Mice deficient in H+-ATPase a4 subunit have severe hearing impairment associated with enlarged endolymphatic compartments within the inner ear.

Mutations in the ATP6V0A4 gene lead to autosomal recessive distal renal tubular acidosis in patients, who often show sensorineural hearing impairment. A first Atp6v0a4 knockout mouse model that recapitulates the loss of H(+)-ATPase function seen in humans has been generated and recently reported (Norgett et al., 2012). Here, we present the first detailed analysis of the structure and function of the auditory system in Atp6v0a4(-/-) knockout mice. Measurements of the auditory brainstem response (ABR) showed significantly elevated thresholds in homozygous mutant mice, which indicate severe hearing impairment. Heterozygote thresholds were normal. Analysis of paint-filled inner ears and sections from E16.5 embryos revealed a marked expansion of cochlear and endolymphatic ducts in Atp6v0a4(-/-) mice. A regulatory link between Atp6v0a4, Foxi1 and Pds has been reported and we found that the endolymphatic sac of Atp6v0a4(-/-) mice expresses both Foxi1 and Pds, which suggests a downstream position of Atp6v0a4. These mutants also showed a lack of endocochlear potential, suggesting a functional defect of the stria vascularis on the lateral wall of the cochlear duct. However, the main K(+) channels involved in the generation of endocochlear potential, Kcnj10 and Kcnq1, are strongly expressed in Atp6v0a4(-/-) mice. Our results lead to a better understanding of the role of this proton pump in hearing function.

Occurrence of NaK-ATPase isoforms during rat inner ear development and functional implications.

This study examined the presence of NaK-ATPase isoforms in the developing inner ear of the rat and studied the importance of functional subunit combinations in endolymph homeostasis. The findings were: (a) the combination alpha 1 beta 1 is found in epithelial, mesenchymal, and neural inner ear cells with an early starting expression 14 days postconception (dpc) in some endolymphatic sac cells; (b) from 1 day after birth (dab) expression of alpha 1 beta 2 is observed in marginal cells, vestibular dark cells, and certain vestibular nonsensory cells; (c) a transient expression of alpha 2 beta 1 is found in suprastrial fibrocytes and spiral ligament fibrocytes type II between 10 and 15 dab; (d) starting at 16 dpc the combination alpha 3 beta 1 is uniquely expressed in inner ear neural cells (as in other neural tissues). In conclusion, during development a switch from alpha 2 beta 1 towards alpha 1 beta 1 is observed in suprastrial fibrocytes and in spiral ligament fibrocytes type II. Thus, according to the biochemical characteristics of these combinations, a switch towards a NaK-ATPase with higher capacity takes place. In addition, prominent expression of the alpha 1 beta 2 combination in predominantly K+ ion transporting marginal and dark cells is in accordance with the characteristic of this combination and thus with the presumed function of these cells as important K+ suppliers for the endolymph. We believe this combination in certain vestibular nonsensory cells to be involved in K+ sensing. Early expression of the alpha 1 beta 1 combination in the endolymphatic sac, prior to that in the other parts of the inner ear, suggests that this structure may be involved to some extent in the development of the vestibulum and cochlea.

Expression of connexin 26 and Na,K-ATPase in the developing mouse cochlear lateral wall: functional implications.

The immunohistochemical localization of connexin 26 (a gap junction protein) and Na,K-ATPase in the mouse cochlear lateral wall was studied at different ages between 0 and 30 days after birth (DAB). Connexin 26-like immunoreactivity was sparsely distributed among the connective tissue cells just lateral to the future marginal cells of the stria vascularis on 0 DAB. In the mice of 3-6 DAB, connexin 26 was observed in the strial basal cell area, and was increased in its distribution density on 10 DAB. Connexin 26 was sparsely distributed among the fibrocytes in the spiral ligament and the suprastrial zone on 10 DAB, and its distribution density increased rapidly in the mouse on 12 DAB. The immunohistochemical distribution reached the adult pattern in the cochlear lateral wall on 15 DAB. Weak Na, K-ATPase-like immunoreactivity was observed in the epithelial cells, corresponding to the future strial marginal cells, on 0 DAB. Its staining intensity was enhanced with the increase of age, and reached the adult pattern on 10 DAB. In contrast, Na,K-ATPase-like immunoreactivity in the type II fibrocytes and suprastrial fibrocytes was first detected on 12 DAB, and reached the mature level on 15 DAB. It is well known that the endolymphatic potential (EP) reaches the adult level 2 weeks after birth. The expression patterns of connexin 26 and Na,K-ATPase in the fibrocytes of the spiral ligament and the suprastrial zone coincided with the rapid growth and maturation of EP. These findings may suggest a role for the gap junctional communications and Na,K-ATPase activity of the fibrocytes within the cochlear lateral wall in the generation and maturation of EP.

Effects of epinephrine on ouabain-sensitive, K(+) -dependent P-nitrophenylphosphatase activity in strial marginal cells of guinea pigs.

In strial marginal cells, Na+/K(+)-ATPase activity is abundant, and contributes to maintain the characteristic electrolyte composition of the cochlear endolymph. In the present study, to clarify the relationship between epinephrine and strial Na+/K(+)-ATPase activity, the ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of strial marginal cells was investigated with a cerium-based method in normal guinea pigs and guinea pigs treated with reserpine, epinephrine, and reserpine plus epinephrine. In our previous study, K(+)-NPPase activity had almost completely decreased 3 to 20 days after reserpine administration. In the present study, at 10 days after reserpinization and following repeated epinephrine treatment, enzyme activity was detectable. These results suggest that exogenous epinephrine was able to restore strial K(+)-NPPase activity in the reserpine-treated animals, and that epinephrine might increase strial Na+/K(+)-ATPase activity.

Mineralocorticoid type I receptor in the rat cochlea: mRNA identification by polymerase chain reaction (PCR) and in situ hybridization.

Expression of mineralocorticoid type I receptor (MR) gene in the rat cochlea was determined using molecular biological techniques. We synthesized complementary DNA (cDNA) from rat cochlear total RNA and then amplified MR cDNA fragments by polymerase chain reaction (PCR). The amplified cDNA fragments were subcloned into an expression vector and the nucleotide sequence was analyzed to confirm the expression of mRNA encoding MR in the cochlea. We then synthesized digoxigenin-labeled riboprobes with this cloned DNA template and examined the localization of MR mRNA in the cochlea by in situ hybridization. The amino acid sequence of MR cDNA expressed in the cochlea was identical to that of the MR first cloned in the rat hippocampus. In situ hybridization showed the expression of MR mRNA in marginal cells of the stria vascularis, suggesting that aldosterone may regulate microhomeostasis of the endolymph, presumably by modulating Na, K-ATPase activity. Intense MR signal was also identified in spiral ganglion cells, the function of which remains to be determined.

[Localization of Na/K ATP-ase in the inner ear]. / De lokalisatie van Na/K ATP-ase in het binnenoor.

The perilymphatic compartment of the inner ear resembles plasma ultrafiltrate, whereas the endolymphatic compartment is characterized by a high potassium and low sodium concentration. Perilymph is considered to be the precursor of endolymph. The chemical composition of endolymph and the generation of the transepithelial potential is regulated by Na-K-activated adenosine triphosphatase. The cytochemical localization of Na/K ATP-ase is demonstrated in the guinea pig inner ear. The mechanism for cation regulation in the endolymphatic compartment is discussed.

Antidiuretic hormone stimulation of adenylate cyclase in semicircular canal epithelium.

Basal adenosine 3',5'-cyclic monophosphate (cAMP) content and the modulation of its production were studied in the frog's semicircular canal epithelium. This epithelium secretes endolymph, a K(+)-rich, positively polarized fluid. The basal cAMP content measured by microradioimmunoassay was 244 +/- 14.2 fmol/structure per 5 min (n = 30). This content was increased about 8 times by 10(-5) M forskolin. Vasotocin, the frog antidiuretic hormone, increased the cAMP production by factors of 1.3 and 3.3 at concentrations of 10(-8) M and 10(-7) M, respectively. This stimulatory effect of vasotocin was blunted by the addition of alpha 2-adrenergic agonists, such as 10(-8) M-10(-5) M norepinephrine, in the presence of 10(-5) M propranolol, or 10(-5) M clonidine. Prostaglandin E2 at a concentration of 10(-8) M, which did not affect the cAMP production, did not modify the response to vasotocin. Glucagon (10(-6) M), calcitonin (10(-6) M), and parathyroid hormone (10 units/ml) did not affect the cAMP content. Prostaglandin E2 (10(-7) M) and the beta-adrenergic agonist isoproterenol (10(-6) M) stimulated the cAMP production by a factor of 1.6. These results indicate that the frog semicircular canal is a target of both vasotocin and catecholamines and that catecholamines through alpha 2-receptors modulate vasotocin-induced cAMP generation. Further, this interaction might be of physiological relevance in the modulation of ion transport in this structure.

Ultracytochemical localization of Na+, K(+)-ATPase activity in the tegmentum vasculosum of the developing chick cochlea.

We studied the morphological changes and the precise localization of Na+, K(+)-ATPase activity in the developing chick tegmentum vasculosum (TV) with a cytochemical method utilizing K(+)-p-nitrophenylphosphatase (K(+)-NPPase). At stage 37, epithelial folds (lamellae) were relatively well developed, and the light and the dark cells were clearly identified. At stage 41 to 42, Na+, K(+)-ATPase activity appeared in the folded cytoplasmic processes of the dark cells which interdigitated with those of the light cells. The present results indicate that the TV is involved in the production of the cochlear endolymph prenatally (at least up to stage 41), and that the dark cell plays a central role in the maintenance of the ionic composition of the endolymph.

Early effects of acetazolamide on anionic activities of the guinea pig endolymph: evidence for active function of carbonic anhydrase in the cochlea.

The effect of acetazolamide (ACZ) on HCO3- and Cl- activities in inner ear fluid was investigated by ion-selective microelectrode methods. The endocochlear potential, at 81.6 +/- 1.5 mV under normal conditions, was reduced by 4.9 +/- 0.9 mV in 30 min following the ACZ injection. The HCO3- concentrations by 11.5 +/- 1.7 mM in 30 min, while the change of Cl- level was not consistent but showed a tendency toward slight increase in average. The calculated PCO2 in endolymph was 39.6 mmHg. ACZ induced a constant elevation of the endolymph PCO2, with an increase in the venous PCO2 during the initial 20 min and thereafter the endolymph PCO2 attained a plateau despite the continuous increase of the venous PCO2. We postulate that the HCO3- secretion into the endolymph is mediated by carbonic anhydrase (CAH) and is decreased by ACZ due to the inhibition of CAH.

Aminotransferases in post-mortem cochlear fluids.

Aspartate and alanine aminotransferase (ASAT, ALAT) activities were measured in human post-mortem sera, cerebrospinal fluid (CSF), perilymph and endolymph. Due to heart and/or liver morbidity during a terminal illness, the ASAT and ALAT serum activities were considerably increased as compared with normal and both were 20--30 times higher (p less than 0.001) than in CSF or inner ear fluids. CSF and inner ear fluids showed mutually similar values.

[Animal experiment histological-histochemical studies on the development of hearing disorders related to hypothyroidism]. / Tierexperimentelle histologisch-histochemische Untersuchingen zur Formalgenese der hypothyreotisch bedingten Schwerhörigkeit

It has been known for a long time that hearing deficits may coexist in patients with thyroid disease, but without definite morphologic evidence present to correlate gland dysfunction with hearing disturbances. To clarify this relationship between thyroid dysfunction and hearing disturbances, the guinea pig was employed as an experimental model. 70 animals were thyroidectomized, and maintained in a hypothyroid state for varying periods of time. The animals were then sacrificed, and various histochemical studies then performed. These studies included analysis for glycosidase (beta-galactosidase, beta-glucuronidase, n-acetyl-beta-glucosamide), non-specific esterases, sulfatases, sulghydryl groups as well as mucous substances within the cochlea and saccus endolymphaticus of the experimental animals. Results indicated that hyaluronidase-sensitive mucous substances were increased in the scala of the inner ear. As a consequence of increased deposition of acid mucopolysaccharides, the relationship of potassium to sodium in endolymph and perilymph was found markedly altered. Marked swelling of the chambers of the inner ear was noted, and believed to represent hydropic induction by acid mucopolysaccharide-with consequent alteration of electrolyte relationships ("Electrochemical Theory").