Formation Of Otoconia Research Articles

The NADPH Oxidase Nox3 Constitutively Produces Superoxide in a p22phox-dependent Manner: ITS REGULATION BY OXIDASE ORGANIZERS AND ACTIVATORS

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.

Partial behavioral compensation is revealed in balance tasked mutant mice lacking otoconia

We describe for the first time behavioral tests which show that mammals with congenital absence of otoconia can learn a motor task that normally relies on gravity perception. The mouse mutation tilted ( tlt) occurs in the otopetrin 1 gene ( Otop1 tlt/tlt ) and eliminates an essential component necessary for the formation of otoconia. Our data show that even in the absence of otoconia, tlt mutant mice, like normal mice, learn to cross a bar suspended between two boxes and, with practice, improve their speed of crossing. Despite this learned compensatory skills, tlt mutant mice show balance impairments, such as falling from the bar, not observed in wild type (WT) or heterozygous (het) Otop1 +/ tlt littermates. The tlt mutant mice also use their tail as additional support, a behavior that is rarely exhibited in the control littermates. Interestingly, the Otop1 +/ tlt heterozygous littermates show in many aspects an intermediate phenotype between wild type and tlt mutant mice, suggestive of a gene dosage effect. Overall, these data support the notion that mammals can use other otic and extraotic receptors such as semicircular canals and limb proprioreceptors, respectively, to compensate for the absence of otoconia-mediated gravity perception in a balance task.

Morphometry of otoconia in the utricle and saccule of developing Japanese quail

The development of otoconia in the utricular and saccular maculae from initial embryonic formation to adult stages was examined in Japanese quails. Both the morphology and size of the otoconia were quantified at different developmental stages. It was observed that the otoconia were initially formed on embryologic stage E5 in the saccule and E6 in the utricle. Otolith mass areas increased in a sigmoidal growth pattern, with saccular otolith areas being smaller than the utricular mass areas. Saccular otolith masses reached adult values at embryonic stage E12 and utricular areas reached adult values at post-hatch day 7. Mature individual otoconia were characterized by a barrel shape with two trihedral faceted ends. However, initial formation of otoconia at E5 (saccular) and E6 (utricular) maculae was characterized by a double fluted morphology that consisted of an hourglass shape with extended fins forming trihedral angles of 120°. Double fluted otoconia rapidly filled, so that by embryonic day 8 mature otoconia dominated the maculae for the remainder of development through adulthood. Thus, a progression from double fluted to mature forms was noted. Mature utricular otoconia in adult quails averaged 11 μm in length and 5 μm in width, with length/width ratios of approximately 2.5:1, for all size ranges. Saccular otoconia were smaller, having about 70% the size of utricular otoconia in both length and width. During development, the average size and range of individual otoconia increased nearly linearly for both otolith organs. In the utricular macula, large otoconia were concentrated in the lateral regions of the epithelium. In contrast, otoconia of various sizes were distributed uniformly across the surface of the saccular macula.

Calbindin D28K is a component of the organic matrix of lizard Podarcis sicula otoconia

The factors controlling otoconia growth are not well known but it seems that the type of proteins contained in the otoconia regulates the initiation and/or the subsequent rates of crystal growth determining the morphology and the size of the final crystal. In order to clarify the mechanism of otoconia formation and their turnover, major proteins contained in the otoconia from the maculae of the saccule, utricle and lagena of inner ear of lizard Podarcis sicula were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Coomassie staining of SDS–PAGE resulted in a major broad band of 15 kDa and four other bands of 21, 28, 45 and 97 kDa. The proteins of 15, 21, 28 and 45 kDa were separated by high-pressure liquid chromatography on a C-4-reverse-phase column and the incubation of blots with monoclonal anti-Calbindin D28K antibodies indicated that the band of 28 kDa was Calbindin D28K, a calcium-binding protein.

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I.

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646–8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152–14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59–61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693–18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA–NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

Development and maintenance of otoconia: biochemical considerations.

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.

Calcification processes in the chick otoconia and calcium binding proteins: patterns of tetracycline incorporation and calbindin-D28K distribution

In order to clarify the otoconia formation and turnover, tetracycline, an antibiotic that precipitates at calcifying fronts and serves as a fluorescent marker, was injected into eggs at different stages of chick embryonic development, as well as into postnatal chicken and into adult animals. The changes in the intensity, location patterns and time course of fluorescent labelling in each examined stage in the otolithic organs was studied. The presence and distribution of calbindin (CB)-D28K, one of the calcium-binding proteins constantly found in the mammalian and chicken cochlea and also in otolithic membrane of some adult mammals, was studied. Results in embryonal stages, postnatal and adult animals allow us to postulate that otoliths are mainly produced during the embryonal phase, but they may also be produced throughout the whole life span. Results also indicate that otoconia are dynamic structures which undergo turnover. The correspondence between the patterns of CB-D28K immunoreactivity and tetracycline fluorescence may indicate that CB-D28K participates in the formation of otoconia.

Otolithic apparatus in Black Sea elasmobranchs

Using light, transmission and scanning electron microscopy, and morphometry, we examined the otolithic organs of 25 Raja clavata (35–93 cm), 12 Dasyatis pastinaca (34–50 cm), and two Squalus acanthias specimens (100 and 145 cm). In D. pastinaca all three otolithic membranes merge into one another, and the otolithic apparatus as a whole is relatively poorly developed. In R. clavata and S. acanthias the large saccular otolithic membrane and the small lagenar otolithic membrane are continuous. The mass of the otolithic membrane and the length of the animal are power related. The otoconia of rays tend to be lemon shaped or spherical. The dogfish has large, cuboidal (parallelepiped-shaped) endogenous otoconia, exogenous sand grains, and crystal-containing globules. The size of the endogenous otoconia does not depend on the size of the animals. No specialized zones were found in rays either on the surface or inside the otolithic membranes containing otoconia of one type or size. The data indicate that the mass of the otolithic apparatus in rays increases on account of the formation of new otoconia. The otolithic apparatus of the dogfish can also increase in mass by addition of small grains of sand, which enter the labyrinth and are incorporated into the otolithic membrane.

PH regulation of the globular substance in the otoconial membrane of the guinea-pig inner ear

Physiological and pharmacological characteristics of the globular substance, a precursor of otoconia, are not well understood. In the present study, we investigated the variations and regulation of internal pH (pH i) of the globular substance of the guinea-pig inner ear. The otoconial membrane was dissected out from the utricular macula and loaded with the pH-sensitive fluorophore, carboxy-seminaphthorhodafluor-1. Dynamic changes of fluorescence were directly observed under a confocal laser scanning microscope, and pH i was calculated from dual emission ratio. In the NaCl standard solution buffered with 5 mM HEPES/Tris at pH 7.4, the pH i of the globular substance varied from 6.26 to 8.55 with an average of 7.21 ( n=270). Exposure to 25 mM NH + 4 induced a rapid increase of the pH i followed by a slow relaxation. Then, wash-out of NH + 4 caused a prompt and pronounced acidification followed by a gradual pH i recovery to the initial level. This gradual pH i recovery was significantly inhibited by the absence of external Na +, indicating the presence of an external Na +-dependent H + extrusion mechanism. This pH i recovery was also inhibited by 1 mM amiloride and 10 μM 3-amino- N-(aminoiminomethyl)-6-[ethyl(2-propyl)amino]pyrazine-2-carboxamide. These results suggest the presence of an Na +-H + exchanger in the globular substance of the guinea pig. However, HCO − 3-transporting mechanisms were not determined. The working hypothesis for the otoconial formation is discussed.

Balance and Hearing Deficits in Mice with a Null Mutation in the Gene Encoding Plasma Membrane Ca2+-ATPase Isoform 2

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

Production of Otoconia in the Endolymphatic Sac in the Japanese Red-bellied Newt, Cynops pyrrhogaster: Light and Transmission Electron Microscopic Study

The formation of otoconia in the endolymphatic sac (ES) of the larval newt, Cynops pyrrhogaster, has been studied by light and transmission electron microscopy. Some of the epithelial cells of the ES contain an abundance of swollen vesicles, Golgi complexes, rough endoplasmic reticula and ribosomes at the late larval stages 50 and 51, approximately 26-30 days after eggs are laid. Five days later, at stage 52, crystals are present in the vacuoles between the epithelial cells. Serial sections indicate that these vacuoles actually form small canals which lie in the wall and join the lumen of the ES. Reconstruction of the ES shows that several canals are contained in the ES wall. At stage 56, about 72 days after eggs are laid, a large number of otoconia are present in the ES lumen, while the otoconia disappear from the canals. It appears that the otoconia are first produced in the canals and then released to the lumen. Some epithelial cells of the ES are thought to expel the organic and inorganic material to the canals to form the otoconia in situ. The process of formation of the otoconia in the ES is discussed.

The Process of Otoconia Formation in Guinea Pig Utricular Supporting Cells

In order to clarify the mechanism of otoconia formation, the pathway of calcium transport in the utricular supporting cells was investigated. Potassium pyroantimonate (PA) precipitation, which indicates the localization of calcium ions, was seen not only in otoconia but also in supporting cell cytoplasm. In the latter, deposits of PA were detected in the secretory granules and endoplasmic reticulum (ER). These deposits were not present in cells pretreated with ethylene glycol-O,O'-bis (2-aminoethyl)-N,N,N',N'-tetra-acetic acid (EGTA). Exposure of the supporting cells to streptomycin sulfate (SM) increased the number of lysosomes. These lysosomes contained many small deposits of PA. The remaining granules and ER in cytoplasm also contained small deposits of PA. The findings suggest that otoconia are formed by the vestibular supporting cells in which calcium ions might be transported via ER-secretory granule-lysosome-cytoplasmic protrusion.

Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica.

To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

Otoconial formation in the chick: changing patterns of tetracycline incorporation during embryonic development and after hatching

The antibiotic tetracycline (TC) is incorporated into calcifying tissues and serves as a fluorescent marker for identifying calcifying sites in bone and otoconia. Fluorescent labeling was performed at different stages in chick embryos and newly hatched chicks. The stagewise changes in the intensity, location and time course of fluorescent labeling were assessed. TC/egg (1–2 mg) was injected into the yolk sacs of embryos on the 4th, 8th and 11th embryonic days (ED), and the embryos were then killed at specified times after injection. In newly hatched chicks, TC was injected daily intraperitoneally with 0.06 mg TC/g body weight for 7 days and the chicks were killed on the 8th day after hatching. Embryos injected on the 4th ED and killed on the 6th ED showed intense fluorescence in the saccular otoconial layer. All maculae from embryos killed after injection on the 4th ED showed uniformly intense fluorescence throughout the otoconial layer. By the 9th ED, otoconia in all three maculae (saccular, utricular, and lagenar) fluoresced. Maculae from embryos killed after injection on the 8th ED showed uniformly intense fluorescence throughout the otoconial layer or intense localized fluorescence mainly in the upper half of the otoconial layer. All maculae from embryos killed after injection on the 11th ED showed moderate to weak fluorescence primarily in the lower half of the otoconial layer. All maculae from posthatched chicks showed very weak fluorescence throughout the otoconial layer. Otoconia at the periphery of the maculae generally showed weak fluorescence in embryos that had been injected on the 4th and 8th ED, but not in embryos injected on the 11th ED. TC, which competes for calcium binding sites, may inhibit the formation of some otoconia. The formation of giant otoconia may reflect subtle changes in the crystallization microenvironment on these occasions. In brief, the results suggest that: (a) otoconial formation in the saccule precedes that of the utricle and lagena; (b) otoconial formation occurs during the early period (beginning the 6th ED); (c) otoconial formation is stratified, with those in the upper layer forming first and those in the lower layer forming last.

Formation and calcium incorporation of giant otoconia of the guinea pig after streptomycin intoxication

The mechanisms for the formation and fate of giant otoconia following streptomycin (SM) intoxication were investigated in adult pigmented guinea pigs by scanning electron microscopy. Calcium turnover into otoconia has also been studied by using tetracycline as a tracer. The administration of SM induced the reduction of otoconia with the formation of giant otoconia. The giant otoconia had a multifaceted morphology in their early developmental period. This type of otoconia showed entire fluorescence indicating existence of calcium uptake. They then grew up to the transitional type and finally to the cylindrical type. The giant otoconia were thought to be formed mainly by dissolution of normal otoconia due to the loss of environmental calcium followed by recrystallization as giant crystals. The transitional type of giant otoconia showed less calcium ion uptake and the removal of calcium from the giant otoconia caused their quick disappearance. These phenomena might be closely related to the otoconial dynamics which may regulate calcium ion homeostasis of endolymph.

Formation and Fate of Giant Otoconia of the Guinea Pig following Streptomycin Intoxication

Formation and fate of abnormal (giant) otoconia of the guinea pig following streptomycin intoxication were investigated using scanning electron microscopy. The giant otoconia formed as multifaceted morphology in their early developmental period. They grew up the the transitional type and finally to the cylindrical type. It has been suggested that the giant otoconia found following streptomycin intoxication may be formed mainly by dissolution of normal otoconia due to the loss of environmental calcium, followed by recrystallization as giant crystals. These phenomena seemed to be closely related to the otoconial dynamics which may regulate calcium ion homeostasis of the endolymph.

Effect of Streptomycin on the Otoconial Layer of the Guinea Pig

The effects of streptomycin on the otoconial layer of the adult guinea pig were investigated using scanning electron microscopy. Administration of streptomycin induced a reduction of otoconia with formation of giant otoconia. These phenomena reached their maximum 4 weeks after the cessation of streptomycin intoxication in the utricle and between 2 and 6 weeks after in the saccule. Otoconia began to recover in the utricle 4 weeks and in the saccule 6 weeks after the cessation of streptomycin intoxication with an increasing number of small otoconia. Eight or 10 weeks after the cessation of streptomycin intoxication, the otoconial layer has completely recovered in both the utricle and saccule.

The mechanism of formation of the otoconia in the utricular supporting cells of the guinea pig

The mechanism of formation of the otocania in the utricular supporting cells of the guinea pig was investigated by means of an organ culture system using the potassium pyroantimonate (PA) precipitation method. The utricular otoconia and the secretory granules and mitochondria in the utricular supporting cells immediately after removal from the animal were positive to PA. When the specimens were treated with ethylene-glycol-0, 0-bis (2-aminoethyl)-N, N, N', N'-tetraacetic acid (EGTA) as a chelater prior to the PA staining, almost all deposits disappeared. This indicates that calcium is the main ion precipitated by the PA method. The utricules of the guinea pig were exposed to 30 mg of streptomycin sulfate per ml for 3 days in culture. The number of large lysosomes which contained vesicles and myeloid bodies in the supporting cells increased. On the other hand, the secretory granules were reduced in the cytoplasm of the supporting cells. Acid phosphatase activity in the lysosomes and the Golgi apparatus decreased. As a result of the PA treatment, these large lysosomes, especially the vesicles which were in them, contained a large quantity of calcium ion. From these findings, I conclude that the mechanism of formation of otoconia is as follows: The area of formation of otoconia is the supporting cells. Globular substances are the precursors of otoconia, and they contain calcium ion which was taken into the supporting cells. These globular substances are made in close relationship among the endplasmic reticula, secretory granules and lysosomes. They are secreted from the supporting cells and form the mature otoconia on the otoconial membrane.

Otoconial alterations after embryonic development in hypergravity

The relation between prolonged hypergravity and structural adaptation of otoconia was studied in hamsters ( n = 56). Three groups of hamsters ( n = 27), were conceived and born in a centrifuge: group 1 ( n = 10) 1 month under 2.5 G, group (2 n = 9) 5 months under 2.5 G and 4 months under 1 G, group 3 ( n = 8) 1 month under 2.5 G and 8 moths under 1 G. Control hamsters ( n = 29) were conceived and born under 1 G (1 month old, n = 7; 9 months old, n = 22). Histological study of the otoconial layers (energy dispersive x-ray element analysis and scanning electron microscopy) showed similar calcium content, size, and shape in utricular and saccular otoconia in all groups. Different were the utricular otoconial size classes, large, medium-sized, a and small. The area with small otoconia increased in group 1 ( p = 0.002). In group 2, the large otoconial area decreased ( p < 0.001) and the medium-sized one increased ( p < 0.001). In group 3, the large otoconial area decreased ( p = 0.003) and the medium-sized one increased ( p = 0.007). For age-related effects we found group 1 with an increased area of large otoconia ( p = 0.001) and a decreased medium-sized one compared to groups 2 ( p < 0.001) and 3 ( p = 0.02). Hypergravity during formation of otoconia does not affect calcium content, size, or shape, but changes relative size of the areas with large, medium-sized, or small otoconia and the development of these areas. This resulted in a structural adaptation to hypergravity.

Calbindin-D28K localization in the primate inner ear.

The distribution of one of the calcium-binding proteins, calbindin-D28K (CB-D28K), was studied in the adult human and squirrel monkey inner ear by means of immunocytochemical methods. Inner and outer hair cells in the organ of Corti and vestibular hair cells showed CB-D28K immunoreactivity, though some vestibular hair cells were devoid of immunoreactivity. In the spiral and vestibular ganglion, immunoreactive cells were found in both the squirrel monkey and human. The present results indicate that CB-D28K is localized within afferent neuronal components in these sensory organs and may regulate Ca++ levels for optimal neurotransmission in the primate auditory and vestibular systems. This study also provides evidence of two nonneuronal localizations of CB-D28K in the squirrel monkey. Subpopulations of fibrocytes in the spiral ligament and vestibular end organs were enriched with CB-D28K, suggesting that these cells are possibly equipped with the function to regulate Ca++ concentration in the perilymphatic fluid. In the maculae, many CB-D28K-immunoreactive particles were found in the otoconial membrane, indicating that CB-D28K may participate in the formation of otoconia.