Glutamate-induced production of nitric oxide in guinea pig vestibular sensory cells

Glutamate-induced production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with glutamate, NMDA and AMPA. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role in the glutamate-induced ototoxicity and also be involved in disease of the inner ear.     

Direct evidence of nitric oxide production in the guinea pig organ of Corti.

Production of nitric oxide (NO) in the organ of Corti of the guinea pig was investigated using the new fluorescence indicator 4,5-diaminofluorescein diacetate for direct detection of NO. The organ of Corti, lateral wall of the cochlea and isolated outer and inner hair cells were examined to locate NO production sites. The fluorescence intensities were augmented by stimulation with L-arginine or glutamate, and significantly increased after inoculation with lipopolysaccharide. This is the first direct evidence of NO production in the cochlea. NO may play an important role in the physiology of the organ of Corti and may also be involved in hearing disorders.     

Simultaneous detection of both nitric oxide and reactive oxygen species in guinea pig vestibular sensory cells

Gentamicin-induced production of reactive oxygen species (ROS) and of nitric oxide (NO) in the vestibular end organs of the guinea pig was investigated by applying two new fluorescence indicators, 4,5-diaminofluorescein diacetate for direct detection of NO and dihydrotetramethylrosamine for ROS. The vestibular sensory cells produced both NO and ROS after exposure to gentamicin. A nonspecific inhibitor of NO synthase, L-NAME, inhibited the production of NO but did not appear to affect the production of ROS following exposure to gentamicin. In contrast, a radical scavenger, D-methionine, or the neurotrophin BDNF suppressed the production of ROS, in turn stimulating NO production. These findings could indicate that both NO and ROS play an important role in aminoglycoside ototoxicity.     

Nitric oxide in guinea pig vestibular sensory cells following gentamicin exposure in vitro.

Gentamicin-induced production of nitric oxide (NO) in the vestibular end organs of the guinea pig was investigated using the new fluorescence indicator 4,5-diaminofluorescein diacetate for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with gentamicin. This increase in fluorescence was inhibited by the presence of the non-specific inhibitor for nitric oxide synthase, L-N(G)-nitroarginine methylester, and by the non-specific N-methyl-D-aspartic acid antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate. These findings indicate that NO may play an important role in the ototoxicity of aminoglycoside.     

Doxycycline reduces nitric oxide production in guinea pig inner ears

Objective

Gentamicin application is an important therapeutic option to control vertigo spells in Ménière's disease. However, even in the case of low-dose intratympanic application, gentamicin might contribute to a pathological NO-increase leading to cochlear damage and hearing impairment. The study was performed to evaluate the nitric oxide (NO) reducing capacity of doxycycline in the inner ear after NO-induction by gentamicin.

Methods

In a prospective animal study, a single dose of gentamicin (10 mg/kg body weight) was injected intratympanically into male guinea pigs (n = 48). The auditory brainstem responses (ABRs) were recorded prior to application and 3, 5 and 7 days afterwards. The organ of Corti and the lateral wall of 42 animals were isolated after 7 days and incubated separately for 6 h in cell culture medium. Doxycycline was adjusted to organ cultures of 5 animals. Two NOS inhibitors, N^G-Nitro-l-arginine methyl ester (l-NAME) and NG-monomethyl-l-arginine monoacetate (l-NMMA), were applied in three different concentrations to the organ cultures of 30 animals in total (5 animals per concentration). As controls, seven animals received no further substance except gentamicin. The NO-production was quantified by chemiluminescence. Additional six gentamicin-treated animals were used for immunohistochemical studies.

Results

The ABRs declined continuously from the first to the seventh day after gentamicin application. Doxycycline reduced NO-production in the lateral wall by 54% (p = .029) comparable to the effect of the applied nitric oxide inhibitors. In the organ of Corti, NO-production was reduced by about 41% showing no statistical significance in respect to great inter-animal variations.

Conclusion

The application of doxycycline might offer a new therapeutic approach to prevent NO-induced cochlea damage through ototoxic substances.     

Hyposmotic stimulation-induced nitric oxide production in outer hair cells of the guinea pig cochlea

Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca(2+)-free solution. L-N(G)-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca(2+) response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca(2+)](i) increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca(2+) response via the NO-cGMP-PKG pathway by a feedback mechanism.     

Lipopolysaccharide-induced expression of nitric oxide synthase II in the guinea pig vestibular end organ

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the vestibular organ of the guinea pig might induce formation of nitric oxide synthase (NOS) II. Forty-eight hours after the animals were injected with 1 mg transtympanic LPS, varying degrees of impaired caloric responses were observed with similar degeneration of vestibular hair cells. These effects could be blocked with N-nitro-l-arginine methylester, a competitive inhibitor of NOS. Findings suggested that NOS II, which was not normally detectable in the guinea pig vestibular organ but was present following inoculation of LPS, produced the nitric oxide as the toxic factor causing cell damage. If true, LPS may represent a reproducible method for studying the vestibular pathogenesis of inner ear disease. Received: 22 July 1997 / Accepted: 18 September 1997     

Ascorbic acid reduces noise-induced nitric oxide production in the guinea pig ear

OBJECTIVES: Noise-induced hearing loss can be caused, among other causes, by increased nitric oxide (NO) production in the inner ear leading to nitroactive stress and cell destruction. Some studies in the literature suggest that the degree of hearing loss (HL) could be reduced in an animal model through ascorbic acid supplementation. To identify the effect of ascorbic acid on tissue-dependent NO content in the inner ear of the guinea pig, we determined the local NO production in the organ of Corti and the lateral wall separately 6 hours after noise exposure. STUDY DESIGN: Prospective animal study in guinea pigs. METHODS: Over a period of 7 days, male guinea pigs were supplied with minimum (25 mg/kg body weight/day) and maximum (525 mg/kg body weight/day) ascorbic acid doses, and afterwards exposed to noise (90 dB sound pressure level for 1 hour). The acoustic-evoked potentials were recorded before and after noise exposure. The organ of Corti and the lateral wall were incubated differently for 6 hours in culture medium, and the degree of NO production was determined by chemiluminescence. RESULTS: Ascorbic acid treatment reduced the hearing threshold shift after noise exposure depending on concentration. When the maximum ascorbic acid dose was substituted, NO production was significantly reduced in the lateral wall after noise exposure and slightly reduced in the organ of Corti. CONCLUSIONS: Oral supplementation of the natural radical scavenger ascorbic acid reduces the NO-production rate in the inner ear in noisy conditions. This finding supports the concept of inner ear protection by ascorbic acid supplementation.     

Carbohydrates of the guinea pig vestibular supporting cells

Ultrastructural localization of glycoconjugates with special reference to the synthesizing process was studied in the guinea pig vestibular supporting cells using the tannic acid and ruthenium red staining technique. The extracellular glycoconjugate (glycocalyx) was well stained by the ruthenium red which may be synthesized by GERL complex. The supporting cell has a numerous number of granules which also contain tannic acid positive glycoconjugate. These granules were closely related to the Golgi complex as well as the apical cell surface. It may be indicated that the glycoconjugate of the supporting cells is glycosylated at the Golgi complex, stored in the granules, transported, secreted, and form otoconial membrane and the cupula.     

Freeze-fracturing of vestibular sensory epithelia in a strain of the waltzing guinea pig

The waltzing guinea pig exhibits genetically induced inner ear disease which develops into deafness and vestibular disorder. The type I hair cell in vestibular end organs shows specific pathology, featuring protrusion of the hair cell apex, sensory hair fusion and an intracellular actin filament rod. With the freeze-fracturing technique, consecutive stages of degeneration of the apical part of the type I hair cell are shown. Different possible mechanisms underlying these changes are discussed. It is probable that the genetically induced prenatally developed hair cell pathology is related to a pathological development of actin filaments and their connections in the apical cytoskeleton.     

Gentamicin induced nitric oxide-related oxidative damages on vestibular afferents in the guinea pig

Gentamicin is a well-known ototoxic aminoglycoside. However, the mechanism underlying this ototoxicity remains unclear. One of the mechanisms which may be responsible for this ototoxicity is excitotoxic damage to hair cells. The overstimulation of the N-methyl-d-aspartate (NMDA) receptors increases the production of nitric oxide (NO), which induces oxidative stress on hair cells. In order to determine the mechanism underlying this excitotoxicity, we treated guinea pigs with gentamicin by placing gentamicin (0.5 mg) pellets into a round window niche. After the sacrifice of the animals, which occurred at 3, 7 and 14 days after the treatment, the numbers of hair cells in the animals were counted with a scanning electron microscope. We then performed immunostaining using neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS) and nitrotyrosine antibodies. The number of hair cells in the animals was found to decrease significantly after 7 days. nNOS and iNOS expression levels were observed to have increased 3 days after treatment. Nitrotyrosine was expressed primarily at the calyceal afferents of the type I hair cells 3 days after treatment. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining revealed positive hair cells 3 days after treatment. Our results suggest that inner ear treatment with gentamicin may upregulate nNOS and iNOS to induce oxidative stress in the calyceal afferents of type I hair cells, via nitric oxide overproduction.     

豚鼠耳蜗一氧化氮合酶异型体的定位

目的研究一氧化氮合酶(NOS)的异型体在豚鼠耳蜗的定位分布,以探讨一氧化氮(NO)在内耳听觉生理和病理生理机制中的作用。方法使用特异性NOS异型体抗体,采用ABC免疫组化染色法,观察NOS异型体在正常豚鼠耳蜗的定位表达。结果NOS Ⅰ主要分布在内骨膜、螺旋神经节的核周体、螺旋韧带和Corti's器的细胞。NOSⅢ是耳蜗的主要NOS异型体免疫染色,其主要免疫染色分布于耳蜗神经、螺旋神经节核周体、螺旋韧带和耳蜗毛细血管球的内皮细胞,也见于Corti's器的细胞和神经纤维。NOS Ⅱ在正常豚鼠耳蜗内不表达。 结论结构型NOS(cNOS)表达在耳蜗的多个部位,表明NO参与内耳的正常生理功能,包括神经突触的神经传导、耳蜗血流的调节和耳蜗的骨代谢。     

Distribution of tyrosine hydroxylase-like immunofluorescence in guinea pig vestibular ganglia and sensory areas.

The distribution of tyrosine hydroxylase (TH)-like immunofluorescence was analyzed in the guinea pig vestibular ganglia and end organs using a monoclonal antibody to TH. TH was chosen as a marker for the sympathetic fibers because TH regulates the first step of catecholamine synthesis by converting tyrosine to dopa. In the vestibular ganglia, there were TH-positive nerve fibers having distinct varicosities surrounding the vestibular ganglion cells. In the sensory areas, there was a sympathetic plexus in the subepithelial tissue of the saccule, the utricle, and the crista ampullaris. We speculated that the sympathetic innervation has a direct influence on the vestibular ganglion cells and diffuse influence on the capillary permeability.     

Glutamate toxicity induced degeneration of outer hair cells with a temporal increase of nitric oxide production in the guinea pig cochlea

The aim of this study was to examine the roles of glutamate (GLU) toxicity and involvement of nitric oxide (NO) in the pathogenesis of cochlear degeneration. We examined guinea pig cochleae following chronic exposure to GLU. Trypan blue extrusion and transmission electron microscopy were performed to evaluate degeneration in the organ of Corti. In parallel, nitric oxide synthase (NOS) activity was demonstrated by histochemical staining of NADPH diapholase. GLU treatment caused time-dependent degeneration of outer hair cells (OHCs) in conjunction with a temporal increase of NOS activity in the organ of Corti. This suggests that GLU may be involved in OHC degeneration under toxic conditions, with NO production possibly playing a role in this process. Received: 2 April 1998 / Accepted: 4 December 1998     

离体豚鼠前庭神经节细胞的分离及胞内钙离子活动的观察

目的进一步了解前庭神经节细胞的形态和电生理特性.方法用体重250～300g的豚鼠,断头后取出颞骨,剔除面神经和听神经,取出前庭神经节.经酶消化后用机械法分离成单细胞,用钙离子敏感的荧光染料Fura-2染色,于数字荧光显微镜下观察.结果分离出的单个前庭神经节细胞形状为园形、椭园形和不规则型,直径从20μm到30μm不等,分为有微绒毛的Ⅰ型细胞和无绒毛的Ⅱ型细胞.在体外室温22～24℃下可存活5～6h.用150 mmol/L KCl灌流1分钟,灌流开始20秒后,前庭神经节细胞胞内钙离子荧光强度比率从静止时的0.47±0.04,增至1.18±0.19,40秒后达峰值1.43±0.18,然后缓慢下降.300秒后降至峰值一半,约10分钟后基本恢复至正常,重复刺激可得到类似的结果.在无钙外环境中,150 mmol/L KCl不能诱发胞内钙离子浓度增加.1μM ionomycin可诱发不可逆的胞内钙离子浓度增加.结论本研究使用酶消化后机械分离法分离出活性良好的单个神经节细胞,前庭神经节细胞膜存在电压依赖性钙离子通道.     

Role of nitric oxide in focal microcirculation disorder of guinea pig cochlea

This study was designed to evaluate the role of endogenous nitric oxide (NO) in focal microcirculation disorder of the guinea pig cochlea. Focal microcirculation disorder was induced by a photochemical reaction at the lateral wall of the second cochlear turn. Saline or N omega-nitro-L-arginine methyl ester (L-NAME) was administered before the onset of photochemical reaction. Cochlear blood flow (CBF) was measured at the focal lesion (ischemic core), 1 mm from the lesion in the apical and basal direction (ischemic border zone) by using a novel non-contact laser blood flowmeter. NO synthase activities were measured by radioenzymeassay. In the saline pretreatment group, CBF was significantly decreased to 58.8+/-4.4% of the baseline at the ischemic core 30 min after the onset of photochemical reaction (P<0.01), while CBF showed no significant change at the ischemic border zone. In the L-NAME pretreatment group, CBF was significantly decreased not only at the focal lesion (48.3+/-6.5%, P<0.01), but also at the ischemic border zone (apical, 49.3+/-2.3%, P<0.05; basal, 58.7+/-7.1%, P<0.05, respectively). NO synthase III activity of cochlea was increased significantly (P<0.01) 15 min after microcirculation disorder. These findings suggest that formation of endogenous NO plays a key role in the maintenance of CBF in acute focal cochlear microcirculation disorder.     

Nitric oxide distribution and production in the guinea pig cochlea

Production sites and distribution of nitric oxide (NO) were detected in cochlear lateral wall tissue, the organ of Corti and in isolated outer hair cells (OHCs) from the guinea pig using the fluorescent dye, 4,5-diaminofluorescein diacetate. Fluorescent signal, indicating the presence of NO, was found in the afferent nerves and their putative endings near inner hair cells (IHCs) and putative efferent nerve endings near OHCs, the IHCs and OHCs, the endothelial cells of blood vessels of the spiral ligament, the stria vascularis, and the spiral blood vessels of the basilar membrane. An increased NO signal was observed following exposure to the substrate for NO, L-arginine, while exposure to NO synthase inhibitors resulted in a decrease in NO signal. Observation of OHCs at the subcellular level revealed differentially strong fluorescent signals at the locations of cuticular plate, the subcuticular plate region, the infranuclear region, and the region adjacent to the lateral wall. The findings indicate the presence of NO in the cochlea and suggest that NO may play an important role in both regulating vascular tone and mediating neurotransmission in guinea pig cochlea.     

Fine structure of guinea pig vestibular kinocilium

The fine structure of the utricular kinocilium of the guinea pig was examined with transmission electron microscopy after treatment with tannic acid to enhance resolution of internal morphology. The utricular kinocilium was devoid of inner dynein arms and a central pair of microtubules, while a set of outer dynein arms and radial spokes was found. This supports the hypothesis that the vestibular kinocilium is non-motile. Internal electron-dense particles at the attachment sites of the stereo-kinociliar bonds were situated in the immediate periphery of the outer dynein arms, although no visible connection existed between these structures. Findings obtained in the present study seem to give insight on the mechanism of mechanosensory transduction in the vestibular sensory cells.     

Basal nitric oxide production contributes to membrane potential and vasotone regulation of guinea pig in vitro spiral modiolar artery

Nitric oxide (NO) is a potent vasodilating agent implicated in cochlear blood flow regulation. We recently demonstrated that exogenously applied NO donor DPTA-NONOate hyperpolarizes both endothelial and smooth muscle cells of in vitro spiral modiolar artery (SMA) via activation of ATP-sensitive K+ channels (K(ATP)). Also, NO was detected in the SMA cells by NO indicator dye in the in vitro basal condition. Using intracellular recording techniques, electrochemical NO-sensing measurement, and a vaso-diameter video tracking method, we investigated the basal release of NO from the in vitro SMA and its role in the vascular function. We found that (1) 300 microM L-NAME, a NO synthase inhibitor, and 3 microM glipizide caused a depolarization of approximately 4.5 and approximately 3.2 mV, respectively, in cells with a resting potential less negative than -60 mV; (2) NO sensor in the close vicinity of the SMA detected a NO concentration of approximately 50 nM that was suppressed by L-NAME and enhanced by L-arginine (1-1000 microM); (3) NO donor DPTA-NONOate (0.1-30 microM) applications produced about 8-245 nM of NO in the recording bath. These data indicate a NO concentration-hyperpolarization relation, with an EC50 of 22 nM. (4) Finally, L-NAME but not glipizide produced a 4.8% reduction in SMA diameter (approximately 50 microm) in the majority of SMAs, whereas NONOate (10 microM) always caused a dilation. Both the induced constriction and dilation were not significantly affected by 3 microM glipizide. We conclude that a significant amount of NO (> 50 nM) is tonically released from the in vitro SMA, which is above the EC50 for activation of K(ATP), and thus contributes to the membrane polarization. The basal release of NO also contributes to vasotone relaxation, but the K(ATP) activation appears to play little role in the relaxation of the in vitro SMA.