Differential expression of apoptosis-related genes in the cochlea of noise-exposed rats.

Exposure to intense noise induces apoptosis in hair cells in the cochlea. To identify the molecular changes associated with noise-induced apoptosis, we used quantitative real-time PCR to evaluate the changes in 84 apoptosis-related genes in cochlear samples from the sensory epithelium and lateral wall. Sprague-Dawley rats exposed to a continuous noise at 115 dB SPL for 2 h. The exposure caused a 40-60 dB threshold shift 4 h post-exposure that decreased to 20-30 dB 7 days post-exposure. These functional changes were associated with apoptotic markers including nuclear condensation and fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Immediately after the noise exposure, 12 genes were downregulated, whereas only one gene (Traf4) was upregulated. At 4 h post-exposure, eight genes were upregulated; three (Tnrsf1a, Tnfrsf1b, Tnfrst5) belonged to the Tnfrsf family, three (Bir3, Mcl1 and Prok2) have anti-apoptotic properties and one (Gadd45a) is a target of p53. At 7 days post-exposure, all the upregulated genes returned to pre-noise levels. Interestingly, the normal control cochlea had high constitutive levels of several apoptosis-related genes. These constitutively expressed genes, together with the inducible genes, may participate in the induction of cochlear apoptotic activity.

The use of Preyer's reflex in evaluation of hearing in mice.

Preyer's reflex, the elicitation of startle response to auditory stimuli, has been widely used for the evaluation of hearing in rodents and other animals. Surprisingly, however, the sensitivity and specificity of Preyer's reflex in the assessment of hearing has not been adequately studied. The aim of this study was to investigate the utility of Preyer's reflex in the evaluation of auditory function in mice. Forty-six adult albino mice on an FVB background with variable hearing loss were used for this study. Two different methods for eliciting a Preyer's reflex were tested: a handclap and a sharp metallic sound. The reflex was considered positive when a rapid movement of the whole body of the animal was clearly noticed. Thereafter, the mice underwent auditory brain stem response (ABR) testing with broadband clicks. The presence or absence of Preyer's reflex was compared with the corresponding ABR thresholds. Five of the 46 animals studied (11%) showed a negative Preyer's reflex, while the remaining 41 animals demonstrated a positive Preyer's reflex. There was no difference between the abilities of the two different stimuli to elicit a Preyer's reflex. The click-evoked ABR thresholds in the test animals varied between 8 and 136 (mean 50) dB sound pressure level (SPL). Preyer's reflex was positive in all animals with an ABR threshold of < or = 76 dB SPL, but was absent in animals with an ABR threshold of > or = 81 dB SPL. Preyer's reflex is effective for identifying profound sensorineural hearing loss in experimental mice, but is insensitive for detecting less severe auditory dysfunction. For definitive hearing assessment, and for defining the hearing thresholds. objective electroacoustical methods such as ABR should be used.

Cochlear gene delivery through an intact round window membrane in mouse.

Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade.

Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H, K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H, K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H, K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.

Localization of myosin-V in the centrosome.

The perinuclear localization of myosin-V was investigated in a variety of cultured mammalian cells and in primary cultures of rat hippocampus. In all cells investigated, myosin-V immunoreactivity was associated with the centrosome. In interphase cells, myosin-V was found in pericentriolar material, and in both mother and daughter centrioles. These results were obtained by using two different fixation protocols with three different affinity-purified antibodies that recognized a single band in Western blots. During cell division, myosin-V staining was intense throughout the cytoplasm and was concentrated in a trail between migrating centrioles and in the mitotic spindle poles and spindle fibers. The centrosome targeting site was determined to reside within the globular tail domain, because centrosome association also was observed in living cells transfected with DNA encoding the tail domain fused with a green fluorescent protein tag, but not in cells transfected with the vector encoding green fluorescent protein by itself.

Inhibition of calcium-dependent motility of cochlear outer hair cells by the protein kinase inhibitor, ML-9.

The calcium ionophore ionomycin has been shown to induce length increases of guinea pig outer hair cells (Dulon et al., 1990). We have demonstrated that these length increases can be inhibited by a 30 min preincubation of the cells with the protein kinase inhibitor ML-9. At either 30 or 60 s after ionomycin application, the effect of ML-9 was dose-dependent with a half maximal response at approximately 0.3 microM. No effect on cell length was detected after 30 min incubation with 0.5 and 5 microM ML-9 alone. However, with 50 and 500 microM ML-9, significant contraction in cell length was observed. 50 microM ML-9 did not interfere with the ability of ionomycin to elevate fluorescence of the calcium indicator Fluo-3, nor did it alter the ability of cells to exclude propidium iodide from their nuclei. Treatment with 500 microM ML-9 resulted in impaired cell morphology. The data support the hypothesis that protein kinase activity regulates calcium-dependent processes that affect shape changes of outer hair cells. They are consistent with the involvement of the calcium/calmodulin-dependent enzyme, myosin light chain kinase, a known target of ML-9, but do not preclude the possibility of another intracellular target for ML-9.

Cellular distribution of myosin-V in the guinea pig cochlea.

The importance of unconventional myosins to hearing has recently been revealed by the identification of myosins-VI and -VII as the defective genes in mouse mutations and in a human syndrome which lead to profound hearing loss. Another class of novel myosins (V) has been implicated in the trafficking of intracellular vesicles in neurons and other secretory cells. We used affinity-purified antibodies to determine the localization of myosin-V in the guinea pig inner ear. In the sensory epithelium of the cochlea, myosin-V epitopes were recognized in neuronal and supporting cells. Neuronal labelling was most intense in the afferent innervation of inner and outer hair cells. Supporting cells labelled were cells of Hensen and Deiters, and inner border, inner phalangeal, inner sulcus and interdental cells. In the vascular tissue of the cochlea, we observed staining of intermediate cells of the stria vascularis and of border cells between the stria and the spiral prominence. Staining of afferent chalice nerve endings was observed on type I vestibular hair cells. The results suggest that, like myosins VI and VII, myosin-V is localized in positions that may be critical to auditory function.

Second messengers in the cochlea.

Second messengers are vital to the regulation of nearly every aspect of cellular physiology. Evidence is reviewed for the role of second messengers in cochlear fluid homeostasis, hair cell adaptation, motility and synaptic activity. We suggest that the elucidation of the role of second messengers in the regulation of cochlear physiology will be important for the therapeutic management of otopathologies and the side effects of ototoxic agents.

Detection and characterization of nitric oxide synthase in the mammalian cochlea.

The messenger molecule nitric oxide (NO) is involved in blood flow regulation, cytotoxicity, and neural signalling, processes that are important in the physiology and pathophysiology of the mammalian cochlea. However, neither the presence of NO nor its synthetic enzyme, NO synthase, has been established in the peripheral auditory system. NO synthase activity, measured as the enzymatic conversion of radioactive arginine to citrulline, was predominantly soluble in the auditory nerve, lateral wall, vestibule and cochlear neuroepithelium. N-methyl-L-arginine and trifluoperazine inhibited NO synthase activity in the lateral wall and auditory nerve. Histochemical staining by NADPH-diaphorase localized NOS activity to the lateral wall and the neuronal elements of the organ of Corti. Based on these results, the predominant NO synthase isoform in the cochlea is the neuronal type-I isoform.

Calcium and calmodulin inhibit phosphorylation of a novel auditory nerve protein.

The growing use of cochlear prosthetic devices and demonstrations of direct ototoxic insult to spiral ganglion neurons make it imperative to gain an understanding of intracellular biochemical regulation in primary sensory neurons. Calcium and calmodulin regulate many aspects of neuronal cellular physiology through stimulation of protein kinase activity. We have previously demonstrated the presence of calmodulin-dependent protein kinase substrates in the guinea pig modiolus and, additionally, the presence of two proteins (12 kDa and 81 kDa, designated as p12 and p81) whose phosphorylation is blocked by calcium and calmodulin (Coling and Schacht, 1991). Here, we investigate three models for this unusual regulatory mechanism. The effects of calcium, calmodulin and trifluoperazine on dephosphorylation of both proteins suggests that calmodulin inhibits protein kinase activity. P81 was identified by immunoprecipitation as the myristoylated alanine-rich C kinase substrate (MARCKS), a ubiquitous actin-binding protein. Two observations indicate that MARCKS may be regulated differently in acoustic nerve than in cerebral cortex. 32P incorporation was significantly higher in acoustic nerve than in brain. The calmodulin-dependent block of MARCKS phosphorylation was observed only in acoustic nerve. p12 shares several characteristics with myelin basic protein (MBP). We used a double label assay with 32P autoradiography and immunoblotting to show that p12 is in fact distinct from MBP. We suggest that either p12 or p12 kinase may be either specific to the peripheral auditory system or novel marker proteins for that tissue.

Characterization of the calcium-binding contractile protein centrin from Tetraselmis striata (Pleurastrophyceae).

Centrin is a major protein of the contractile striated flagellar roots of the green alga Tetraselmis striata. We present a newly modified procedure for the preparation of centrin in sufficient quantity and purity to allow for detailed biochemical characterization. We establish that centrin purified by differential solubility, followed by phenyl-Sepharose and DEAE-Sephacel chromatography is identical with the protein extracted directly from striated flagellar roots with regard to molecular weight, isoelectric point, and calcium-dependent behavior in SDS-PAGE. We also compare the biochemical properties of purified centrin with calmodulin isolated from Tetraselmis and calmodulin isolated from mammalian brain. Centrin can be fully distinguished from either algal or mammalian calmodulin on the basis of molecular weight, isoelectric point, calcium-dependent behavior in SDS-PAGE, proteolytic peptide maps, amino acid composition, ability to activate bovine brain phosphodiesterase, and reactivity with specific antibodies.

Regulation of expression and phosphorylation of A9/alpha 6 beta 4 integrin in normal and neoplastic keratinocytes.

The A9 antigen is a basement membrane antigen of normal squamous epithelial cells that is strongly expressed in many squamous carcinomas. High expression of this antigen is associated with early relapse in squamous cell carcinomas of the head and neck. We now know that the A9 antigen is structurally, immunologically, and functionally similar to the alpha 6 beta 4 integrin that has been shown to be linked to metastatic behavior in murine tumor models. The alpha 6 and beta 4 genes have been cloned and sequenced, and a model has been constructed from the deduced amino acid composition. In this study we present a hypothetical model and use it to design experiments to assess the factors that influence the expression of the A9/alpha 6 beta 4 integrin in normal and malignant keratinocytes. High calcium induces down regulation of A9/alpha 6 beta 4 antigen in normal but not malignant keratinocytes within 24 hours. Although calcium can down-regulate beta 4 message in tumor cells in the absence of epidermal growth factor (EGF), transcription of beta 4 increased in the tumor cells under the conditions we used for assessing antigen expression (calcium plus EGF). Retinoic acid also stimulated transcription of beta 4 in tumor cells, but this was partially inhibited by the presence of high calcium. Phosphorylation of the beta 4 chain was stimulated by epidermal growth factor and calcium in normal keratinocytes, but in the malignant cells phosphorylation was constant regardless of the culture conditions. Our results indicate that high expression of the alpha 6 beta 4 integrin is associated with conditions that favor migration and undifferentiated proliferation of normal keratinocytes and that malignant keratinocytes differ from normal keratinocytes by constitutive phosphorylation of beta 4 and by failure to downregulate beta 4 transcription in response to calcium in the presence of EGF.

Protein phosphorylation in the organ of Corti: differential regulation by second messengers between base and apex.

Major aspects of cellular physiology are regulated by the phosphorylation state of proteins through the action of protein kinases and protein phosphatases. Phosphorylation of proteins by endogenous protein kinase activity was assayed in homogenates from guinea pig inner ear tissues with [gamma-32P] ATP. Phosphoproteins showed distinct distributions in organ of Corti, lateral wall and spiral ganglion. In the organ of Corti, several protein kinase activities were distinguished by their activation by appropriate agonists: protein kinase C, calmodulin-dependent protein kinases and cyclic nucleotide-dependent protein kinases. Twelve putative substrates for these kinases were identified in organ of Corti on the basis of increased 32P-incorporation with addition of lipids, calmodulin, and cyclic nucleotides, respectively. In addition, differences in phosphorylation were observed between the base and apex of the organ of Corti. 32P-incorporation into proteins of molecular weights between 45 and 100 kDa was significantly higher in apical tissue than in tissue from the base. In contrast, phosphate incorporation into proteins of around 29 kDa was much lower in apical tissues than in basal tissues. Furthermore, labeling of both the high and low molecular weight proteins from the apex but not the base markedly increased in response to calcium. These data indicate the presence of differential modes of regulation that may underlie structural and functional gradients along the sensory epithelium.

Calcium-modulated contractile proteins associated with the eucaryotic centrosome.

Affinity-purified antibodies that recognize the 20,000-dalton molecular weight (20 kd) striated flagellar root protein of Tetraselmis striata have been used to identify antigenic homologs in other eucaryotic organisms of diverse evolutionary origins. Among the green algae, Tetraselmis and Chlamydomonas, and their colorless relative, Polytomella, the 20-kd homologs appear associated with basal bodies. This occurs most prominently in the form of flagellar roots of both striated and microtubule subtended types. Among cultured mammalian cells (PtK2 and primary mouse macrophage cell lines), flagellar root protein homologs appear as basal feet, pericentriolar fibrils, and pericentriolar satellites. Mammalian sperm cells also show flagellar root protein homologs associated with their basal bodies. We envisage a functional role for these fibrous calcium-sensitive contractile proteins in altering the orientation of centrioles or basal bodies with their associated MTOCs by responding to topological calcium fluxes.