RESUMEN
BACKGROUND: Claudins are major components of tight junctions, which form the paracellular barrier between the cochlear luminal and abluminal fluid compartments that supports the large transepithelial voltage difference and the large concentration differences of K+, Na+ and Ca2+ needed for normal cochlear function. Claudins are a family of more than 20 subtypes, but our knowledge about expression and localization of each subtype in the cochlea is limited. RESULTS: We examined by quantitative RT-PCR the expression of the mRNA of 24 claudin isoforms in mouse cochlea during postnatal development and localized the expression in separated fractions of the cochlea. Transcripts of 21 claudin isoforms were detected at all ages, while 3 isoforms (Cldn-16, - 17 and - 18) were not detected. Claudins that increased expression during development include Cldn-9, - 13, - 14, - 15, and -19v2, while Cldn-6 decreased. Those that do not change expression level during postnatal development include Cldn-1, - 2, - 3, - 4, - 5, - 7, - 8, -10v1, -10v2, - 11, - 12, -19v1, - 20, - 22, and - 23. Our investigation revealed unique localization of some claudins. In particular, Cldn-13 expression rapidly increases during early development and is mainly expressed in bone but only minimally in the lateral wall (including stria vascularis) and in the medial region (including the organ of Corti). No statistically significant changes in expression of Cldn-11, - 13, or - 14 were found in the cochlea of Slc26a4 -/- mice compared to Slc26a4 +/- mice. CONCLUSIONS: We demonstrated developmental patterns of claudin isoform transcript expression in the murine cochlea. Most of the claudins were associated with stria vascularis and organ of Corti, tissue fractions rich in tight junctions. However, this study suggests a novel function of Cldn-13 in the cochlea, which may be linked to cochlear bone marrow maturation.
Asunto(s)
Claudinas/metabolismo , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Animales , Proteínas de Transporte de Anión/genética , Femenino , Masculino , Ratones Noqueados , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Transportadores de SulfatoRESUMEN
BACKGROUND: Disturbance of acid-base balance in the inner ear is known to be associated with hearing loss in a number of conditions including genetic mutations and pharmacologic interventions. Several previous physiologic and immunohistochemical observations lead to proposals of the involvement of acid-base transporters in stria vascularis. RESULTS: We directly measured acid flux in vitro from the apical side of isolated stria vascularis from adult C57Bl/6 mice with a novel constant-perfusion pH-selective self-referencing probe. Acid efflux that depended on metabolism and ion transport was observed from the apical side of stria vascularis. The acid flux was decreased to about 40 % of control by removal of the metabolic substrate (glucose-free) and by inhibition of the sodium pump (ouabain). The flux was also decreased a) by inhibition of Na,H-exchangers by amiloride, dimethylamiloride (DMA), S3226 and Hoe694, b) by inhibition of Na,2Cl,K-cotransporter (NKCC1) by bumetanide, and c) by the likely inhibition of HCO3/anion exchange by DIDS. By contrast, the acid flux was increased by inhibition of gastric H,K-ATPase (SCH28080) but was not affected by an inhibitor of vH-ATPase (bafilomycin). K flux from stria vascularis was reduced less than 5 % by SCH28080. CONCLUSIONS: These observations suggest that stria vascularis may be an important site of control of cochlear acid-base balance and demonstrate a functional role of several acid-base transporters in stria vascularis, including basolateral H,K-ATPase and apical Na,H-exchange. Previous suggestions that H secretion is mediated by an apical vH-ATPase and that basolateral H,K-ATPase contributes importantly to K secretion in stria vascularis are not supported. These results advance our understanding of inner ear acid-base balance and provide a stronger basis to interpret the etiology of genetic and pharmacologic cochlear dysfunctions that are influenced by endolymphatic pH.
Asunto(s)
Equilibrio Ácido-Base , Endolinfa/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Estría Vascular/metabolismo , Animales , Femenino , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos C57BL , Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estría Vascular/enzimologíaRESUMEN
Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 (Δ/Δ) mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 (Δ/Δ) line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.
Asunto(s)
Oído Interno/metabolismo , Saco Endolinfático/metabolismo , Pérdida Auditiva/genética , Proteínas de Transporte de Membrana/genética , Animales , Proteínas de Transporte de Anión/metabolismo , Oído Interno/patología , Endolinfa/metabolismo , Saco Endolinfático/patología , Femenino , Pérdida Auditiva/patología , Humanos , Ratones , Ratones Transgénicos , Mutación , Embarazo , Transportadores de Sulfato , ATPasas de Translocación de Protón Vacuolares/genética , Acueducto Vestibular/metabolismo , Acueducto Vestibular/fisiopatologíaRESUMEN
BACKGROUND: The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under ß2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids. RESULTS: Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 µM), 8-Br-cAMP (EC50: 180 µM), 8-pCPT-cAMP (100 µM), IBMX (250 µM), and RO-20-1724 (100 µM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 µM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 µM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 µM), flufenamic acid (5 µM) and diphenylamine-2,2'-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/- mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR-/- mice had no responses. Nonetheless, CFTR-/- mice showed no difference from CFTR+/- mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 µM), prednisolone (0.3, 1 & 3 µM), hydrocortisone (0.01, 0.1 & 1 µM), and corticosterone (0.1 & 1 µM) and mineralocorticoid aldosterone (1 µM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined. CONCLUSIONS: These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.
Asunto(s)
Bumetanida/farmacología , Cloruros/metabolismo , AMP Cíclico/metabolismo , Epitelio/efectos de los fármacos , Glucocorticoides/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Epitelio/metabolismo , Transporte Iónico/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Inhibidores de Fosfodiesterasa/farmacología , Potasio/metabolismo , Ratas , Ratas Wistar , Receptores de Glucocorticoides/metabolismo , Canales Semicirculares , Sodio/metabolismoRESUMEN
Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)-mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU-induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9-defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9-defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.
Asunto(s)
Pérdida Auditiva/metabolismo , Iones/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Animales , Transporte Biológico , Claudinas , Cóclea/química , Cóclea/metabolismo , Modelos Animales de Enfermedad , Femenino , Células Ciliadas Auditivas/química , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva/genética , Humanos , Iones/química , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos A , Ratones Transgénicos , Mutagénesis , Permeabilidad , Uniones Estrechas/química , Uniones Estrechas/genéticaRESUMEN
BACKGROUND: Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC), which is composed of the three subunits α-,ß- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. RESULTS: We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196), RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b) nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3). By contrast, α-,ß- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. CONCLUSIONS: These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αßγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala media.
Asunto(s)
Cóclea/metabolismo , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/metabolismo , Sodio/metabolismo , Animales , Transporte Biológico Activo/fisiología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Cóclea/citología , Células Epiteliales/clasificación , Células Epiteliales/citología , Ratones , Ratones Endogámicos C57BLRESUMEN
The saccule is a vestibular sensory organ that depends upon regulation of its luminal fluid, endolymph, for normal transduction of linear acceleration into afferent neural transmission. Previous studies suggested that endolymph in the saccule was merely derived from cochlear endolymph. We developed and used a preparation of isolated mouse saccule to measure transepithelial currents from the extramacular epithelium with a current density probe. The direction and pharmacology of transepithelial current was consistent with Na(+) absorption by the epithelial Na(+) channel (ENaC) and was blocked by the ENaC-specific inhibitors benzamil and amiloride. Involvement of Na(+),K(+)-ATPase and K(+) channels was demonstrated by reduction of the current by ouabain and the K(+) channel blockers Ba(2+), XE991, and 4-AP. Glucocorticoids upregulated the current via glucocorticoid receptors. Dexamethasone stimulated the current after 24 h and the stimulation was blocked by mifepristone but not spironolactone. No acute response was observed to elevated cAMP in the presence of amiloride nor to bumetanide, a blocker of Na(+),K(+),2Cl(-) cotransporter. The results are consistent with a canonical model of corticosteroid-regulated Na(+) absorption that includes entry of luminal Na(+) through apical membrane Na(+) channels and active basolateral exit of Na(+) via a Na(+) pump, with recycling of K(+) at the basolateral membrane via K(+)-permeable channels. These observations provide our first understanding of the active role played by saccular epithelium in the local regulation of the [Na(+)] of endolymph for maintenance of our sense of balance.
Asunto(s)
Endolinfa/fisiología , Epitelio/fisiología , Homeostasis/fisiología , Sáculo y Utrículo/fisiología , Sodio/fisiología , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Sensory transduction in the cochlea depends on regulated ion secretion and absorption. Results of whole-organ experiments suggested that Reissner's membrane may play a role in the control of luminal Cl(-). We tested for the presence of Cl(-) transport pathways in isolated mouse Reissner's membrane using whole-cell patch clamp recording and gene transcript analyses using RT-PCR. The current-voltage (I-V) relationship in the presence of symmetrical NMDG-Cl was strongly inward-rectifying at negative voltages, with a small outward current at positive voltages. The inward-rectifying component of the I-V curve had several properties similar to those of the ClC-2 Cl(-) channel. It was stimulated by extracellular acidity and inhibited by extracellular Cd2+, Zn2+ and intracellular ClC-2 antibody. Channel transcripts expressed include ClC-2, Slc26a7 and ClC-Ka, but not Cftr, ClC-1, ClCa1, ClCa2, ClCa3, ClCa4, Slc26a9, ClC-Kb, Best1, Best2, Best3 or the beta-subunit of ClC-K, barttin. ClC-2 is the only molecularly-identified channel present that is a strong inward rectifier. This study is the first report of conductive Cl(-) transport in epithelial cells of Reissner's membrane and is consistent with an important role in endolymph anion homeostasis.
Asunto(s)
Canales de Cloruro/fisiología , Cóclea/fisiología , Células Epiteliales/fisiología , Audición/fisiología , Animales , Canales de Cloruro CLC-2 , Células Cultivadas , Canales de Cloruro/genética , Cloruros/fisiología , Cóclea/citología , Endolinfa/fisiología , Transporte Iónico , Ratones , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
BACKGROUND: The low luminal Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca2+-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium. RESULTS: We now identify this system in native vestibular and cochlear tissues by qRT-PCR, immunoblots and confocal immunolocalization. Transcripts were found and quantified for several isoforms of epithelial calcium channels (TRPV5, TRPV6), calcium buffer proteins (calbindin-D9K, calbindin-D28K), sodium-calcium exchangers (NCX1, NCX2, NCX3) and plasma membrane Ca2+-ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca2+ channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH)2vitamin D3 (VitD) significantly up-regulated transcripts of TRPV5 in SCCD, calbindin-D9K in SCCD and LW, NCX2 in LW, while PMCA4 in SCCD and PMCA3 in LW were down-regulated. The expression of TRPV5 relative to TRPV6 was in the sequence SV > Neonatal SCCD > Adult SCCD > LW > primary culture SCCD. Expression of TRPV5 protein from primary culture of SCCD did not increase significantly when cells were incubated with VitD (1.2 times control; P > 0.05). Immunolocalization showed the distribution of TRPV5 and TRPV6. TRPV5 was found near the apical membrane of strial marginal cells and both TRPV5 and TRPV6 in outer and inner sulcus cells of the cochlea and in the SCCD of the vestibular system. CONCLUSIONS: These findings demonstrate for the first time the expression of a complete Ca2+ absorptive system in native cochlear and vestibular tissues. Regulation by vitamin D remains equivocal since the results support the regulation of this system at the transcript level but evidence for control of the TRPV5 channel protein was lacking.
Asunto(s)
Calcio/metabolismo , Cóclea/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Catiónicos TRPV/metabolismo , Vestíbulo del Laberinto/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Calbindina 1 , Calbindinas , Epitelio/metabolismo , Técnica del Anticuerpo Fluorescente , Glicosilación , Transporte Iónico , Microscopía Confocal , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína G de Unión al Calcio S100/genética , Intercambiador de Sodio-Calcio/genética , Canales Catiónicos TRPV/genéticaRESUMEN
Unipolar brush cells (UBCs) are excitatory granular layer interneurons in the vestibulocerebellum. Here we assessed motor coordination and balance to investigate if deletion of acid-sensing ion channel 5 (Asic5), which is richly expressed in type II UBCs, is sufficient to cause ataxia. The possible cellular mechanism underpinning ataxia in this global Asic5 knockout model was elaborated using brain slice electrophysiology. Asic5 deletion impaired motor performance and decreased intrinsic UBC excitability, reducing spontaneous action potential firing by slowing maximum depolarization rate. Reduced intrinsic excitability in UBCs was partially compensated by suppression of the magnitude and duration of delayed hyperpolarizing K+ currents triggered by glutamate. Glutamate typically stimulates burst firing subsequent to this hyperpolarization in normal type II UBCs. Burst firing frequency was elevated in knockout type II UBCs because it was initiated from a more depolarized potential compared to normal cells. Findings indicate that Asic5 is important for type II UBC activity and that loss of Asic5 contributes to impaired movement, likely, at least in part, due to altered temporal processing of vestibular input.
Asunto(s)
Canales Iónicos Sensibles al Ácido/genética , Potenciales de Acción , Ataxia Cerebelosa/metabolismo , Neuronas/metabolismo , Animales , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/patología , Ácido Glutámico/metabolismo , Masculino , Ratones , Neuronas/fisiología , Potasio/metabolismoRESUMEN
Purinergic receptors protect the cochlea during high-intensity stimulation by providing a parallel shunt pathway through non-sensory neighboring epithelial cells for cation absorption. So far, there is no direct functional evidence for the presence and type/subunit of purinergic receptors in the utricle of the vestibular labyrinth. The goal of the present study was to investigate which purinergic receptors are expressed and carry cation-absorption currents in the utricular transitional cells and macula. Purinergic agonists induced cation-absorption currents with a potency order of ATP > bzATP = αßmeATP â« ADP = UTP = UDP. ATP and bzATP are full agonists, whereas αßmeATP is a partial agonist. ATP-induced currents were partially inhibited by 100 µM suramin, 10 µM pyridoxal-phosphate-6-azo-(benzene-2,4-disulfonic acid (PPADS), or 5 µM 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1, 4-diazepin-2-one (5-BDBD), and almost completely blocked by 100 µM Gd3+ or by a combination of 10 µM PPADS and 5 µM 5-BDBD. Expression of the P2RX2 and P2RX4 receptor was detected by immunocytochemistry in transitional cells and macular supporting cells. This is the first study to demonstrate that ATP induces cation currents carried by a combination of P2RX2 and P2RX4 in utricular transitional and macular epithelial cells, and supporting the hypothesis that purinergic receptors protect utricular hair cells during elevated stimulus intensity levels.
Asunto(s)
Adenosina Trifosfato/metabolismo , Células Laberínticas de Soporte/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Sáculo y Utrículo/metabolismo , Animales , Agonismo Parcial de Drogas , Células Laberínticas de Soporte/efectos de los fármacos , Potenciales de la Membrana , Ratones Endogámicos C57BL , Ratones Transgénicos , Agonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X2/efectos de los fármacos , Receptores Purinérgicos P2X4/efectos de los fármacos , Sáculo y Utrículo/citología , Sáculo y Utrículo/efectos de los fármacos , Transducción de Señal , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMEN
Mutations of SLC26A4 cause an enlarged vestibular aqueduct, nonsyndromic deafness, and deafness as part of Pendred syndrome. SLC26A4 encodes pendrin, an anion exchanger located in the cochlea, thyroid, and kidney. The goal of the present study was to determine whether developmental delays, possibly mediated by systemic or local hypothyroidism, contribute to the failure to develop hearing in mice lacking Slc26a4 (Slc26a4(-/-)). We evaluated thyroid function by voltage and pH measurements, by array-assisted gene expression analysis, and by determination of plasma thyroxine levels. Cochlear development was evaluated for signs of hypothyroidism by microscopy, in situ hybridization, and quantitative RT-PCR. No differences in plasma thyroxine levels were found in Slc26a4(-/-) and sex-matched Slc26a4(+/-) littermates between postnatal day 5 (P5) and P90. In adult Slc26a4(-/-) mice, the transepithelial potential and the pH of thyroid follicles were reduced. No differences in the expression of genes that participate in thyroid hormone synthesis or ion transport were observed at P15, when plasma thyroxine levels peaked. Scala media of the cochlea was 10-fold enlarged, bulging into and thereby displacing fibrocytes, which express Dio2 to generate a cochlear thyroid hormone peak at P7. Cochlear development, including tunnel opening, arrival of efferent innervation at outer hair cells, endochondral and intramembraneous ossification, and developmental changes in the expression of Dio2, Dio3, and Tectb were delayed by 1-4 days. These data suggest that pendrin functions as a HCO3- transporter in the thyroid, that Slc26a4(-/-) mice are systemically euthyroid, and that delays in cochlear development, possibly due to local hypothyroidism, lead to the failure to develop hearing.
Asunto(s)
Antiportadores de Cloruro-Bicarbonato/fisiología , Cóclea/crecimiento & desarrollo , Enfermedades Cocleares/etiología , Audición/fisiología , Hipotiroidismo/complicaciones , Animales , Antiportadores de Cloruro-Bicarbonato/genética , Cóclea/patología , Enfermedades Cocleares/patología , Electrofisiología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Expresión Génica/fisiología , Concentración de Iones de Hidrógeno , Hipotiroidismo/patología , Hibridación in Situ , Yoduro Peroxidasa/biosíntesis , Yoduro Peroxidasa/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportadores de Sulfato , Tiroxina/sangre , Yodotironina Deyodinasa Tipo IIRESUMEN
Epithelial cells of the inner ear coordinate their ion transport activity through a number of mechanisms. One important mechanism is the autocrine and paracrine signaling among neighboring cells in the ear via nucleotides, such as adenosine, ATP and UTP. This review summarizes observations on the release, detection and degradation of nucleotides by epithelial cells of the inner ear. Purinergic signaling is thought to be important for endolymph ion homeostasis and for protection from acoustic over-stimulation.
Asunto(s)
Vías Auditivas/metabolismo , Oído Interno/metabolismo , Nucleótidos de Purina/metabolismo , Receptores Purinérgicos/metabolismo , Transducción de Señal , Estimulación Acústica/efectos adversos , Animales , Oído Interno/lesiones , Endolinfa/metabolismo , Células Epiteliales/metabolismo , Humanos , Hidrólisis , Mecanotransducción Celular , Ruido/efectos adversos , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismoRESUMEN
Mutations in the gene encoding the gap junction protein connexin26 (Cx26) are responsible for the autosomal recessive isolated deafness, DFNB1, which accounts for half of the cases of prelingual profound hereditary deafness in Caucasian populations. To date, in vivo approaches to decipher the role of Cx26 in the inner ear have been hampered by the embryonic lethality of the Cx26 knockout mice. To overcome this difficulty, we performed targeted ablation of Cx26 specifically in one of the two cellular networks that it underlies in the inner ear, namely, the epithelial network. We show that homozygous mutant mice, Cx26(OtogCre), have hearing impairment, but no vestibular dysfunction. The inner ear developed normally. However, on postnatal day 14 (P14), i.e., soon after the onset of hearing, cell death appeared and eventually extended to the cochlear epithelial network and sensory hair cells. Cell death initially affected only the supporting cells of the genuine sensory cell (inner hair cell, IHC), thus suggesting that it could be triggered by the IHC response to sound stimulation. Altogether, our results demonstrate that the Cx26-containing epithelial gap junction network is essential for cochlear function and cell survival. We conclude that prevention of cell death in the sensory epithelium is essential for any attempt to restore the auditory function in DFNB1 patients.
Asunto(s)
Apoptosis , Cóclea/fisiología , Conexinas/fisiología , Uniones Comunicantes/metabolismo , Pérdida Auditiva/metabolismo , Animales , Cóclea/metabolismo , Conexina 26 , Conexinas/genética , Oído Interno/metabolismo , Oído Interno/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Potasio/metabolismoRESUMEN
The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K(+)-secreting vestibular dark cells and ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K(+) and low in Na(+). Direct measurement of this fluid in live animals revealed significant decreases in K(+) concentration and endolymphatic potential in both EphB2 and ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.
Asunto(s)
Endolinfa/fisiología , Efrina-B2/fisiología , Receptor EphB2/fisiología , Animales , Efrina-B2/deficiencia , Efrina-B2/genética , Femenino , Heterocigoto , Homeostasis , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Mutación , Potasio/metabolismo , Embarazo , Receptor EphB2/genética , Transducción de Señal , Sodio/metabolismo , Vestíbulo del Laberinto/patología , Vestíbulo del Laberinto/fisiologíaRESUMEN
The lumen of the inner ear has an unusually low concentration of endolymphatic Na+, which is important for transduction processes. We have recently shown that glucocorticoid receptors (GR) stimulate absorption of Na+ by semicircular canal duct (SCCD) epithelia. In the present study, we sought to determine the presence of genes involved in the control of the amiloride-sensitive Na+ transport pathway in rat SCCD epithelia and whether their level of expression was regulated by glucocorticoids using quantitative real-time RT-PCR. Transcripts were present for alpha-, beta-, and gamma-subunits of the epithelial sodium channel (ENaC); the alpha1-, alpha3-, beta1-, and beta3-isoforms of Na+-K+-ATPase; inwardly rectifying potassium channels [IC50 of short circuit current (Isc) for Ba2+: 210 microM] Kir2.1, Kir2.2, Kir2.3, Kir2.4, Kir3.1, Kir3.3, Kir4.1, Kir4.2, Kir5.1, and Kir7.1; sulfonyl urea receptor 1 (SUR1); GR; mineralocorticoid receptor (MR); 11beta-hydroxysteroid dehydrogenase (11beta-HSD) types 1 and 2; serum- and glucocorticoid-regulated kinase 1 (Sgk1); and neural precursor cell-expressed developmentally downregulated 4-2 (Nedd4-2). On the other hand, transcripts for the alpha4-subunit of Na+-K+-ATPase, Kir1.1, Kir3.2, Kir3.4, Kir6.1, Kir6.2, and SUR2 were found to be absent, and Isc was not inhibited by glibenclamide. Dexamethasone (100 nM for 24 h) not only upregulated the transcript expression of alpha-ENaC (approximately 4-fold), beta2-subunit (approximately 2-fold) and beta3-subunit (approximately 8-fold) of Na+-K+-ATPase, Kir2.1 (approximately 5-fold), Kir2.2 (approximately 9-fold), Kir2.4 (approximately 3-fold), Kir3.1 (approximately 3- fold), Kir3.3 (approximately 2-fold), Kir4.2 (approximately 3-fold), Kir7.1 (approximately 2-fold), Sgk1 (approximately 4-fold), and Nedd4-2 (approximately 2-fold) but also downregulated GR (approximately 3-fold) and 11beta-HSD1 (approximately 2-fold). Expression of GR and 11beta-HSD1 was higher than MR and 11beta-HSD2 in the absence of dexamethasone. Dexamethasone altered transcript expression levels (alpha-ENaC and Sgk1) by activation of GR but not MR. Proteins were present for the alpha-, beta-, and gamma-subunits of ENaC and Sgk1, and expression of alpha- and gamma-ENaC was upregulated by dexamethasone. These findings are consistent with the genomic stimulation by glucocorticoids of Na+ absorption by SCCD and provide an understanding of the therapeutic action of glucocorticoids in the treatment of Meniere's disease.
Asunto(s)
Amilorida/farmacología , Canales Epiteliales de Sodio/metabolismo , Epitelio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Canales Semicirculares/metabolismo , Sodio/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Animales Recién Nacidos , Transporte Biológico/efectos de los fármacos , Proteínas de Transporte de Catión/genética , Células Cultivadas , Dimetilsulfóxido/farmacología , Complejos de Clasificación Endosomal Requeridos para el Transporte , Epitelio/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Ubiquitina-Proteína Ligasas Nedd4 , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Glucocorticoides/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Generation of a strong electrical potential in the cochlea is uniquely mammalian and may reflect recent evolutionary advances in cellular voltage-dependent amplifiers. This endocochlear potential is hypothesized to dramatically improve hearing sensitivity, a concept that is difficult to explore experimentally, because manipulating cochlear function frequently causes rapid degenerative changes early in development. Here, we examine the deafness phenotype in adult Claudin 11-null mice, which lack the basal cell tight junctions that give rise to the intrastrial compartment and find little evidence of cochlear pathology. Potassium ion recycling is normal in these mutants, but endocochlear potentials were below 30 mV and hearing thresholds were elevated 50 dB sound pressure level across the frequency spectrum. Together, these data demonstrate the central importance of basal cell tight junctions in the stria vascularis and directly verify the two-cell hypothesis for generation of endocochlear potential. Furthermore, these data indicate that endocochlear potential is an essential component of the power source for the mammalian cochlear amplifier.
Asunto(s)
Sordera/genética , Sordera/patología , Proteínas del Tejido Nervioso/genética , Estría Vascular/fisiología , Uniones Estrechas/ultraestructura , Animales , Supervivencia Celular , Claudina-1 , Claudinas , Sordera/fisiopatología , Difusión , Electrofisiología , Endolinfa/metabolismo , Células Endoteliales/ultraestructura , Potenciales Evocados Visuales , Células Ciliadas Auditivas/patología , Técnicas In Vitro , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Emisiones Otoacústicas Espontáneas , Potasio/metabolismo , Transporte de Proteínas , Ganglio Espiral de la Cóclea/patología , Estría Vascular/metabolismo , Estría Vascular/patología , Uniones Estrechas/metabolismoRESUMEN
BACKGROUND: It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6) leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization. RESULTS: The transepithelial short circuit current (Isc) represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP) and diadenosine tetraphosphate (Ap4A) but not uridine 5'-diphosphate (UDP) at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB), an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 microM) did not inhibit the effect of UTP (1 microM). The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization. CONCLUSION: The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.
RESUMEN
BACKGROUND: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-). METHODS: Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. RESULTS: Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2. CONCLUSIONS: These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.
Asunto(s)
Cóclea/química , Sordera/etiología , Proteínas de Transporte de Membrana/análisis , Canales de Potasio de Rectificación Interna/análisis , Vestíbulo del Laberinto/química , Animales , Conexina 26 , Conexinas , Endolinfa/química , Potenciales Evocados Auditivos/fisiología , Bocio , Ratones , Canales de Potasio de Rectificación Interna/metabolismo , ARN Mensajero/análisis , Transportadores de Sulfato , SíndromeRESUMEN
The CD-1 mouse strain is known to have early onset of hearing loss that is progressive with aging. We sought to determine whether a disturbance of K+ homeostasis and pathological changes in the cochlear lateral wall were involved in the age-related hearing loss (AHL) of CD-1 as compared to the CBA/CaJ strain which has minimal AHL. In the present study, the endocochlear potential (EP) and endolymphatic K+ concentration ([K+]e) were measured in both strains of mice with double-barrel microelectrodes at "young" (1-2 mo) and "old" (5-9 mo) ages. CBA/CaJ mice displayed no changes with aging in EP and [K+]e of the basal turn. In the apical turn, there was a small positive shift of the EP (10 mV) with aging under both normoxic and acute anoxic conditions (-EP), without any change of [K+]e. Further, there were no obvious pathological changes in the lateral wall of CBA/CaJ mice. By contrast, old CD-1 mice displayed a significantly reduced [K+]e by 30% in both basal and apical turns with no significant changes in normoxic EP. The -EP in the apical turn was significantly reduced in magnitude by 6 mV. A severe loss of cells with aging was observed in the region of type IV fibrocytes of the apical and basal turns and of type II fibrocytes in the basal turn. A complete degeneration of organ of Corti was also observed at the basal turn of old CD-1 mice, as well as a basalward decline of spiral ganglion neuron density. The pathological changes in spiral ligament of CD-1 mice were similar to those of an inbred mouse strain C57BL/6J that expresses an AHL gene (ahl) and might be a primary etiology of AHL of CD-1 mice. These findings have ramifications for our understanding of AHL and for interpretation of genetic mutations in a CD-1 background.