Isolde Thalmann

Effect of ethacrynic acid, furosemide, and ouabain upon the endolymphatic potential and upon high energy phosphates of the stria vascularis.

The loop diuretics ethacrynic acid (EA) and furosemide (FU) were applied systemically to guinea pigs at dosages from 10–100 mg/kg. At high dosages the endolymphatic potential (EP) invariably turned negative. When the EP had reached maximum negative values due to EA, the ATP levels of the stria vascularis were moderately reduced, but P‐creatine levels were normal. In the case of FU both high energy phosphates remained at normal levels. When EA and FU intoxicated ears were subjected to ischemia, the rate of decline of ATP and P‐creatine was markedly less than the ischemic decline in nonintoxicated ears. These results suggest a strong interference with energy utilization, and in the case of EA a moderate impairment of energy generation. In severe intoxication by perilymphatically applied ouabain (10−3 M) strial ATP remained normal but P‐creatine was significantly increased. The reduction of the ischemic decline rate in ouabain intoxicated ears was even more marked than in the case of EA or FU, indicating a very strong interference with energy utilization, presumably due to complete inhibition of Na+K+‐ATPase. The I50 of the endolymphatic potential with regard to perilymphatically applied EA and FU was found to be 10−5 M and 2 x 10−4 M respectively. By K contrast, strial Na+K+‐ATPase was 50% inhibited with 5 x 10−3 M EA and not inhibited at all by FU. It is therefore unlikely that the effect of loop diuretics upon the endolymphatic potential is due to interference with strial Na+K+‐ATPase.

The influence of ischemia upon the energy reserves of inner ear tissues

Ischemic changes in the levels of glucose, glycogen, ATP and P‐creatine are determined under “closed system” conditions in the organ of Corti, stria vascularis, ganglion spirale, cochlear nerve and vestibular sensory epithelia. From the resting levels of these compounds the total energy reserve in terms of equivalents of high energy phosphate, both preformed and potentially available from anaerobic glycolysis, is computed. The energy reserves are highest in the organ of Corti, intermediate in stria vascularis, spiral ganglion and vestibular structures, and lowest in the cochlear nerve. The rate of depletion of these energy reserves in ischemia is used as an indicator of the energy requirements of the respective tissues. The metabolic rate is by far the highest in the stria vascularis, intermediate in ganglion spirale and cochlear nerve and lowest in the organ of Corti and vestibular structures. There is no correlation between the total energy reserve and the initial energy use rates. The obtained data are compared with the dynamic patterns of the corresponding biopotentials and with pertinent results of enzymatic and respirometric studies; in addition, ischemic changes in glucose and lactate levels of perilymph are described.

Development and Maintenance of Otoconia

Abstract: The first part of this review deals with recent advances in the understanding of biochemical mechanisms of otoconial morphogenesis. Most important in this regard is the molecular characterization of otoconin 90, the principal matrix protein of mammalian calcitic otoconia, which was found to be a homologue of the phospholytic enzyme PLA2. The unique and unexpected expression pattern of this protein required radical rethinking of traditional concepts. The new data, when integrated with existing information, provide a rational basis for an explanation of the mechanisms leading to crystal nucleation and growth. Based on this information, a hypothetical model is presented that posits interaction of otoconin 90 with microvesicles derived from the supporting cells as a key event in the formation of otoconia. The second part of the review is directed at the controversial subject of maintenance of mature otoconia and systematically analyzes the available indirect information on this topic. A synthesis of these theoretical considerations is viewed in relation to the pathogenesis of the important otoneurologic entities of BPPN and senile otoconial degeneration. The last part of the review deals with several animal models that promise to help elucidate normal and abnormal mechanisms of otoconial morphogenesis, including mineral deficiencies, mutations with selective otoconial agenesis, as well as targeted disruption of essential genes.

Cyclic amp and adenylate cyclase in the inner ear

The activity of adenylate cyclase and the steady state levels of cyclic AMP (cAMP) were determined in stria vascularis (SV) and organ of Corti (OC) of the guinea pig cochlea. The activities are 12 and 19 pmoles/mg dry weight/minute for OC and SV, respectively. The activity was increased two to four-fold by NaF. The base level of cAMP is 4.2 and 4.4 nmoles/g dry weight in OC and SV, respectively. In contrast to brain, neither ischemia nor barbiturates produced major changes of the steady state levels of cAMP. No in vitro effect of cAMP upon the state of activation of glycogen phosphorylase was noticeable in either tissue. cAMP did not exert a significant in vitro inhibition of strial Na+K+-ATPase. Perilymphatic perfusion of cAMP (10-3 M) and of theophylline (5 times 10-3 M) did not produce changes in the endolymphatic potential (EP), but dibutyryl cAMP (10-3 M) led to a significant increase of EP. The alpha adrenergic blocking agent, phentolamine, produced very complex changes of the cochlear potentials. A possible role of catecholamines and cAMP in the secretory phenomena of the SV and in the transduction and/or transmission processes of the auditory sense organ are discussed.

Otoconial agenesis in tilted mutant mice

The sense of balance is one of the phylogenetically oldest sensory systems. The vestibular organs, consisting of sensory hair cells and an overlying extracellular membrane, have been conserved throughout vertebrate evolution. To better understand mechanisms regulating vestibular development and mechanisms of vestibular pathophysiology, we have analyzed the mouse mutant, tilted (tlt), which has dysfunction of the gravity receptors. The tilted mouse arose spontaneously and has not been previously analyzed for a developmental or physiological deficit. Here we demonstrate that the tilted mouse, like the head tilt (het) mouse, specifically lacks otoconia and consequently does not sense spatial orientation relative to the force of gravity. Unlike other mouse mutations affecting the vestibular system (such as pallid, mocha and tilted head), the defect in the tilted mouse is highly penetrant, results in the nearly complete absence of otoconia, exhibits no degeneration of the sensory epithelium and has no apparent abnormal phenotype in other organ systems. We further demonstrate that protein expression in the macular sensory epithelium is qualitatively unaltered in tilted mutant mice.

Oncomodulin Is Expressed Exclusively by Outer Hair Cells in the Organ of Corti

Oncomodulin (OM) is a small, acidic calcium-binding protein first discovered in a rat hepatoma and later found in placental cytotrophoblasts, the pre-implantation embryo, and in a wide variety of neoplastic tissues. OM was considered to be exclusively an oncofetal protein until its recent detection in extracts of the adult guinea pigs organ of Corti. Here we report that light and electron microscopic immunostaining of gerbil, rat, and mouse inner ears with a monoclonal antibody against recombinant rat OM localizes the protein exclusively in cochlear outer hair cells (OHCs). At the ultrastructural level, high gold labeling density was seen overlying the nucleus, cytoplasm, and the cuticular plate of gerbil OHCs. Few, if any, gold particles were present over intracellular organelles and the stereocilia. Staining of a wide range of similarly processed gerbil organs failed to detect immunoreactive OM in any other adult tissues. The mammalian genome encodes one α- and one β-isoform of parvalbumin (PV). The widely distributed α PV exhibits a very high affinity for Ca2+ and is believed to serve as a Ca2+ buffer. By contrast, OM, the mammalian β PV, displays a highly attenuated affinity for Ca2+, consistent with a Ca2+-dependent regulatory function. The exclusive association of OM with cochlear OHCs in mature tissues is likely to have functional relevance. Teleological considerations favor its involvement in regulating some aspect of OHC electromotility. Although the fast electromotile response of OHCs does not require Ca2+, its gain and magnitude are modulated by efferent innervation. Therefore, OM may be involved in mediation of intracellular responses to cholinergic stimulation, which are known to be Ca2+ regulated.

Mixing model systems: Using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development

Human vestibular dysfunction is an increasing clinical problem. Degeneration or displacement of otoconia is a significant etiology of age-related balance disorders and Benign Positional Vertigo (BPV). In addition, commonly used antibiotics, such as aminoglycoside antibiotics, can lead to disruption of otoconial structure and function. Despite such clinical significance, relatively little information has been compiled about the development and maintenance of otoconia in humans. Recent studies in model organisms and other mammalian organ systems have revealed some of the proteins and processes required for the normal biomineralization of otoconia and otoliths in the inner ear of vertebrates. Orchestration of extracellular biomineralization requires bringing together ionic and proteinaceous components in time and space. Coordination of these events requires the normal formation of the otocyst and sensory maculae, specific secretion and localization of extracellular matrix proteins, as well as tight regulation of the endolymph ionic environment. Disruption of any of these processes can lead to the formation of abnormally shaped, or ectopic, otoconia, or otoconial agenesis. We propose that normal generation of otoconia requires a complex temporal and spatial control of developmental and biochemical events. In this review, we suggest a new hypothetical model for normal otoconial and otolith formation based on matrix vesicle mineralization in bone which we believe to be supported by information from existing mutants, morphants, and biochemical studies.

Protein profile of human perilymph: in search of markers for the diagnosis of perilymph fistula and other inner ear disease.

Recent developments in high-resolution two-dimensional polyacrylamide gel electrophoresis, combined with amino acid sequencing and computer-assisted image analysis, have allowed separation of approximately 100 proteins and identification and quantitation of some 30 proteins in human perilymph. The majority of proteins were found to be present in perilymph at levels in basic agreement with the total protein gradient between perilymph and plasma (1:35). However, several striking differences were observed: (1) β2-transferrin, known to be absent from normal plasma but present in cerebrospinal fluid, was detected in perilymph at a concentration roughly equal to that in cerebrospinal fluid; and (2) two high-density lipoprotein-associated apolipoproteins—apo D (formerly PLS:33) and apo J or NA1 and NA2 (formerly PSL:29/30), the latter showing identity with 5P40/40, or cytolysis inhibitor—were found to be present at concentrations 1 to 2 orders of magnitude higher when examined in terms of total protein and to be comparable with or higher than plasma levels when examined in terms of absolute concentrations. The functional significance of the extremely high levels of the two apolipoproteins is not known at this time. An attempt was made to use β2-transferrin, as well as apo D and apo J (NA1/NA2), as markers for the diagnosis of perilymph fistula, one of the most controversial and challenging problems for the otologist today. It was determined that the technique is indeed applicable when relatively pure fistula samples are analyzed. Limitations and potential improvements of the technique are discussed. In addition, the potential usefulness of two-dimensional polyacrylamide gel electrophoresis in other pathologic conditions of the inner ear is discussed briefly.

The cochlear F-box protein OCP1 associates with OCP2 and connexin 26.

OCP1 and OCP2 are the most abundant proteins in the organ of Corti. Their distributions map identically to the epithelial gap-junction system, which unites the supporting cell population. Sequence data imply that OCP1 and OCP2 are subunits of an SCF E3 ubiquitin ligase. Consistent with that hypothesis, electrophoretic mobility-shift assays and pull-down assays with immobilized OCP1 demonstrate the formation of an OCP1-OCP2 complex. Sedimentation equilibrium data indicate that the complex is heterodimeric. The coincidence of the OCP1-OCP2 distribution and the epithelial gap-junction system suggests that one or more connexin isoforms may be targets of an SCF(OCP1) complex. Significantly, immobilized OCP1 binds (35)S-labeled connexin 26 (Cx26) produced by in vitro transcription-translation. Moreover, Cx26 can be co-immunoprecipitated from extracts of the organ of Corti by immobilized anti-OCP1, implying that OCP1 and Cx26 may associate in vivo. Given that lesions in the Cx26 gene (GJB2) are the most common cause of hereditary deafness, the OCP1-Cx26 interaction has substantial biomedical relevance.

Amino acid profiles in inner ear fluids and cerebrospinal fluid

The levels of 19 amino acids in utricular endolymph, vestibular and cochlear perilymph, and cerebrospinal fluid of guinea pigs were determined using gradient elution reverse phase high performance liquid chromatography of the o‐phthaldialdehyde‐ethanethiol adducts with fluorescence detection. Aspartate and glutamate were significantly higher in endolymph than in perilymph, in agreement with earlier results on cochlear fluids based on enzymatic fluorometric techniques. All other amino acids tested were significantly lower in the endolymph, in most cases by an order of magnitude. Vestibular perilymph and perilymph of scala vestibuli are virtually identical. Amino acid levels were all higher in perilymph of scala vestibuli than in cerebrospinal fluid; two by an order of magnitude. All differences were statistically significant, with the exception of aspartate. Amino acid levels in perilymph of scala tympani were highly variable, dependent upon sampling technique, and no definite values are therefore presented. Comparisons with results from other laboratories, technical pitfalls, and possible implications and interpretations of the results are presented.