Cellular localization of adenylate cyclase in the developing and mature inner ear of the mouse

The localization of adenylate cyclase in the developing and mature inner ear of the CBA/CBA mouse, in Shaker-1 and Shaker-2 mutants and in organ culture was determined by a histochemical procedure with adenylyl imidodiphosphate as substrate and Sr²⁺ as capture ion. Enzymatic activity was associated with cell membranes in stria vascularis and Reissner's membrane but could not be demonstrated in other cochlear structures. In stria vascularis, adenylate cyclase was found on the contraluminal infoldings of marginal cells but not on their luminal surface, while Reissner's membrane showed activity on its endolymphatic surface. Activity was also associated with utricular dark cells. Essentially the same localization but differing in quantitative aspects was observed during development, in organ culture and in the mutant strains. The speculative role of adenylate cyclase in cochlear ion and fluid balance is discussed.     

Expression pattern of adenylyl cyclase isoforms in the inner ear of the rat by RT-PCR and immunochemical localization of calcineurin in the organ of Corti.

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.e. in the stria vascularis and endolymphatic sac. In the organ of Corti and vestibulum the Ca2+-modulated isoforms types 1, 6 and 9 were expressed. The regulation of adenylyl cyclase 9, which is the major isoform expressed in the organ of Corti, proceeds via the Ca2+-activated protein phosphatase 2B (calcineurin, PPP3). PCR with specific primers for calcineurin demonstrated its abundant expression in the organ of Corti. Using a monoclonal antibody we localized calcineurin immunochemically to the cochlear nerve, the nerve fibers and the inner hair cells. In the cochlear and vestibular nerves a characteristic neuronal expression pattern of adenylyl cyclase isoforms was observed, i.e. adenylyl cyclases types 2, 3 and 8. The functional consequences of the adenylyl cyclase expression pattern in the inner ear are discussed in conjunction with its unique sensory performance.     

Binding sites of atrial natriuretic peptide (ANP) in the mammalian cochlea and stimulation of cyclic GMP synthesis.

The distribution of binding sites for atrial natriuretic peptide (ANP) has been examined in frozen sections of the guinea pig inner ear by means of autoradiography. The highest density was found in the stria vascularis of all cochlear turns. In membrane preparations of stria vascularis in vitro, the production of the second messenger cGMP was strongly stimulated by synthetic ANP in a dose dependent manner. Adenylate cyclase was neither stimulated nor inhibited by ANP, thus suggesting, that the binding sites coincide with an ANP receptor, which is coupled to guanylate cyclase but not negatively coupled to an adenylate cyclase molecule. The production of cyclic GMP could not be reduced by GDP-beta S, a strong inhibitor of the Gs protein. We conclude the existence of an ANP receptor-guanylate cyclase signal transfer system, similar to the beta 2 receptor-adenylate cyclase system in the inner ear, without coupling to a G protein. ANP might play a role in sodium and water regulation of the endolymph and might antagonize the action of vasopressin.     

Adenylate cyclase and G-proteins as a signal transfer system in the guinea pig inner ear

In many eukaryotic cells G-proteins play a key role in signal transduction through outer cell membranes. To study this pathway in the auditory organ of mammals we examined tissue preparations from the stria vascularis and the organ of Corti from the guinea pig inner ear. The activity of adenylate cyclase was measured by stimulation at the site of the enzyme, the hormone receptors and the modulating G-proteins. In the organ of Corti we found a low enzyme activity in all cochlear turns. The stria vascularis, however, showed a constant high concentration of beta 2-adrenergic receptors and of stimulating G-proteins in all cochlear turns. In contrast, the activity of the enzyme increased from the apical to the basal turn. Adenylate cyclase could be stimulated or inhibited in a concentration-dependent manner by drugs selectively effecting the G-proteins. Our results suggest a structure of the adenylate cyclase complex in the inner ear similar to other organs. Pathophysiological correlations to hearing loss associated with pseudohypoparathyroidism are discussed.     

Arguments against a mediating role of the adenylate cyclase--cyclic AMP system in the ototoxic action of loop diuretics

Previous studies in our laboratory have indicated that adenylate cyclase of the stria vascularis is strongly inhibited in vitro by ethacrynic acid and furosemide. In order to test whether the in vitro effects upon the enzyme are also present under in vivo conditions, ethacrynic acid was perfused perilymphatically for 15 min and 20 min at a concentration of 10(-3) M. Cyclic AMP of the stria vascularis was reduced by 27% and 34%, respectively, but ATP also declined significantly, suggesting unspecific effects. When ethacrynic acid was applied intravenously at a dosage of 50 mg/kg, and the endolymphatic potential allowed to decline to -10mV, no significant changes in cyclic AMP and ATP were seen. The absence of effects upon cyclic AMP in the early stage of systemic intoxication with ethacrynic acid is strong evidence against a mediating role of adenylate cyclase in the ototoxic action of ethacrynic acid. When a bolus of 3 x 10(-2) M furosemide was applied intra-arterially the endolymphatic potential declined at the exceedingly rapid rate of about 10 mV/sec, strongly suggesting that the action of the drug takes place in the vicinity of the capillaries of the stria vascularis. In view of the proposition that adenylate cyclase appears to be located primarily at eh luminal aspect of the stria vascularis, this constitutes further evidence against a role of the enzyme in the mediation of the specific ototoxic effects of loop diuretics. Other recent evidence against a mediatory role of the adenylate cyclase--cyclic AMP system is discussed.     

The influence of ischemia upon the energy reserves of inner ear tissues

Ischemic changes in the levels of glucose, glycogen, ATP and P-creatine are determined under “closed system” conditions in the organ of Corti, stria vascularis, ganglion spirale, cochlear nerve and vestibular sensory epithelia. From the resting levels of these compounds the total energy reserve in terms of equivalents of high energy phosphate, both preformed and potentially available from anaerobic glycolysis, is computed. The energy reserves are highest in the organ of Corti, intermediate in stria vascularis, spiral ganglion and vestibular structures, and lowest in the cochlear nerve. The rate of depletion of these energy reserves in ischemia is used as an indicator of the energy requirements of the respective tissues. The metabolic rate is by far the highest in the stria vascularis, intermediate in ganglion spirale and cochlear nerve and lowest in the organ of Corti and vestibular structures. There is no correlation between the total energy reserve and the initial energy use rates. The obtained data are compared with the dynamic patterns of the corresponding biopotentials and with pertinent results of enzymatic and respirometric studies; in addition, ischemic changes in glucose and lactate levels of perilymph are described.     

Localization of soluble guanylate cyclase activity in the guinea pig inner ear

The aim of this study was to characterize the nitric oxide (NO) receptor soluble guanylate cyclase (sGC), to determine the cells targeted by NO and to elucidate the function of the NO/cGMP pathway in the inner ear. sGC activity in the inner ear was localized by immunohistochemical detection of NO-stimulated cGMP. Soluble guanylate cyclase activity in the cochlea was detected in the nerve endings underneath the outer and inner hair cells, supporting cells, stria vascularis and vessels. In the vestibular organs, sGC activity was detected in the cytoplasm of sensory cells, nerve fibres, dark cells and transitional cells and vessels. These findings suggest that the NO/cGMP pathway may be involved in regulatory processes in neurotransmission, blood flow and inner ear fluid homeostasis.     

Localization of Soluble Guanylate Cyclase Activity in the Guinea Pig Inner Ear

The aim of this study was to characterize the nitric oxide (NO) receptor soluble guanylate cyclase (sGC), to determine the cells targeted by NO and to elucidate the function of the NO/cGMP pathway in the inner ear. sGC activity in the inner ear was localized by immunohistochemical detection of NO-stimulated cGMP. Soluble guanylate cyclase activity in the cochlea was detected in the nerve endings underneath the outer and inner hair cells, supporting cells, stria vascularis and vessels. In the vestibular organs, sGC activity was detected in the cytoplasm of sensory cells, nerve fibres, dark cells and transitional cells and vessels. These findings suggest that the NO/cGMP pathway may be involved in regulatory processes in neurotransmission, blood flow and inner ear fluid homeostasis.     

Influence of “loop” diuretics upon Na+K+-ATPase and adenylate cyclase of the stria vascularis

Summary We have previously found that the inhibition of strial Na⁺K⁺-ATPase by ethacrynic acid occurs at much higher concentrations (I₅₀=5×10^–3 M) than the inhibition of the endolymphatic potential (I₅₀=1×10^–5 M), which in itself is evidence against the concept that Na⁺K⁺-ATPase is a target of ethacrynic acid action in the stria vascularis. In order to check whether the stria vascularis accumulates the drug selectively (as is the case in tissue slices of kidney) we perfused the perilymphatic space with ¹⁴C-ethacrynic acid for 30 min. No accumulation of the drug above the concentration in the perfusion medium (5×10^–5 to 5×10^–3M) occurred in the stria vascularis. Since 30 min of perfusion produces maximal alterations of the endolymphatic potential at 1×10^–4 M and above, and near maximal alterations at 5×10^–5 M, Na⁺K⁺-ATPase can be excluded as a primary target of ethacrynic acid action upon the stria vascularis. No inhibition of Na⁺K⁺-ATPase by furosemide is detectable at 1×10^–2 M.Adenylate cyclase of the stria vascularis is strongly inhibited by both ethacrynic acid and furosemide. In the case of ethacrynic acid the I₅₀ (1×10^–5 M) coincides with that of the endolymphatic potential. The I₅₀ of adenylate cyclase with respect to furosemide is equally low (9×10^–6 M), but the I₅₀ of the endolymphatic potential is considerably higher (2×10^–4 M). These results are consistent with the concept that interference with adenylate cyclase is a primary mode of action of ethacrynic acid and furosemide upon the stria vascularis. However, these data in themselves do not prove that this concept is valid under in vivo conditions. Both ethacrynic acid and furosemide are strong inhibitors of adenylate cyclase in the organ of Corti (I₅₀= 8×10^–6 and 3×10^–6 M, respectively).Basal activities of adenylate cyclase in spiral ganglion, Reissner's membrane and macula sacculi are reported, as well as revised results for organ of Corti and stria vascularis.A brief version of this paper was presented at the 90th Meeting of the Acoustical Society of America, November 1975 (Paloheimo and Thalmann, 1976).Supported by NIH grant R01 NS 06575We thank Dr. J. E. Baer from the Merck, Sharp and Dohme, Research Laboratories (Division of Merck and Co., Inc., West Point, Pennsylvania 19486) for his generous gift of ¹⁴C-ethacrynic acid.     

川芎嗪对庆大霉素耳中毒耳蜗外毛细胞和血管纹的保护作用

目的：探讨在庆大霉素（gentamycin，GM）耳中毒情况下，川芎嗪（tetramethylpyrazine，TMP）对豚鼠耳蜗外毛细胞和血管纹边缘细胞的保护作用。方法：12只豚鼠随机分为GM组、联合用药组、TMP组及对照组，用药十天后处死，采用透射电镜观察耳蜗外毛细胞及血管纹边缘细胞超微结构，扫描电镜观察血管纹边缘细胞表面形态。结果：透射和扫描电镜显示，联合用药组外毛细胞及血管纹边缘细胞超微及表面结构破坏不均明显轻于庆大霉素组，特别是其中的线粒体结构破坏与数目减少更显著轻于庆大霉素组。结论：川芎嗪具有保护庆大霉素耳中毒耳蜗外毛细胞和血管纹结构的作用，从而拮抗庆大霉素耳毒性。     

Distribution of beta-tubulin in guinea pig inner ear

Our previous research had suggested that beta-tubulin might be an autoantigen for autoimmune inner ear disease. In this study, the expression of beta-tubulin in inner ears of normal and tubulin-immunized guinea pigs was examined by immunohistochemical staining. Strong immunoreactivity to beta-tubulin monoclonal antibody was found in stria vascularis, neurons of the spiral ganglion, cochlear nerve fibers and spiral ligament. Diffuse staining was found in the stria vascularis and the neurons of the spiral ganglion, while dense network staining was found in the spiral ligament, the nerve fibers and the vestibular end organs. The semicircular canals, endolymphatic duct and sac were also positively stained. In inner ears of guinea pigs challenged with beta-tubulin, staining intensity was diminished in the stria vascularis, the spiral ligament, and the neurons of the spiral ganglion. The results suggest that beta-tubulin is distributed to most structures of guinea pig inner ear. A challenge to the inner ear by tubulin could change the beta-tubulin distribution and cause degeneration in the spiral ganglion. The results support the hypothesis that beta-tubulin might be an autoantigen for autoimmune inner ear disease.     

Viral labyrinthitis: early pathology in the human

The histologic findings in the temporal bones of three patients who died from viral encephalopathy are presented. Pathology was restricted to the scala media, vestibular labyrinth, and internal auditory canal and was considered to be expressions of viral labyrinthitis. The changes were different degrees of degeneration of the organ of Corti, early encapsulation of the tectorial membrane, degeneration of the stria vascularis, and round cell infiltration of the modiolus and contents of the internal auditory canal. A new finding in the organ of Corti and early stages of cystic degeneration of the stria vascularis are documented. In all cases, the saccule was degenerated with sloughing of the otolithic membrane and vestibular labyrinth was involved in varying degrees.     

胎儿耳蜗血管纹的扫描电镜观察

目的：借助扫描电镜技术观察血管纹的表面结构,以便对血管纹的结构和功能提供新的信息。方法：标本取自尸检3个足月胎儿的6个颢骨,在死后尽可能快速取出耳蜗,经处理后,借助扫描电镜技术观察人胎儿耳蜗血管纹的超微结构。结果：借助扫描电镜技术可从耳蜗基底圈到顶圈,上起前庭膜嵴,下到基底膜的基底嵴全面观察血管纹的各个部分,血管纹边缘细胞表面结构呈圆球形．细胞表面有许多微绒毛。螺旋凸部位有一个细胞移行区,这个区域的边缘细胞表面形态明显不同,细胞细K或呈不规则的多角形细胞,细胞边界微绒毛丰富,显出明显的细胞界限,细胞表面分布均匀的微绒毛。血管纹断面的观察还可获得中间细胞、基底细胞和毛细血管结构。结论：扫描电镜观察胎儿耳蜗血管纹,可获得整个血管纹全貌的表面精细结构,尤其是边缘细胞的表面形态,通过对血管纹断面的观察还可获得中间细胞、基底细胞的精细结构特征,为认识血管纹的结构和功能提供新的信息。     

Volumetric and dimensional analysis of the guinea pig inner ear

The objective of this study was to provide accurate volumetric data on the fluid spaces and soft tissue in the guinea pig inner ear by measuring all histologic serial sections by means of Metamorph Imaging Software at 400x to 1,000x magnification. The total endolymph volume of the inner ear was 4.691 mm3, of which 1.501 mm3 was in the cochlea, 3.090 mm3 in the vestibular labyrinth, and 0.100 mm3 in the endolymphatic duct and sac. The total perilymph volume was 15.938 mm3, of which 8.867 mm3 was in the cochlea and 7.071 mm3 in the vestibular labyrinth. The volume of the organ of Corti per millimeter length increased toward the apex, but the volumes of the stria vascularis, spiral ligament, and spiral limbus decreased. The volume of the macula utriculi was larger than that of the macula sacculi. The measurement of the luminal surface area of the stria vascularis was 3.944 mm2, and that of the vestibular dark cells was 5.772 mm2.     

Acoustic stimulation alters deoxyglucose uptake in the mouse cochlea and inferior colliculus

Deoxyglucose uptake and activities of hexokinase and glucose-6-phosphatase in auditory structures (organ of Corti, stria vascularis and spiral ligament, modiolar section of VIIIth nerve, inferior colliculus) and non-auditory tissues (heart, kidney, liver) of the mouse were analyzed. [³H]Deoxyglucose was given as a pulse into the tail vein and uptake was quantitated by microdissection of tissues and scintillation counting. Radioactivity in cochlear tissues was maximal after 45–60 min and declined with a half-life of 30–60 min. Deoxyglucose 6-phosphate represented ca. 60% of total radioactivity (heart, inferior colliculus. > 80%). The ratio of hexokinase to glucose-6-phosphatase activity was considerably lower in the auditory periphery than in brain. The rank order was inferior colliculus > VIIIth nerve ≈ heart > stria vascularis and spiral ligament > kidney > organ of Corti ≈ liver.Exposure to broadband noise increased glucose utilization in all auditory structures. Uptake was maximally (2- to 3-fold) stimulated at moderate noise intensity (55–85 dBA). In addition, the auditory system showed two salient features: at high intensities (100 and 115 dBA) deoxyglucose uptake decreased from the maximum; and the non-sensory tissues of the cochlea (stria vascularis and spiral ligament) responded to sound parallel to the sensory structures at all levels of stimulus intensity. There were no effects of acoustic stimulation on serum glucose levels, serum kinetics of deoxyglucose. or deoxyglucose uptake into other body tissues.     

Adenylate cyclase and G-proteins as a signal transfer system in the guinea pig inner ear

Summary In many eukaryotic cells G-proteins play a key role in signal transduction through outer cell membranes. To study this pathway in the auditory organ of mammals we examined tissue preparations from the stria vascularis and the organ of Corti from the guinea pig inner ear. The activity of adenylate cyclase was measured by stimulation at the site of the enzyme, the hormone receptors and the modulating G-proteins. In the organ of Corti we found a low enzyme activity in all cochlear turns. The stria vascularis, however, showed a constant high concentration of 2-adrenergic receptors and of stimulating G-proteins in all cochlear turns. In contrast, the activity of the enzyme increased from the apical to the basal turn. Adenylate cyclase could be stimulated or inhibited in a concentration-dependent manner by drugs selectively effecting the G-proteins. Our results suggest a structure of the adenylate cyclase complex in the inner ear similar to other organs. Pathophysiological correlations to hearing loss associated with pseudohypoparathyroidism are discussed.     

水通道蛋白-2在大鼠内耳的定位及其意义

目的：探讨水通道蛋白-2(Aqp-2)在大鼠内耳中的定位，分析其在内耳水代谢中的作用机制。方法：使用正常成年SD大鼠15只，制备颞骨组织标本，采用SP免疫组织化学技术，检测Aqp-2蛋白在大鼠内耳的表达情况。结果：Aqp-2在大鼠的内淋巴囊、血管纹、螺旋神经节有较强表达，在Corti器、基底膜、螺旋缘的前庭唇和鼓唇、盖膜、螺旋凸也有表达。结论：Aqp-2主要分布在与内淋巴代谢有关的结构(内淋巴囊和血管纹)，它在Corti器的表达为膜迷路积水时伴有的听力下降提供了又一解释；Aqp-2在螺旋神经节的表达，提示它可能与正常听觉的维持有关。     

Inhibition of adenylate-cyclase-coupled G protein complex by ototoxic diuretics andcis-platinum in the inner ear of the guinea pig

Summary The enzyme adenylate cyclase produces the second messenger cAMP and is located in the mammalian inner ear, predominantly in the stria vascularis and to a lesser extent in the organ of Corti. It is coupled to hormone receptors and regulating G proteins in the outer cell membrane. By means of immunofluorescence in cryostat sections of the guinea pig cochlea, we could demonstrate the G proteins GS and Gi, which belong to the adenylate cyclase complex. These proteins had their highest density in the stria vascularis. In membrane preparations of this tissue, the adenylate cyclase complex was inhibited by ototoxic drugs (furosemide, ethacrynic acid and cis-platinum). Stimulation at different sites of the enzyme system showed that the target of these drugs was probably the regulating G protein complex and not the enzyme molecule itself. Inhibition depended on the concentration of the drug and the incubation time.Extracts reported at the 61st meeting of the German Society of Otorhinolaryngology, Würzburg, FRG, 27–31 May 1990     

Otopathology in a case of type I Waardenburg's syndrome

We report a case of type I Waardenburg's syndrome that provides insight into the etiopathogenesis of sensorineural hearing loss (SNHL) in this syndrome. The subject, a 76-year-old woman with type I Waardenburg's syndrome (dystopia canthorum, heterochromia irides, and white hair), had congenital low-frequency SNHL in her right ear only, which had remained relatively stable throughout her life. Blood leukocyte DNA studies revealed a PAX-3 mutation with a 1 base pair C-to-A substitution in exon 5 at base 602. Light microscopic studies of the right cochlea showed intact neurosensory structures in only the lower basal turn, with the remainder of the cochlea showing absence of melanocytes, absence of stria vascularis, missing hair cells, dysmorphogenesis of the tectorial membrane, and lack of peripheral processes of the spiral ganglion cells. There was pathological alteration of the vestibular dark cells with marked reduction of melanocytes associated with these dark cells. The left inner ear was normal, with a full complement of neurosensory structures, including melanocytes. Because the PAX-3 gene is involved in neural crest development and melanocytes migrate from the neural crest to the ear, the findings in this case are consistent with the hypothesis that defective melanocyte migration or defective melanocyte function results in defective development of the stria vascularis (and perhaps other structures of the ear), leading to SNHL.     

Adenylate cyclase activity in the fetal and the early postnatal inner ear of the mouse

The adenylate cyclase activity was analyzed in fetal, early postnatal and adult inner ears of the CBA/CBA mouse and also in approximately one month old inner ears from Shaker ?1 and Shaker ?2 mice. A comparison was made with the maturation of potassium levels in endolymph as investigated with the X-ray energy dispersive technique.Adenylate cyclase activity in the developing normal inner ear shows two significant periods of increases: from the 16th to the 19th gestational day in both the cochlear and vestibular parts of the labyrinth, and from birth to day 6 after birth in the lateral wall tissues of the scala media. During the first period the anatomical boundaries of the secretory epithelia are developing. The postnatal rise in adenylate cyclase activity correlates with the morphological maturation of stria vascularis at the cellular and subcellular levels and the rise in potassium content of endolymph. The rise of enzyme activity in the cochlea during the maturation of endolymph supports a link between adenylate cyclase and the control of inner ear fluids. Adenylate cyclase activity in stria vascularis/spiral ligament of Shaker ?1 and Shaker ?2 mice were at normal levels and correlated better with the rather normal morphology of the tissues than the abnormal composition of endolymph in these mutants.