Mitochondria modulate the spatio-temporal properties of intra- and intercellular Ca signals in cochlear supporting cells

In the cochlea, cell damage triggers intercellular Ca²⁺ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca²⁺ waves are thought to convey information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt), and yet little is known about their role during the propagation of such intercellular Ca²⁺ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell [Ca²⁺]_cyt signals that are triggered by ATP or by hair cell damage. ATP application (0.1–50 μM) caused a dose dependent increase in [Ca²⁺]_cyt which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca²⁺ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, enhanced the peak amplitude and duration of ATP-induced [Ca²⁺]_cyt transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca²⁺ waves was significantly increased. Thus, mitochondria function as spatial Ca²⁺ buffers during agonist-evoked [Ca²⁺]_cyt signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca²⁺ waves.     

ATP-mediated potassium recycling in the cochlear supporting cells

Gap junction-mediated K⁺ recycling in the cochlear supporting cell has been proposed to play a critical role in hearing. However, how potassium ions enter into the supporting cells to recycle K⁺ remains undetermined. In this paper, we report that ATP can mediate K⁺ sinking to recycle K⁺ in the cochlear supporting cells. We found that micromolar or submicromolar levels of ATP could evoke a K⁺-dependent inward current in the cochlear supporting cells. At negative membrane potentials and the resting membrane potential of −80 mV, the amplitude of the ATP-evoked inward current demonstrated a linear relationship to the extracellular concentration of K⁺, increasing as the extracellular concentration of K⁺ increased. The inward current also increased as the concentration of ATP was increased. In the absence of ATP, there was no evoked inward current for extracellular K⁺ challenge in the cochlear supporting cells. The ATP-evoked inward current could be inhibited by ionotropic purinergic (P2X) receptor antagonists. Application of pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or pre-incubation with an irreversible P2X7 antagonist oxidized ATP (oATP, 0.1 mM) completely abolished the ATP-evoked inward current at the negative membrane potential. ATP also evoked an inward current at cell depolarization, which could be inhibited by intracellular Cs⁺ and eliminated by positive holding potentials. Our data indicate that ATP can activate P2X receptors to recycle K⁺ in the cochlear supporting cells at the resting membrane potential under normal physiological and pathological conditions. This ATP-mediated K⁺ recycling may play an important role in the maintenance of cochlear ionic homeostasis.     

Postnatal changes in the ultrastructure of the rat olfactory epithelium: the supranuclear region of the supporting cells

Summary The present electron-microscopical study reports ultrastructural changes occurring in the supranuclear region of the supporting cells of the rat olfactory epithelium during the first 16 days of postnatal life. These changes are concerned with the enclosure of receptor cell dendrites and an increase in the amount of smooth endoplasmic reticulum, which has a specific distribution in the supporting cell. An increase in microvillous projections at the free cell surface is also observed. Moreover, this report demonstrates that the cytoarchitecture of the apical portion of the olfactory epithelium at the 16th day of postnatal life is similar, with respect to the relationship between supporting cells and receptor cell dendrites, to that of adult animals.     

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca<Superscript>2+</Superscript> waves

Intercellular Ca²⁺ waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca²⁺ waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca²⁺ waves are triggered during damage to cochlear explants. Both Ca²⁺ waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca²⁺, their velocity, their extent of spread and the cell type through which they propagate. A slower Ca²⁺ wave (14 μm/s) communicates between Deiters’ cells and is mediated by P2Y receptors and Ca²⁺ release from IP₃-sensitive stores. In contrast, a faster Ca²⁺ wave (41 μm/s) propagates through sensory hair cells and is mediated by Ca²⁺ influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca²⁺ wave is mediated by P2X₄ receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.     

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea

Correct patterning of the inner ear sensory epithelium is essential for the conversion of sound waves into auditory stimuli. Although much is known about the impact of the developing cytoskeleton on cellular growth and cell shape, considerably less is known about the role of cytoskeletal structures on cell surface mechanical properties. In this study, atomic force microscopy (AFM) was combined with fluorescence imaging to show that developing inner ear hair cells and supporting cells have different cell surface mechanical properties with different developmental time courses. We also explored the cytoskeletal organization of developing sensory and non-sensory cells, and used pharmacological modulation of cytoskeletal elements to show that the developmental increase of hair cell stiffness is a direct result of actin filaments, whereas the development of supporting cell surface mechanical properties depends on the extent of microtubule acetylation. Finally, this study found that the fibroblast growth factor signaling pathway is necessary for the developmental time course of cell surface mechanical properties, in part owing to the effects on microtubule structure.     

Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea

Summary The distribution and polarity of actin in sensory hair cells of the chinchilla cochlea has been determined by decoration of actin filaments with myosin sub fragment S₁. Decorated actin filaments of the same polarity were present within the stereocilia above the cuticular plate. However the filaments in the rootlets and the thin filaments projecting laterally from the rootlets into the cuticular plate did not decorate with S₁. Decorated actin filaments were present within the cuticular plate, and near the plasma-membrane filaments of opposite polarity were observed. In the cross-striated region at the base of the cuticular plate of inner hair cells, decorated filaments were present in the dense bands of the cross-striations but the thin filaments perpendicular to the dense bands were not decorated. These results are discussed with respect to the two mechanisms that have been suggested for actin-myosin mediated movement of the stereocilia of inner-ear sensory cells.     

Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.     

Appearance of B and H blood group antigens in the developing cochlear hair cells

Summary The presence of human blood-group antigens was analyzed in the rat cochlea during its postnatal development, using anti-A, anti-B and anti-H antibodies. At no stage was reactivity with anti-A antibody observed. With the anti-H antibody, a strong reactivity was observed from 1 to 9 days after birth within hair cells and some other surface epithelial cells of the cochlear duct. After postnatal day 9, only a faint reactivity persisted in a few non-sensory cells. With the anti-B antibody, only hair cells were selectively labeled. At early stages (postnatal day 1 and 3), the reactivity was intense and observed both around the cell surface and within the supranuclear region of cytoplasm. Later on, the reactivity decreased; it was limited at postnatal day 9 to a reactive spot below the cuticular plate. Results are compared with a preliminary finding describing the first appearance of B and H antigens in the organ of Corti at a prenatal stage, and with data concerning other sensory and neural structures. The appearance and progressive disappearance of B and H antigens on sensory and non-sensory cells can be correlated with significant events in the development of the cochlea. The transient expression of B and H antigens in cochlear sensory cells may correspond to developmental changes in their surface glycoconjugates.     

Electromotility of outer hair cells from the cochlea of the echolocating bat,Carollia perspicillata

Isolated outer hair cells (OHCs) and explants ot the organ of Corti were obtained from the cochlea of the echolocating bat, Carollia perspicillata, whose hearing range extends up to about 100 kHz. The OHCs were about 10–30 m long and produced resting potentials between-30 to -69 mV. During stimulation with a sinusoidal extracellular voltage field (voltage gradient of 2 mV/m) cyclic length changes were observed in isolated OHCs. The displacements were most prominent at the level of the cell nucleus and the cuticular plate. In the organ of Corti explants, the extracellular electric field induced a radial movement of the cuticular plate which was observed using video subtraction and photodiode techniques. Maximum displacements of about 0.3–0.8 m were elicited by stimulus frequencies below 100 Hz. The displacement amplitude decreased towards the noise level of about 10–30 nm for stimulus frequencies between 100–500 Hz, both in apical and basal explants. This compares well with data from the guinea pig, where OHC motility induced by extracellular electrical stimulation exhibits a low pass characteristic with a corner frequency below 1 kHz. The data indicate that fast OHC movements presumably are quite small at ultrasonic frequencies and it remains to be solved how they participate in amplifying and sharpening cochlear responses in vivo.Abbreviations BM basilar membrane - FFT fast Fourier Transfer - IHC inner hair cell - OHC outer hair cell     

Postnatal morphological changes in neurons and glial cells in neocortex of mice developing at the background of serotonin deficit

It has been shown that deficit of serotonin during embryogenesis in rodents is accompanied by changes of morphological characteristics of neurons and glial cells at the period of postnatal development. A characteristic peculiarity of these changes is cell vacuolization that is of different expression in various cortical layers. In the experimental animals as compared with control ones, neurons of all neocortex layers have changed nuclei and a reduced volume of the cytoplasm. In neurons of upper layers, nuclei and cytoplasm contain occasional small vacuoles. In deep layers, vacuolization both of nuclei and of the cytoplasm is expressed to the much greater degree and vacuoles of large size are predominant. Results of immunocytochemical study have shown that in animals developing at the background of serotonin deficit there takes place a delay of the rates of formation and differentiation of astrocytic glia.     

Intercellular cross-linkages between the stereociliary bundles of adjacent hair cells in the guinea pig cochlea

Summary Hair cells of the guinea pig organ of Corti have been examined using high resolution scanning electron microscopy. In addition to the extensive array of cross-links between the stereocilia of individual hair cells which have been reported previously, we have seen examples of attachments between the stereocilia of both adjacent inner and adjacent outer hair cells. The implications of these observations are discussed.     

Possible Ca2+-dependent mechanism of apical outer hair cell modulation within the cochlea of the guinea pig

Calcium ions were precipitated with potassium antimonate after injection of the inorganic calcium channel blocker MnCl₂ or the inorganic potassium channel blockers BaCl₂ or CsCl into the perilymph of the scala vestibuli of the guinea pig. The spatial distribution of the formed histochemical reaction products within the organ of Corti was studied by energy-filtering transmission-electron microscopy. Compared with untreated control ears, the number of the formed precipitates drastically increased at the extracellular side of the lamina reticularis after application of the various inorganic channel blockers. The apical side of the outer hair cells and the intervening Deiter cells were covered by a thick layer of calcium precipitates, whereas the number of histochemical reaction products was clearly reduced in the nearby acellular tectorial membrane. The high calcium content within the formed reaction products at the lamina reticularis could be demonstrated by elemental mapping and by electron energy-loss spectroscopy. To ascertain the alterations in the amounts of the calcium precipitates within the tectorial membrane after application of the various inorganic channel blockers, the precipitate densities were determined semiquantitatively by an image processing system and the values obtained compared with those of untreated control specimens. The observed histochemical results are in good agreement with published electrophysiological findings concerning the spatial distribution of ion channels located at the apical outer hair cell membrane. The detected alterations in the spatial distribution of calcium precipitates might correspond to calcium-dependent processes involved in outer hair cell modulation. Received: 4 November 1996 / Accepted: 28 October 1997     

ATP-induced calcium signalling in acinar cells of the submandibular salivary gland

To study changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular solution decreased. The action of ATP induced a dose-dependent increase in the [Ca²⁺]_i; the EC₅₀ was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results in a rise in the [Ca²⁺]_i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the [Ca²⁺]_i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in the control of functioning of this gland. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005.     

Ca-independent effects of BAPTA and EGTA on single-channel Cl currents in brown adipocytes

The Cl⁻ channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl⁻ channels (ORCC). To study tentative Ca²⁺ regulation of these channels, we attempted to control Ca²⁺ levels at the cytoplasmic side of the inside-out membrane patches with Ca²⁺-chelating agents. However, we found that the commonly used Ca²⁺-chelators EGTA and BAPTA by themselves influenced the Cl⁻ channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca²⁺-chelators (acting from the cytoplasmic side) on the single Cl⁻ channel currents in patch-clamp experiments. Using fixed (1-2 mM) concentrations of chelators, two types of Cl⁻ channels were identified, as discriminated by their reaction to the Ca²⁺-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca²⁺. The anionic classical Cl⁻ channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca²⁺ level control in themselves could fundamentally affect the behaviour of Cl⁻ channels.     

Differences between adrenomedullary adrenaline and noradrenaline cells: quantitative electron-microscopic evaluation of their differential cellular association with supporting cells

Quantitative differences in cellular association of adrenomedullary chromaffin cells with other types of cells, mainly supporting cells, were studied. Adrenaline (A) and noradrenaline (NA) cells were compared. Electron micrographs (12000 x) of profiles of A and NA cells, bordering against other types of cells, were used for quantitative evaluation. Supporting cells constituted the majority of the non-chromaffin cell types. Occurrence frequencies of chromaffin cells contiguous with other types of cells were: (1) higher for A cells (68.9%, 199/289) than for NA cells (11.0%, 34/309) in case of small contact regions (²-test: P<0.001) and (2) higher for NA cells (68.3%, 211/309) than for A cells (9.7%, 28/289) in case of extended contact regions (P<0.001). In conclusion, the extent of cellular association with supporting cells was remarkably lower in A cells than in NA cells. Such an arrangement is likely to be appropriate for the extensive, homogeneous control and amplified response characteristic of A cells, and for the close range, complex control and more diverse responses characteristic of NA cells.Supported in part by The Karoji Memorial Fund for Medical Research in Hirosaki University, JapanThe authors, TK, TS and GT, wish to dedicate their part of this work to Dr. W.B. Quay on the occasion of his 65th birthday     

Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

Disordered calcium crystal handling in antisense CLC-5-treated collecting duct cells

Dent's disease, an X-linked tubulopathy secondary to defects in chloride channel CLC-5, is characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Mechanisms leading to nephrocalcinosis are unknown. Using a murine collecting duct cell line (mIMCD-3), we confirm endogenous expression of mCLC-5. During transfection of antisense CLC-5, we observe a reduction in CLC-5 protein expression that correlates with a reduction in the number of acidic endosomal compartments, as determined by quantitative analysis of confocal microscope images using LysoTracker Red. Using wheat germ agglutinin-lectin as an endocytic marker, an arrest of endocytosis is observed in antisense CLC-5 treated cells. Exposure of the cell surface to calcium oxalate crystals results in crystal agglomeration in a minority of sense CLC-5 transfectants (45%) and all antisense CLC-5 transfectants. We conclude that expression of CLC-5 in mIMCD-3 cells allows acidification of endosomes and endocytosis, and that disruption of CLC-5 expression causes abnormal crystal agglomeration.     

Shape changes in goose erythrocytes

Goose erythrocytes were subjected to agents and treatments that produce echinocytosis in human erythrocytes. In the presence of the ionophore A23187 and calcium at greater than micromolar concentrations, goose red cells retained their normal ellipsoidal symmetry, but developed extensive semiregular membrane wrinkles or corrugations. Metabolic NTP (nucleoside triphosphate) depletion, induced either by iodoacetamide or by incubating the cells without a substrate, initially produced a similar cell corrugation, but after prolonged incubation most cells became spherical with the nucleus displaced to the cell periphery. The echinocytic agents indomethacin and dimyristoylphosphatidylcholine had no effect on the gross morphology of goose erythrocytes.