the sacculus was 5.4 units/mg protein, while little or no activity was detected in the nuclear and mitochondrial fractions. The enzyme activity was highly inhibited by acetazolamide. The concentration of 50% inhibition was 8.16 nM and the inhibition constant of the activity was 8.25 nM. Transitional and squamous epithelial cells of the sacculus were immunopositive with an anti-CA II antibody, but sensory epithelial cells and mitochondria-rich cells in the transitional epithelium were not. These results suggest that transitional epithelial cells other than mitochondria-rich cells and squamous epithelial cells play an important role in otolith calcification by supplying bicarbonate to otoliths and/or by eliminating protons from endolymph.

Sodium- and potassium-activated ATPase in the mammalian vestibular system.

Autoradiographic and cytochemical procedures were employed to determine the cellular distribution of the Na,K-ATPase enzyme in the mammalian vestibular system. A light-microscope survey of vestibular tissues incubated with [(3)H]ouabain shows high densities of ouabain binding sites within the dark cell epithelium (DC) of the ampullae of the semi-circular canals, and to a lesser extent, the DC of the utricular macula. A moderate number of binding sites was found in nerve fibers penetrating the connective tissue beneath the sensory epithelium (SE) of the ampullae and the maculae. A small number of binding sites is distributed in the deep portion of the SE, both in the ampullae and in the maculae. These latter binding sites seem to be associated with nerve terminals and receptor cells. At the ultrastructural level, the vestibular dark cells exhibit extensive basolateral membrane infolding, a morphological hallmark of cells engaged in trans-epithelial ion transport. The cytochemical reaction product is K(+)-dependent, ouabain inhibitable, and is restricted to the basolateral membrane extensions, with little or no product on the luminal membrane. The extent of membrane infolding in dark cells of the utricle is less pronounced than that of the ampullar dark cells and the intensity of the cytochemical reaction appears to correlate with the extent of membrane infolding. The results support the widely held hypothesis that the vestibular dark cells play a role in endolymph production. They also suggest that the vestibular sensory epithelia may be a site of ion exchange.

Caspase activation in hair cells of the mouse utricle exposed to neomycin.

Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating neomycin-induced hair cell death in this preparation.

Meshwork arrangement of mitochondria-rich, Na+,K+-ATPase-rich cells in the saccular epithelium of rainbow trout (Oncorhynchus mykiss) inner ear.

BACKGROUND: Electrolyte composition of the teleost fish inner ear endolymph is characterized by a high potassium concentration. From the ultrastructural characteristics, the mitochondria-rich cells (MRCs) in the inner ear epithelium are suggested to regulate the ionic composition of the endolymph. METHODS: In the present study, the ultrastructure of MRCs in the saccular epithelium of the rainbow trout (Onchorhynchus mykiss) was studied, and the immunocytochemical detection of Na+,K+-ATPase, the key enzyme of the ion-transport, in the saccular epithelium was conducted. Electrolyte composition of the saccular endolymph was also determined. RESULTS: Electron-microscopic observations revealed that MRCs located at the periphery of the sensory macula have numerous elongated mitochondria and a well-developed tubular system. Immunocytochemical detection of Na+,K+-ATPase on paraffin sections showed that immunoreactive (ir-) cells were distributed specifically around the sensory macula. Judging from their shape, size, and localization, the Na+,K+-ATPase ir-cells corresponded to the MRCs. The whole-mount immunocytochemistry using Na+,K+-ATPase as a marker for the MRC revealed that MRCs were connected with one another by extended cellular processes, and thus forming a dense meshwork structure around the macula. In the endolymph, potassium levels were 13 times higher than those in plasma, chloride levels were slightly higher whereas sodium, calcium, magnesium, and phosphate levels were lower. CONCLUSIONS: Thus, the saccular MRCs abundant in Na+,K+-ATPase are distributed around the sensory macula forming a dense meshwork structure, with the suggested function to regulate the electrolyte composition of the saccular endolymph.

Expression of cytochrome oxidase in hair cells of the teleost utricle.

Ultrastructural variation in some cytoplasmic organelles and synaptic structures is one characteristic distinguishing the types of hair cells in the teleost ear. In this study, we explored differences in mitochondria by analyzing mitochondrial reactivity for cytochrome oxidase (COX) in hair cells of the teleost utricle. The reactivity for COX within mitochondria in the subcuticular compartment directly beneath the cuticular plate differentiated among hair cells in utricles of three teleost species, Carassius auratus, Pantodon buchholzi, and Astronotus ocellatus. Mitochondria in the subcuticular region of hair cells in the striola reacted intensely. Within juxtastriola and extrastriolar hair cells near the striola, mitochondria reacted at a lowered intensity than in striolar hair cells. Subcuticular mitochondria of extrastriolar hair cells located distant from the striola reacted negligibly. The reactivity of mitochondria in other cytoplasmic compartments did not provide similar evidence for distinguishing among teleost hair cells. Mitochondria within intraepithelial branches of the eighth nerve terminals in the different utricular regions reacted to COX histochemistry commensurate with their respective presynaptic hair cells. Branches of sensory afferent neurons innervating striolar hair cells displayed a dense COX reaction. Sensory afferents innervating the extrastriolar hair cells did not display many mitochondria at synapses nor, when present, was the staining as dense. The presynaptic side of the hair cell-afferent nerve synapse usually, but not always, contained reactive mitochondria. The presynaptic side of the efferent nerve-hair cell synapse did not necessarily contain mitochondria. Mitochondria filling the cytoplasm in a type of juxtamacula cell revealed uniformly dense COX reactivity.

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.

Cytochemical localization of adenylyl cyclase activity within the sensory epithelium of the trout saccule.

Adenylyl cyclase, the enzyme of synthesis of cAMP, the second messenger molecule mediating signal transduction in response to sensory, neurotransmitter and hormonal stimuli, has been localized in the sensory epithelium of the rainbow trout (Salmo gairdneri R.) saccule by cytochemical detection of enzyme activity. In the sensory receptor cell, or hair cell, reaction product has been visualized in the stereocilia in close association with the outer cell membrane and also at the apical surface of the cuticular plate. A diffuse distribution of precipitate was observed within the cytoplasm of terminal endings of nerve fibers presumed to be efferent on the basis of characteristic synaptic specializations including presynaptic vesicles and a postsynaptic cistern lying within the hair cell. Occasionally, reaction product was observed to be associated with the external cell membrane of these nerve terminals. There appeared to be little or no adenylyl cyclase activity associated with the plasma membrane at the base of the hair cell or in presumptive afferent nerve endings. However, a subpopulation of nerve fiber endings which exhibited both efferent and afferent synaptic specializations contained precipitate. A concentration of adenylyl cyclase activity in hair cell stereocilia and efferent nerve terminals in the sensory epithelium is suggestive of a role for cAMP in second messenger action at these sites, possibly related to mechanosensory transduction and efferent neuromodulation, respectively.

Effect of streptomycin on the supporting cells of the utricular macula.

Morphological changes in utricular supporting cells in the guinea pig following streptomycin sulfate (SM) intoxication were investigated ultrastructurally in vitro, using an organ culture system. The intracellular structures of the supporting cells, such as the nucleus, mitochondria, and Golgi apparati were well preserved after 7 days in culture. After 10 and 14 days in culture, the supporting cells degenerated. The organ culture system was applied to ototoxicity studies on the supporting cells in utricular macula within 7 days after explanation. When the utricles were exposed to 3, 10 and 30 mg/ml of SM for 3 days, the number of granules in the supporting cells decreased markedly and the number of lysosomes increased daily. The lysosomes contained mitochondria, myeloid bodies, granules and vesicles. Acid phosphatase (AcPase) activity decreased in Golgi apparati and lysosomes. When the concentration of SM was reduced to 3 and 10 mg/ml, the damage to the supporting cells was less marked than that in cells exposed to 30 mg/ml. The supporting cells showed a dose-dependent response with respect to morphological damage. After 3 days culture with 30 mg/ml of SM, the specimens were subsequently cultured for 4 days in a medium without SM. After removal of SM from the medium, lysosomes decreased in number, and the granules and the endoplasmic reticulum showed a gradual increase. The AcPase activity was determined in both lysosomes and Golgi apparatus. This study revealed that the morphological changes in the supporting cells can be reversible.

Development of melanosomes and cytochemical observation of tyrosinase activity in the inner ear.

The ultrastructure and tyrosinase activity of melanocytes in the inner ear of pigmented guinea pigs were observed. Melanocytes/melanocyte-like cells were seen in the stria vascularis, the vestibular dark cell area and the endolymphatic sac. In the stria vascularis, melanosomes in several stages of maturation were seen in the cytoplasm of the intermediate cells and melanin-laden endosomes existed in the basal cells. Only the intermediate cells contained tyrosinase-positive cytoorganelles; Golgi sacs and neighboring small vesicles. Numerous melanocytes containing many melanosomes were observed under the epithelium of the vestibular dark cell area, and they showed tyrosinase activity. Melanocytes were seen in the endolymphatic sac, and they also showed tyrosinase activity. However, the epithelial cells of the endolymphatic sac, which had vacuoles containing melanin, did not show tyrosinase activity. Based on these findings, it can be said that (1) most of the intermediate cells of the stria vascularis must be melanocytes, (2) melanogenesis is vigorous in melanocytes of the inner ear, and (3) melanin in the epithelial cells of the endolymphatic sac is transferred in from melanocytes and is never synthesized in the epithelial cells.

Na,K-ATPase subunit isoform expression in the guinea pig endolymphatic sac.

Na,K-ATPase subunit isoform expression was studied by immunocytochemistry in the guinea pig endolymphatic sac, using subunit isoform-specific polyclonal antibodies. Epithelial cells of the guinea pig endolymphatic sac were observed to contain the alpha 1- and beta 2-subunit isoforms, and to a lesser extent the beta 1-subunit isoform, of Na,K-ATPase. The alpha 1- and beta 2-subunit isoforms of Na,K-ATPase have been observed previously in other ion and fluid transporting regions of the membranous labyrinth, e.g., stria vascularis and vestibular dark cells. Combined data indicate that the alpha 1-, beta 2-form of Na,K-ATPase plays a role in the microhomeostasis of endolymph. The alpha 1 beta 2 Na,K-ATPase subunit isoform combination is different from typical ion and fluid transporting tissues, e.g., kidney and colon, and may reflect distinctive characteristics of inner ear Na,K-ATPase.

Cholinesterase activity in vestibular organs of young and old mice.

The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were studied by histochemical methods in the semicircular canal end organs, the utricle and the saccule of young and old mice. AChE was located on the plasma membrane of efferent nerve terminals beneath vestibular hair cells, and along the basement membrane. In the ampulla, stained efferent terminals were more prevalent on the slopes of the crista than in the central region. In all organs examined, there were no discernible differences in AChE activity between young and old mice. BChE activity was observed in the epithelial light cells and supporting cells of the saccule, utricle, and ampulla. Its distribution was similar in both young and old mice in the ampulla, but decreased significantly with age in the utricle. Preliminary data suggest that BChE activity is also weak in old saccular supporting cells. Unlike the utricle, old saccular light cells retained intense BChE activity.

Cytochemical studies of Ca++-ATPase activity in the vestibular epithelia of the guinea pig.

We used ultracytochemistry to examine Ca++-ATPase activity in the vestibular epithelia of the guinea pig. Many reaction products were found along the basolateral plasma membrane of the vestibular dark cell. There were also marked reaction deposits on the apical and lateral cell membranes of the transitional cells, and the utricular and saccular wall cells. Both sensory and supporting cells showed Ca++-ATPase activity along their ciliary membrane and apical-lateral cell surfaces. Our findings indicate that the Ca++-ATPase activity found on the plasma membrane is closely related to Ca++-transport across the plasma membrane. When either Ca++ or ATP was omitted from the incubation medium, enzyme activity (as seen by the staining reaction present) was completely abolished. Our present results suggest that Ca++-ATPase located in the vestibular epithelia plays a significant role in the regulation of the Ca++-concentration in the vestibular endolymph.

Is the Na+,K+-ATPase symmetrically distributed in the neuroepithelium of the vestibular system in the axolotl (Ambyostoma mexicanum)?

This study was undertaken to assess the localization of the Na+,K+-ATPase in the neuroepithelial cells of the macula sacculi. In vitro perilymphatic (basolateral) perfusion with ouabain produced a significant drop in the membrane potential. Endolymphatic (apical) application of ouabain had practically no effect on membrane potentials. This suggests that Na+,K+-ATPase is asymmetrically distributed in the neuroepithelial cells.