Age-related changes in Na, K-ATPase expression, subunit isoform selection and assembly in the stria vascularis lateral wall of mouse cochlea.

Due to the critical role of cochlear ion channels for hearing, the focus of the present study was to examine age-related changes of Na, K-ATPase (NKA) subunits in the lateral wall of mouse cochlea. We combined qRT-PCR, western blot and immunocytochemistry methodologies in order to determine gene and protein expression levels in the lateral wall of young and aged CBA/CaJ mice. Of the seven NKA subunits, only the mRNA expressions of α1, ß1 and ß2 subunit isoforms were detected in the lateral wall of CBA/CaJ mice. Aging was accompanied by dys-regulation of gene and protein expression of all three subunits detected. Hematoxylin and eosin (H&E) staining revealed atrophy of the cochlear stria vascularis (SV). The SV atrophy rate (20%) was much less than the ∼80% decline in expression of all three NKA isoforms, indicating lateral wall atrophy and NKA dys-regulation are independent factors and that there is a combination of changes involving the morphology of SV and NKA expression in the aging cochlea which may concomitantly affect cochlear function. Immunoprecipitation assays showed that the α1-ß1 heterodimer is the selective preferential heterodimer over the α1-ß2 heterodimer in cochlea lateral wall. Interestingly, in vitro pathway experiments utilizing cultured mouse cochlear marginal cells from the SV (SV-K1 cells) indicated that decreased mRNA and protein expressions of α1, ß1 and ß2 subunit isoforms are not associated with reduction of NKA activity following in vitro application of ouabain, but ouabain did disrupt the α1-ß1 heterodimer interaction. Lastly, the association between the α1 and ß1 subunit isoforms was present in the cochlear lateral wall of young adult mice, but this interaction could not be detected in old mice. Taken together, these data suggest that in the young adult mouse there is a specific, functional selection and assembly of NKA subunit isoforms in the SV lateral wall, which is disrupted and dys-regulated with age. Interventions for this age-linked ion channel disruption may have the potential to help diagnose, prevent, or treat age-related hearing loss.

The gastric H,K-ATPase in stria vascularis contributes to pH regulation of cochlear endolymph but not to K secretion.

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.

All Akt isoforms (Akt1, Akt2, Akt3) are involved in normal hearing, but only Akt2 and Akt3 are involved in auditory hair cell survival in the mammalian inner ear.

The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear-especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear.

Prenatally exposure to exogenous glucocorticoids and stress may affect the inner ear.

OBJECTIVES: This study aims to investigate the effect of exogenous glucocorticoid exposure in the prenatal period on hearing and to evaluate the effectiveness of caffeic acid phenethyl ester (CAPE), an antioxidant, on the prevention of the inner ear injury. MATERIALS AND METHODS: Dexamethasone was given to half of twelve Sprague-Dawley pregnant rats and the distilled water was given to the remaining half. The real subjects were obtained by born of the offsprings. When the all subjects were two months of age, they were exposed to 110 dB noise during four hours as a stressor effect. These subjects were divided into three groups. Group 1: subjects to whose mothers were given distilled water; Group 2: subjects to whose mothers were given dexamethasone; Group 3: subjects to whose mothers were given dexamethasone and CAPE. RESULTS: While there was no statistical significance in hearing thresholds which exposed and not exposed to exogenous dexamethasone before noise exposure (p>0.05) between the groups, the elevation of hearing thresholds of subjects which exposed to exogenous dexamethasone was statistically significant after noise exposure (p<0.05). CONCLUSION: Prenatally exposure to exogenous glucocorticoids may cause the inner ear susceptible to the effect of noise, and CAPE is effective to prevent the possible damage.

A study of the histologic and enzyme histochemical changes in the cochlea of Guinea pigs after non-focused ultrasound irradiation.

We evaluated the histologic and histochemical (succinate dehydrogenase, SDH) changes in cochleas of guinea pigs after non-focused ultrasound (NFU) irradiation. For this purpose, the cochleas of 50 guinea pigs (both ears in Groups 1-10) were irradiated at 2.5-8.0 MHz NFU for 6 h and the histologic/histochemical changes were determined. Our data show that after NFU irradiation for 6 h, no histological changes were observed in the cochleas by silver staining and scanning electron microscopic examination; however, the SDH activity of the basilar membranes and stria vascularises at the corresponding position decreased significantly. The SDH values of basilar membranes and stria vascularis were higher at 8 h than at 30 min after the NFU irradiation. SDH activity of the outer hair cells presented more declination than that of the inner hair cells. It was, therefore, concluded that a certain dosage of NFU irradiation at various frequencies could lead to metabolic changes in the basilar membrane and stria vascularis at different areas of cochlea. Moreover, these changes were found to be reversible or partially reversible. These changes also suggest that the cochlear hair cells located at different areas might be related to ultrasonic perception.

Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin.

Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.

Age-related changes in antioxidant enzymes related to hydrogen peroxide metabolism in rat inner ear.

Oxidative stress is a pervasive factor in aging and has been implicated in noise-induced cochlear pathology. In this study, we measured the activities of two enzymes that catalyze the removal of hydrogen peroxide (H(2)O(2)), catalase and glutathione peroxidase (Gpx), in 3- and 24-month-old Fisher-344 rats, and reduced and oxidized glutathione in 3-, 12-, and 24-month-old rats. There was an increase in Gpx activity in vascular tissue (spiral ligament and stria vascularis), but no change in modiolar, sensory or vestibular tissue of the cochlea. The elevation in vascular tissue was age-related. We observed a significant elevation of catalase activity in vestibular tissue, a tendency for age-related elevation in the modiolus, but no change in vascular or sensory cochlear tissue. These findings suggest that increased Gpx activity in vascular cochlear tissue may be an age-related compensation for a decrease in glutathione and a decline in the redox state measured by the ratio of reduced to oxidized glutathione.

Endocytosis of microperoxidase in the marginal cells of stria vascularis.

OBJECTIVE: Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis. METHODS: We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5-60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy. RESULTS: Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed. CONCLUSION: The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.

Gastric type H+,K+-ATPase in the cochlear lateral wall is critically involved in formation of the endocochlear potential.

Cochlear endolymph has a highly positive potential of approximately +80 mV known as the endocochlear potential (EP). The EP is essential for hearing and is maintained by K(+) circulation from perilymph to endolymph through the cochlear lateral wall. Various K(+) transport apparatuses such as the Na(+),K(+)-ATPase, the Na(+)-K(+)-2Cl(-) cotransporter, and the K(+) channels Kir4.1 and KCNQ1/KCNE1 are expressed in the lateral wall and are known to play indispensable roles in cochlear K(+) circulation. The gastric type of the H(+),K(+)-ATPase was also shown to be expressed in the cochlear lateral wall (Lecain E, Robert JC, Thomas A, and Tran Ba Huy P. Hear Res 149: 147-154, 2000), but its functional role has not been well studied. In this study we examined the precise localization of H(+),K(+)-ATPase in the cochlea and its involvement in formation of EP. RT-PCR analysis showed that the cochlea expressed mRNAs of gastric alpha(1)-, but not colonic alpha(2)-, and beta-subunits of H(+),K(+)-ATPase. Immunolabeling of an antibody specific to the alpha(1) subunit was detected in type II, IV, and V fibrocytes distributed in the spiral ligament of the lateral wall and in the spiral limbus. Strong immunoreactivity was also found in the stria vascularis. Immunoelectron microscopic examination exhibited that the H(+),K(+)-ATPase was localized exclusively at the basolateral site of strial marginal cells. Application of Sch-28080, a specific inhibitor of gastric H(+),K(+)-ATPase, to the spiral ligament as well as to the stria vascularis caused prominent reduction of EP. These results may imply that the H(+),K(+)-ATPase in the cochlear lateral wall is crucial for K(+) circulation and thus plays a critical role in generation of EP.

Cu/Zn superoxide dismutase and age-related hearing loss.

Mice, in which the genetics can be manipulated and the life span is relatively short, enable evaluation of the effects of specific gene expression on cochlear degeneration over time. Antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1) protect cells from toxic, reactive oxygen species and may be involved in age-related degeneration. The effects of SOD1 deletion and over-expression on the cochlea were examined in Sod1-null mice, Sod1 transgenic mice and in age- and genetics-matched controls. Auditory brainstem responses (ABR) were measured and cochleae were histologically examined. The absence of SOD1 resulted in hearing loss at an earlier age than in wildtype or heterozygous mice. The cochleae of the null mice had severe spiral ganglion cell degeneration at 7-9 months of age. The stria vascularis in the aged, null mice was thinner than in the heterozygous or wildtype mice. Over-expression of SOD1 did not protect against hearing loss except at 24 months of age. In conclusion, SOD1 seems important for survival of cochlear neurons and the stria vascularis, however even half the amount is sufficient and an over abundance does not provide much protection from age-related hearing loss.

Matrix metalloproteinase dysregulation in the stria vascularis of mice with Alport syndrome: implications for capillary basement membrane pathology.

Alport syndrome results from mutations in genes encoding collagen alpha3(IV), alpha4(IV), or alpha5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.

Probable function of Boettcher cells based on results of morphological study: localization of nitric oxide synthase.

Boettcher cells lie on the basilar membrane beneath Claudius cells. The cells are considered supporting cells for the organ of Corti, and present only in the lower turn of the cochlea, which responds to high-frequency sound. Boettcher cells interdigitate with each other, and project microvilli into the intercellular space. Their structural specialization suggests that Boettcher cells may play a significant role in the function of the cochlea. Nitric oxide synthase (NOS) has previously been detected in substructures of the cochlea. In the cochlea, it is believed that nitric oxide plays an important role in neurotransmission, blood flow regulation, and induction of cytotoxicity under pathological conditions. Findings concerning detection of NOS on Boettcher cells are rare. We demonstrated here the localization of NOS on Boettcher cells of the rat by immunohistochemistry using polyclonal antibody to NOS. On observation with the light microscope using DAB staining, positive immunostaining to NOS was observed in Boettcher cells. In immunoelectron micrographs, NOS was detected abundantly in the cytoplasm of the interdigitations. This suggests that the interdigitations may play significant roles by using NOS. It follows from this that the nitric oxide (NO) on Boettcher cells may influences neighboring Boettcher cells. The ultrastructure of Boettcher cells suggests that they may be active cells, which perform both secretory and absorptive functions.

Upregulated iNOS and oxidative damage to the cochlear stria vascularis due to noise stress.

Our previous work has revealed increased nitric oxide (NO) production in the cochlear perilymph following noise stress. However, it is not clear if the increase of NO is related to iNOS and whether NO-related oxidative stress can cause vascular tissue damage. In this study, iNOS immunoreactivity, NO production, and reactive oxygen species (ROS) in the lateral wall were examined in normal mice and compared with similar animals exposed to 120 dBA broadband noise, 3 h/day, for 2 consecutive days. In the normal animals, iNOS expression was not observed in the vascular endothelium of the stria vascularis and only weak iNOS immunoactivity was detected in the marginal cells. However, expression of iNOS in the wall of the blood vessels of stria vascularis and marginal cells was observed after loud sound stress (LSS). Relatively low levels of NO production and low ROS activity were detected in the stria vascularis in the unstimulated condition. In contrast, NO production was increased and ROS activity was elevated in the stria vascularis after LSS. These changes were attenuated by the iNOS inhibitor, GW 274150. To explore whether noise induces apoptotic processes in the stria vascularis, we examined morphological changes in endothelial- and marginal-cells. In vitro, annexin-V phosphatidylserine (PS) (to label and detect early evidence of apoptosis) was combined with propidium iodide (PI) (to probe plasma membrane integrity). PI alone was used in fixed tissues to detect later stage apoptotic cells by morphology of the nuclei. Following LSS, PS was expressed on cell surfaces of endothelial cells of blood vessels and marginal cells of the stria vascularis. Later stage apoptosis, characterized by irregular nuclei and condensation of nuclei, was also observed in these cells. The data indicate that increased iNOS expression and production of both NO and ROS following noise stress may lead to marginal cell pathology, and the dysfunction of cochlear microcirculation by inducing blood vessel wall damage.

Nuclear-factor kappa B (NF-kappa B)-inducible nitric oxide synthase (iNOS/NOS II) pathway damages the stria vascularis in cisplatin-treated mice.

BACKGROUND: Cisplatin is reported to damage the stria vascularis of the cochlea. Free radicals, especially large amounts of nitric oxide catalyzed by inducible nitric oxide synthase, are considered to have an important role in this toxicity. The induction of inducible nitric oxide synthase is regulated by nuclear-factor kappa B (NF-kappa B). We examined the damage of the stria vascularis by immunohistochemical techniques. MATERIALS AND METHODS: Cisplatin (15 mg/kg b.w.) was injected intraperitoneally into the mice. Three days after the injection, the cochleas were immunohistochemically-stained using specific antibodies for nuclear-factor kappa B (NF-kappa B), inducible nitric oxide synthase (iNOS) or single-stranded DNA. RESULTS: NF-kappa B was expressed in the cisplatin-treated cochlea, especially in the stria vascularis and the spiral ligament. iNOS was also expressed in the stria vascularis and the spiral ligament. Fragments of DNA were observed only in the stria vascularis. CONCLUSION: The large amounts of NO catalyzed by iNOS led to inner ear dysfunction. Our results indicate that apoptosis is triggered by iNOS and that it mediates the ototoxicity induced by cisplatin.

Localization of nitric oxide synthase isoforms in the human cochlea.

The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.

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.

[Effect of injectio Salvia Miltiorrhiza on gentamicin ototoxicity-induced activity of nitric oxide synthase in cochlear stria vascularis of guinea pig].

AIM: to investigate the change of nitric oxide synthase (NOS) activity in cochlear stria vascularis (SV) of guinea pig after gentamicin (GM) and Salvia Miltiorrhiza (SM) injection, and to explore the protective role of injectio SM on GM ototoxicity. METHODS: NADPH-diaphorase histochemistry staining and image quantitative analysis technique, combined with auditory brainstem response (ABR) measurement. RESULTS: SM + GM significantly reduced NOS activity in cochlear SV and ABR threshold as compared with CM along (P < 0.01); and ABR threshold shift was in high correlation with NOS activity (rControl = -0.9464; rGM = -0.9117; rSM + GM = -0.8958; P < 0.01). CONCLUSION: SM can reduce NOS activity in cochlear SV so as to alleviate GM ototoxicity, thus ameliorate hearing function.

Effect of a beta-stimulant on the inner ear stria vascularis.

In vivo and in vitro experiments were conducted to investigate the effect of alpha-isoproterenol on the inner ear stria vascularis with intracellular cytochrome oxidase activity used as an index. Intraperitoneal injection of alpha-isoproterenol (5 mg/kg) was performed in 10 rats, and that of physiological saline in 4 rats, for 21 consecutive days. After the 3-week treatment, bilateral cochleas were excised for frozen sections and stained for cytochrome oxidase. The staining density of the stria vascularis for the enzyme was analyzed with a computer. Electron microscopic observation was also performed for some specimens. As for the in vitro experiments, bilateral cochleas from 6 normal rats were excised for cell culture. Cochlear cells from the right ear were cultured with medium containing alpha-isoproterenol (10-micromol/L concentration), and those from the left ear with medium alone. After 3-day culture, the enzyme activity of cytochrome oxidase in the stria vascularis was quantified by the same method used for the in vivo experiments. Cytochrome oxidase activity was markedly elevated in the alpha-isoproterenol group. The activity tended to be higher in the lower turns of the cochlea. Electron microscopy revealed that numerous mitochondria were present in marginal cells that protruded into the endolymphatic space. The enzyme activity was also elevated in the stria vascularis from cochlear specimens in the alpha-isoproterenol group of the in vitro experiment. The above results suggest that alpha-isoproterenol accelerated the metabolic activity of the cells that constitute the stria vascularis. The increase in activity was probably attributable to direct pharmaceutical effects of the beta-stimulant, rather than an increase in blood flow. It is possible that the cells that constitute the stria vascularis may have beta-receptors.

Lipopolysaccharide-induced expression of inducible nitric oxide synthase in the guinea pig organ of Corti.

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the cochlea of the guinea pig could elicit formation of inducible nitric oxide synthase (iNOS). Immunohistochemical study revealed that immunoreactivity to iNOS was seen below outer hair cells representing nerve fibers and synaptic nerve endings. iNOS-staining could also be observed in phalangeal dendrites of Deiter's cells pointing to the cuticular membrane, Hensen's cells and on stria vascularis 48 h after inoculation with LPS. Immunohistochemical investigation with a specific anti-nitrotyrosine antibody also revealed intense immunoreactivity identical to that of iNOS, suggesting formation of peroxynitrite in the organ of Corti by the reaction of NO with O(2)(-). On the basis of these findings, it can be concluded that NO together with O(2)(-), which form the more reactive peroxynitrite, are the most important pathogenic agents in LPS-induced damage of cochlea in the guinea pig.

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.