豚鼠内淋巴囊碳酸酐酶组化分布测定和超微定位

用组织化学、细胞化学结合图象分析，观察了豚鼠内淋巴囊（ＥＳ）碳酸酐酶（ＣＡＨ）的分布和超微定位。结果发现：在ＥＳ的近侧部仅少数上皮细胞有阳性反应，中间部和远侧部的大多数上皮细胞着色；中间部和远侧部ＣＡＨ阳性产物灰度值明显高于近侧部（Ｐ＜０．０１）。在ＥＳ上皮细胞的阳性产物主要位于内淋巴腔面细胞膜、微绒毛、细胞浆、侧膜皱褶和基侧膜皱褶处；表明ＣＡＨ在调节内淋巴的容量、离子转运及内环境稳定中起重要作用。     

348 Ca^2＋—ATP酶对耳蜗Ca^2＋平衡的调节作用

钙离子（C ^2＋）做为第二信使几乎参与了细胞所有的生理活动。在耳蜗水平,钙与机械－电转换、内耳声感受的频率选择性、基底膜振动非对称性等有密切关系。毛细胞及内淋巴液的Ca^2＋浓度变化与感音神经性耳聋有着密切的联系,而在Ca^2 -ATP酶（又称钙泵）在耳蜗Ca^2 稳态的调控中起了重要作用。Ca^2 －ATP酶的作用及其机理已成为感音神经性耳聋发病机制和防治研究的关键之一。     

正常豚鼠内耳前庭器碳酸酐酶活性观察

为了解前庭器中酶的分布，通过组织化学、细胞化学方法，观察了豚鼠内耳前庭器碳酸酐酶（ＣＡ）的分布和超微定位。结果发现：毛细胞的静纤毛和皮板呈阳性反应，而毛细胞体为阴性；在支持细胞、暗细胞、移行细胞及壁细胞的细胞膜、微绒毛、侧皱褶和基侧皱褶处均见有ＣＡ活性反应产物。认为分布于前庭器的ＣＡ属膜结合ＣＡ，且在维持前庭内环境稳定和耳石代谢中起重要作用。     

正常豚鼠内耳前庭器碳酸酐酶活性观察

为了解前庭器中酶的分布，通过组织化学、细胞化学方法，观察了豚鼠内耳前庭器碳酸酐酶（ＣＡ）的分布和超微定位。结果发现：毛细胞的静纤毛和皮板呈阳性反应，而毛细胞体为阴性；在支持细胞、暗细胞、移行细胞及壁细胞的细胞膜、微绒毛、侧皱褶和基侧皱褶处均见有ＣＡ活性反应产物。认为分布于前庭器的ＣＡ属膜结合ＣＡ，且在维持前庭内环境稳定和耳石代谢中起重要作用。     

豚鼠膜迷路积水模型耳蜗Ca2+-ATP酶的变化

目的了解Ｃａ２＋ＡＴＰ酶在耳蜗活动位点及膜迷路积水后耳蜗Ｃａ２＋ＡＴＰ酶的变化。方法选成年健康、Ｐｒｅｙｅｒ反射正常豚鼠１４只,左耳装圆窗电极后阻塞内淋巴,经声反应阈（ＣＡＰ阈值）证明膜迷路积水形成;右侧为对照耳。用枸橼酸铅细胞组化法测定Ｃａ２＋ＡＴＰ酶,在透射电镜下Ｃａ２＋ＡＴＰ酶以磷酸铅黑色颗粒显示。结果对照耳Ｃａ２＋ＡＴＰ酶活动部位在蜗管前庭膜内淋巴侧、内外毛细胞皮板及静纤毛、血管纹中间细胞指突膜等处;实验性膜迷路积水后,声反应阈提高,前庭膜、内外毛细胞等处之Ｃａ２＋ＡＴＰ酶颗粒明显减少。结论膜迷路积水后声反应阈提高,前庭膜、内外毛细胞等处Ｃａ２＋ＡＴＰ酶活性明显下降,两者呈负相关。     

豚鼠膜迷路积水模型耳蜗Ca^2＋ATP酶的变化

目的 了解Ｃａ＾２＋－ＡＴＰ酶在耳蜗活动位点及膜迷路积水后耳蜗Ｃａ＾２＋－ＡＴＰ酶的变化。方法 选成年健康、Ｐｒｅｙｅｒ反射正常豚鼠１４只，左耳装圆窗电极一阻塞肉淋巴，经声反应阈（ＣＡＰ阀值）证明膜迷路积水形成；右侧为对照耳。用枸橼酸铅细胞组化法测定Ｃａ＾２＋－ＡＴＰ酶，在透射电镜下Ｃａ＾２＋－ＡＴＰ酶以磷酸铅黑色颗粒显示。结果 对照耳Ｃａ＾２＋－ＡＴＰ酶活动部位在蜗管前庭膜内淋巴侧、内外毛细胞皮     

碳酸酐酶在豚鼠耳蜗的分布及其意义

本文应用组织化学和超微细胞化学方法显示碳酸酐酶（ＣＡＨ）在豚鼠血管纹（ＳＶ）和Ｃｏｒｔｉ器中的分布。光镜观察到ＳＶ和Ｃｏｒｔｉ器均含有丰富的ＣＡＨ，电镜观察到ＳＶ的边缘细胞和外毛细胞（ＯＨＣ）呈强阳性反应，ＳＶ的中间细胞和基底细胞、内毛细胞（ＩＨＣ）见到少量反应产物。结果表明耳蜗ＣＡＨ的分布与工春功能有密切关系。     

碳酸酐酶在豚鼠耳蜗的分布及其意义

本文应用组织化学和超微细胞化学方法显示碳酸酐酶（ＣＡＨ）在豚鼠血管纹（ＳＶ）和Ｃｏｒｔｉ器中的分布。光镜观察到ＳＶ和Ｃｏｒｔｉ器均含有丰富的ＣＡＨ，电镜观察到ＳＶ的边缘细胞和外毛细胞（ＯＨＣ）呈强阳性反应，ＳＶ的中间细胞和基底细胞、内毛细胞（ＩＨＣ）见到少量反应产物。结果表明耳蜗ＣＡＨ的分布与其功能有密切关系。     

庆大霉素及其代谢产物对外毛细胞内游离钙浓度的影响

目的：探讨庆大霉素（GM）耳毒性与耳蜗外毛细胞（OHC）内游离钙（[Ca^2 ])之间的关系。方法：离体耳蜗OHC经钙荧光指示剂Fluo-3负载后，用激光扫描共聚焦显微镜测定高钾溶液、GM、GM代谢产物等对OHC[Ca^2 ])i的影响。结果GM使[Ca^2 ]i短暂下降，并阻滞高钾溶液引起的钙内流，GM代谢产物则使[Ca^2 ]i持续升高，对高钾引起的钙内流无明显影响，结论GM耳毒与外毛细胞[Ca^2 ]i持续升高有关。     

Na^＋—D^＋—ATP酶活性在豚鼠内淋巴囊的细胞化学定位

Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶在Ｎａ＾＋－Ｋ＾＋离子主动性跨上皮转运过程中起重要作用。应用对硝基苯酚磷酸（ｐ－ＮＰＰ）为底物的柠檬酸铅一步法Ｋ＾＋－ＮＰＰ酶电细胞化学技术，观察了反映Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶活性的Ｋ＾＋－ＮＰＰ酶在豚鼠内淋巴囊的超微结构定位分布。结果：在透射电镜下观察，内淋巴囊超薄切片上的Ｄ＾＋－ＮＰＰ酶活性反应产物仅分布于上皮细胞底侧面胞膜的胞浆一侧，向上一直延续至上皮细胞间的紧密     

Changes of Ca(2+)-ATPase in the cochlea of guinea pig during labyrinthine hydrop]

OBJECTIVE: To investigate the localization of Ca(2+)-ATPase (Ca2+ pump) in the cochlea and its change in labyrinthine hydrops. METHODS: The left endolymphatic sac was ablated to induce endolymphatic hydrops in fourteen healthy guinea pigs after the sliver ball electrode was placed on the round window. The Ca(2+)-ATPase was studied by the lead citrate reaction in the control and hydropic ears. The reaction product was lead phosphate particles as an expression of Ca(2+)-ATPase activity under the electron-microscope. RESULTS: The Ca(2+)-ATPase activity was found mainly on the endolymphatic surface of the Reissner's membrane, the stereocilia and cuticular plate of inner and outer hair cells, as well as along the infolding plasma membrane of the strial intermediate cells. The Ca(2+)-ATPase activity was significantly decreased during endolymphatic hydrops in the above-mentioned locations. CONCLUSION: The response thresholds of filtered click were increased and the Ca(2+)-ATPase significantly decreased in the cochlea during labyrinthine hydrops. These results suggest that correlation exists between the CAP threshold and the activity of Ca(2+)-ATPase in the model of labyrinthine hydrops.     

Histochemical localization of calcium ATPase in the cochlea of the guinea pig

Summary The activity of Ca²⁺-ATPase in the inner ear of the guinea pig was studied ultracytochemically by the lead citrate reaction. The electron-dense reaction products as an expression of Ca²⁺-ATPase activity were localized in endolymphatic cells of Reissner's membrane, in outer and inner hair cells and in some supporting cells. The main finding was the difference in the localization of Ca²⁺-ATPase in outer and inner hair cells. In the latter cells the activity sites were mainly intracellular and in apical membrane specializations, whereas in the outer hair cells the enzyme was localized in the apical membrane specializations and the basolateral plasma membrane.     

Cytochemical localization of Na-K ATPase in the guinea pig endolymphatic sac

The light microscopical and ultracytochemical localization of Na-K ATPase (ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase) in the guinea pig endolymphatic sac was studied by a newly developed lead citrate one step method. The cytochemical reaction product was detected in the epithelial cell layer of the endolymphatic sac at the light microscopical level. At the electron microscopical level, the reaction product was located to the basolateral plasma membrane of both the dark and the light cells. The results suggest that Na-K ATPase may play a role in the ion transport mechanism in the endolymphatic sac.     

Presence of plasma membrane-bound Ca(2+)-ATPase in the secretory epithelia of the inner ear.

The Ca2+ concentration of the endolymph is low, around 0.023 mM. Yet, because of the positive endocohlear potential, Ca2+ must be actively transported into the endolymphatic space. The mechanisms responsible for the active Ca2+ transport into the endolymph are not known. In this study, the presence of plasma membrane-bound Ca(2+)-ATPase (PMCA ATPase) in the endolymph-producing, secretory epithelia of the inner ear from guinea pig was investigated with immunoblotting and immunohistochemistry. The antibody used was a monoclonal antibody which recognizes an epitope shared by all four known isoforms of PMCA ATPase. With immunoblotting, a band corresponding to PMCA ATPase was found in the stria vascularis, the ampullary tissue, the utricle and the endolymphatic sac in assays from at least three different batches of tissue. With immunohistochemistry, a strong positive staining reaction for PMCA ATPase could be seen in the stria vascularis and the dark cells of the ampullary tissue and the utricle. The epithelial cells in the endolymphatic sac showed a moderate positive staining reaction. Accordingly, in this study the presence of PMCA ATPase was shown in all the endolymph-producing, secretory epithelia of the inner ear. These results indicate that PMCA ATPase plays a role in the regulation of the Ca2+ concentration in the endolymph.     

Cytochemical localization of Ca++-ATPase activity in the lateral cochlear wall of the guinea pig

Ca++-ATPase activity was examined cytochemically in the lateral cochlear wall of the guinea pig. The reaction products showing Ca++-ATPase activity were found along the folded plasma membrane of the strial marginal cells. In contrast, little or no reaction was seen on the apical surfaces of these cells. There were also marked reaction products on the microvilli and the endolymphatic cell surface of Reissner's membrane, and the apical and lateral plasma membranes of the spiral prominence and the external sulcus cells. These reactions completely disappeared when Ca++ or ATP was removed from the incubation medium. Our results strongly suggest that Ca++-ATPase plays an important role in Ca++ transport system for the regulation of Ca++ concentration in the cochlear endolymph.     

Cytochemical studies of Ca++-ATPase activity in the vestibular epithelia of the guinea pig

We used ultracytochemistry to examine Ca++-ATPase activity in the vestibular epithelia of the guinea pig. Many reaction products were found along the basolateral plasma membrane of the vestibular dark cell. There were also marked reaction deposits on the apical and lateral cell membranes of the transitional cells, and the utricular and saccular wall cells. Both sensory and supporting cells showed Ca++-ATPase activity along their ciliary membrane and apical-lateral cell surfaces. Our findings indicate that the Ca++-ATPase activity found on the plasma membrane is closely related to Ca++-transport across the plasma membrane. When either Ca++ or ATP was omitted from the incubation medium, enzyme activity (as seen by the staining reaction present) was completely abolished. Our present results suggest that Ca++-ATPase located in the vestibular epithelia plays a significant role in the regulation of the Ca++-concentration in the vestibular endolymph.     

Na+,K+-ATPase and Ca2+-ATPase activities in the cochlear lateral wall following surgical induction of hydrops

Na+,K+-ATPase and Ca2+-ATPase activities have not been studied quantitatively in the cochlea affected by endolymphatic hydrops. The present study was designed to measure quantitatively the Na+,K+-ATPase and Ca2+-ATPase activities in the cochlear lateral wall and the threshold of auditory brainstem response (ABR) for guinea pigs in the early stages (=2 months) of experimentally induced endolymphatic hydrops. A significant negative association was demonstrated between Ca2+-ATPase activity and the change in ABR threshold for hydropic cochleae (P=0.014), but not for control cochleae (P=0.123), although no such significant association was revealed between Na+,K+-ATPase activity and any change in ABR threshold for both hydropic cochleae (P=0.751) and control cochleae (P=0.352). A significant increase in Ca2+-ATPase activity in the cochlear lateral wall was observed for the hydropic ear, in which normal ABR thresholds were maintained, as compared to the control ear. On the contrary, a mild decrease in Ca2+-ATPase activity in the cochlear lateral wall was observed for the hydropic ear, in which ABR thresholds increased significantly. The present findings suggest that alterations of Ca2+-ATPase activity in the cochlear lateral wall may implicate disturbed calcium-homeostasis in the inner ear, resulting in hearing dysfunction in the early stages of experimentally induced endolymphatic hydrops.     

Na+,K+-ATPase activity and ultrastructural localization in the tegmentum vasculosum in the cochlea of the duckling

The tegmentum vasculosum of the avian cochlear duct mimics the stria vascularis of the mammalian cochlear duct in both location and structure, and previous studies indicate that it may be its functional counterpart with regard to endolymph synthesis. In the present study, we report on the enzymatic activity and ultrastructural localization of the Na+,K+-ATPase in the tegmentum vasculosum of the duckling. Na+,K+-ATPase activity was determined by measuring K+-dependent, ouabain-sensitive p-nitrophenyl phosphatase (p-NPPase) activity in homogenates of dissected regions of the cochlear duct. The ultrastructural localization of the Na+,K+-ATPase was identified using K+-dependent, ouabain-sensitive, p-NPPase cytochemistry. Specific enzyme activity was localized primarily in homogenates of the tegmentum vasculosum (2.27 micromol p-nitrophenyl phosphate/mg protein/min) when compared to homogenates of the entire cochlear duct (0.69 micromol p-nitrophenyl phosphate/mg protein/min). Reaction product for p-NPPase was localized primarily along the basolateral plasma membrane folds of the dark cells. The cytochemical deposits appeared to be located exclusively on the cytoplasmic side of the plasma membrane. The light cells were devoid of reaction product. Biochemical and cytochemical localization of p-NPPase activity on the basolateral plasma membrane folds of the dark cells of the tegmentum vasculosum in conjunction with the ultrastructural morphology of these cells is compatible with a Na+,K+-ATPase-dependent ion transport function related to endolymph synthesis.     

Cytochemical localization of Ca++-ATPase activity in the lateral cochlear wall of the guinea pig

Summary Ca⁺⁺-ATPase activity was examined cytochemically in the lateral cochlear wall of the guinea pig. The reaction products showing Ca⁺⁺-ATPase activity were found along the folded plasma membrane of the strial marginal cells. In contrast, little or no reaction was seen on the apical surfaces of these cells. There were also marked reaction products on the microvilli and the endolymphatic cell surface of Reissner's membrane, and the apical and lateral plasma membranes of the spiral prominence and the external sulcus cells. These reactions completely disappeared when Ca⁺⁺ or ATP was removed from the incubation medium. Our results strongly suggest that Ca⁺⁺-ATPase plays an important role in Ca⁺⁺ transport system for the regulation of Ca⁺⁺ concentration in the cochlear endolymph.