Tomonori Takasaka

Gap junction systems in the mammalian cochlea.

Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Excluding sensory cells, most cells in the cochlea are connected via gap junctions and these gap junctions appear to play critical roles in cochlear ion homeostasis. Connexin 26 occurs in gap junctions connecting all cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions. The first system, the epithelial cell gap junction system, is mainly composed of all organ of Corti supporting cells, and also includes interdental cells in the spiral limbus and root cells within the spiral ligament. The second system, the connective tissue cell gap junction system, consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal dark cells in the supralimbal zone, and fibrocytes in the spiral limbus. One function of these gap junctional systems is the recirculation of K(+) ions from hair cells to the strial marginal cells. Interruption of this recirculation, which may be caused by the mutation in connexin 26 gene, would deprive the stria vascularis of K(+) and result in hearing loss.

Novel mutations in the connexin 26 gene (GJB2) responsible for childhood deafness in the Japanese population

Mutations in the connexin 26 gene (GJB2), which encodes a gap-junction protein and is expressed in the inner ear, have been shown to be responsible for a major part of nonsyndromic hereditary prelingual (early-childhood) deafness in Caucasians. We have sequenced the GJB2 gene in 39 Japanese patients with prelingual deafness (group 1), 39 Japanese patients with postlingual progressive sensorineural hearing loss (group 2), and 63 Japanese individuals with normal hearing (group 3). Three novel mutations were identified in group 1: a single nucleotide deletion (235delC), a 16-bp deletion (176-191 del (16)), and a nonsense mutation (Y136X) in five unrelated patients. The 235delC mutation was most frequently observed, accounting for seven alleles in 10 mutant alleles. Screening of 203 unrelated normal individuals for the three mutations indicated that the carrier frequency of the 235delC mutation was 2/203 in the Japanese population. No mutation was found in group-2 patients. We also identified two novel polymorphisms (E114G and I203T) as well as two previously reported polymorphisms (V27I andV37I). Genotyping with these four polymorphisms allowed normal Japanese alleles to be classified into seven haplotypes. All 235delC mutant alleles identified in four patients resided only on haplotype type 1. These findings indicate that GJB2 mutations are also responsible for prelingual deafness in Japan.

Cellular mechanism of acetylcholine-induced response in dissociated outer hair cells of guinea-pig cochlea.

1. The acetylcholine (ACh)‐induced currents (IACh) in dissociated outer hair cells (OHCs) of guinea‐pig cochlea were investigated using the whole‐cell patch‐clamp technique, in both conventional and nystatin perforated‐patch configurations. 2. ACh and carbamylcholine (CCh) induced outward currents at a holding potential (VH) of ‐60 mV in the perforated‐patch configuration. The IACh increased in a sigmoidal fashion over the concentration range between 3 x 10(‐6) and 10(‐3) M. The dissociation constant (KD) was 1.7 x 10(‐5) M and the Hill coefficient (n) was 2.7. The KD and n for CCh were 8.7 x 10(‐5) M and 2.2, respectively. Neither nicotine nor muscarine induced any detectable current up to a concentration of 10(‐3) M. 3. Various muscarinic agonists such as oxotremorine‐M, McN‐A‐343 and oxotremorine could also induce the outward currents, although these current amplitudes were about one‐third that of ACh, indicating that they were partial agonists. 4. The muscarinic antagonists atropine, 4‐DAMP, AF‐DX 116 and pirenzepine inhibited the IACh in a concentration‐dependent manner. The half‐inhibitory concentrations (IC50) for atropine, 4‐DAMP, AF‐DX 116 and pirenzepine were 4.8 x 10(‐6), 6.2 x 10(‐6), 2.1 x 10(‐5) and 2.9 x 10(‐4) M, respectively. 5. When the extracellular Ca2+ concentration ([Ca2+])o) was reduced to lower than 1 mM, the amplitude of IACh, abruptly decreased. In a nominally Ca(2+)‐free external solution ACh did not induce any current. The increase of [Ca2+]o beyond 1 mM did not change the IACh. 6. When OHCs were perfused intracellularly with a pipette solution containing 10 mM BAPTA in the conventional whole‐cell mode, ACh could not induce outward K+ currents. The Ca2+ ionophore A23187 induced an outward current. These results indicate that intracellular Ca2+ is involved in the ACh response. 7. Calmodulin inhibitors such as chlorpromazine, W‐7 and trifluoperazine inhibited the IACh in a concentration‐dependent manner. 8. When OHCs were dialysed with either 100 microM GDP beta S or 1 micrograms/ml pertussis toxin (PTX) through the patch pipette at a VH of ‐60 mV, the IACh diminished within 10 min, whereas the IACh of the control remained steady for over 20 min, suggesting that a PTX‐sensitive G‐protein is involved in the ACh response.(ABSTRACT TRUNCATED AT 400 WORDS)

Cisplatin-induced apoptotic cell death in Mongolian gerbil cochlea

Cisplatin is well known to cause cochleotoxicity. In order to determine the underlying mechanisms of cisplatin-induced cell death in the cochlea, we investigated the apoptotic changes and the expression of bcl-2 family proteins controlling apoptosis. Mongolian gerbils were administered 4 mg/kg/day cisplatin consecutively for 5 days. The cisplatin-treated animals showed a significant deterioration in the responses of both distortion product otoacoustic emissions and the endocochlear potential as compared with those of the age-matched controls, suggesting outer hair cell and stria vascularis dysfunction. The presence of DNA fragmentation revealed by a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling method was recognized in the organ of Corti, the spiral ganglion, and the stria vascularis in the cisplatin-treated animals whereas almost negative results were obtained in the control animals. The nuclear morphology obtained by Hoechst 33342 staining revealed pyknotic and condensed nuclei, confirming the presence of the characteristic features of apoptosis. A significant increase and reduction in the number of bax- and bcl-2-positive cells, respectively, following cisplatin treatment was observed in the cells of the organ of Corti, the spiral ganglion, and the lateral wall. These findings suggest a critical role for bcl-2 family proteins in the regulation of apoptotic cell death induced by cisplatin. The underlying mechanisms of the cisplatin-induced cell death are discussed.

Endoscopic Sinus Surgery Improves Pulmonary Function in Patients with Asthma Associated with Chronic Sinusitis

We evaluated the clinical efficacy of endonasal endoscopic sinus surgery (ESS) in patients with asthma associated with chronic sinusitis. Twenty-one patients (13 men and 8 women) from 27 to 72 years old were enrolled in this study. All patients had had sinus-related symptoms for more than 3 months and had computed tomographic evidence of paranasal sinus opacification. Fifteen patients underwent bilateral endonasal ESS under local anesthesia, and 6 other patients without surgery were controls. The sinus-related symptoms of the preoperative and postoperative periods were assessed via a questionnaire. The period 6 months prior to surgery was compared with that 6 months postoperatively with regard to peak expiratory flow and total dosage of systemic glucocorticoids. Sinus-related symptoms in the ESS group were significantly improved 6 months postoperatively. The average peak expiratory flow 6 months following surgery was improved in the ESS patients, ranging from 40 to 190 L/min. Seven patients showed a reduction in the need for corticosteroids, whereas 2 patients were unchanged and 2 patients required larger dosages. The remaining 4 patients needed no corticosteroids before or after ESS. No significant changes in sinus-related symptoms or peak expiratory flow were obtained for the control group. Improvement of paranasal sinus disease by successful ESS can alleviate pulmonary dysfunction in asthma associated with chronic sinusitis. We believe that adequate and positive treatment for chronic sinusitis would reduce not only the nasal and sinus-related symptoms evoked by chronic sinusitis, but also some of the signs induced by asthma.

Expression of connexin 31 in the developing mouse cochlea.

Connexin 31 (C×31) mutations cause an autosomal dominant form of high-frequency hearing loss. The immunohistochemical localization of C×31 in mouse cochlea was studied at different ages between 0 and 60 days after birth (DAB). C×31-like immunoreactivity was detected in fibrocytes of spiral ligament and spiral limbus at 12 DAB, gradually enhanced with the increase of age and reached the adult pattern on 60 DAB. Immunoreactivity decreased gradually from the basal to apical turn in all developmental stages. The mRNA of C×31 was also identified by RT-PCR. The distribution of C×31 and connexin 26 were obviously different in the developing mouse cochlea. The expression and distribution of C×31 in the development may explain the progressive hearing loss in human C×31 mutations.

Density of motility-related charge in the outer hair cell of the guinea pig is inversely related to best frequency

Whole cell voltage clamp and freeze fracture were used to study the electrophysiological and ultrastructural correlates of the outer hair cell (OHC) lateral membrane molecular motors. We find that specific voltage-dependent capacitance, which derives from motility-related charge movement, increases as cell length decreases. This increasing non-linear charge density predicts a corresponding increase in sensor-motor density. However, while OHC lateral membrane particle density increases, a quantitative correspondence is absent. Thus, the presumed equivalence of particle and motor is questionable. The data more importantly indicate that whereas the voltage driving OHC motility, i.e. the receptor potential, may decrease with frequency due to the OHCs low-pass membrane filter, the electrical energy (Q x V) supplied to the lateral membrane will tend to remain stable. This conservation of energy delivery is likely crucial for the function of the cochlear amplifier at high frequencies.

The Ca2+ activity of cochlear endolymph of the guinea pig and the effect of inhibitors.

The Ca2+ concentrations in cochlear perilymph and endolymph of the guinea pig were measured with double-barreled Ca2+-selective microelectrodes and showed 1.76 +/- 0.74 X 10(-3) M and 2.20 +/- 0.19 X 10(-5) M, respectively. The electrochemical potential gradient for Ca2+ between perilymph and endolymph was 23.2 mV and the existence of an active transport mechanism from the former to the latter was suggested. Vanadate given perilymphatically decreased the Ca2+ concentration in endolymph with a slight elevation of the endocochlear potential and was suspected of blocking the active transport. The Ca2+ concentration in endolymph was abruptly increased by anoxia or the intravenous administration of 60 mg/kg furosemide and was slightly increased by the intravenous administration of 30 mg/kg furosemide or 100 mg/kg acetazolamide. The endolymphatic pH measured with pH-microelectrodes under various conditions indicates that the mechanism of increase in the Ca2+ concentration is attributed not to the liberation of Ca2+ from the surrounding tissues caused by a fall in pH but to the increased influx of Ca2+ from perilymph due to the depression of the endocochlear potential.

Mechanism of neutrophil recruitment induced by IL-8 in chronic sinusitis

BACKGROUND The mechanism of neutrophil recruitment in patients with chronic sinusitis is unclear. OBJECTIVE This study aims to elucidate the role of IL-8 in inducing neutrophil accumulation in the nasal discharge of patients with chronic sinusitis. METHODS Nasal discharge and mucosal specimens were obtained from two groups of patients, those with chronic sinusitis and those with allergic rhinitis. The samples were subjected to immunohistochemical examination and in situ hybridization. The IL-8 level in the nasal discharge was measured by enzyme immunoassay. RESULTS Immunoreactivity to IL-8 was observed in polymorphonuclear cells of nasal smear, in nasal gland duct cells, and in epithelial cells of the chronic sinusitis group; whereas those of the allergic rhinitis group mostly showed little or no reaction. Similar patterns of localization were shown by in situ hybridization for IL-8 messenger RNA. The IL-8 level in nasal discharge was significantly higher in the chronic sinusitis group than in the allergic rhinitis group. CONCLUSION These results suggest that chemotactic factors in sinus effusion, including IL-8 derived from nasal gland duct cells and epithelial cells, attract neutrophils out of mucosa, and the neutrophils that have emigrated into the sinus effusion secrete IL-8. This induces further neutrophil accumulation in the sinus effusion of patients with chronic sinusitis.

Inhibitory Effect of Macrolides on Interleukin-8 Secretion From Cultured Human Nasal Epithelial Cells

The mechanism of macrolide therapy in chronic sinusitis patients is unclear. The authors studied the effect of macrolides on interleukin (IL)‐8 secretion from cultured human nasal epithelial cells. Epithelial cells harvested from the nasal polyps of patients with chronic sinusitis were primary‐cultured, and secreted IL‐8 in culture media was measured by enzyme immunoassay. The cells secreted considerable amounts of IL‐8 constitutively and in response to lipopolysaccharide. The secretion was significantly inhibited by 10−5 M of erythromycin, clarithromycin, roxithromycin, and josamycin. 10−6 M erythromycin still showed the inhibitory effect, whereas the same concentration of josamycin did not. These results indicate that macrolide antibiotics may act as an immunomodulator to reduce IL‐8 in inflammatory sites and, at least partially, account for the clinically discrepant effects between 14‐ and 16‐membered ring macrolides in long‐term low‐dose therapy for chronic sinusitis.