Endocytosis of microperoxidase in marginal cells is mainly regulated by RhoA signaling cascade, but not by Rho-associated protein kinase, myosin light-chain kinase and myosin phosphatase.

Endocytosis plays an important role in cell function and the activation and propagation of signaling pathways. Signaling occurs on endocytic pathways and signaling endosomes, and endocytosis is subjected to high-order regulation by cellular signaling mechanisms. Marginal cells showed active endocytosis of microperoxidase (MPO) via the clathrin-independent pathway. We examined the signaling pathway that regulates MPO endocytosis in marginal cells using specific inhibitors and activators of signaling molecules. The results showed that pertussis toxin - which inhibits the ribosylation of G-protein-coupled receptor - did not affect MPO endocytosis, but Clostridium botulinum C3 toxin - which induces RhoA inactivation resulting in extracellular-signal-related kinase inactivation - inhibited MPO endocytosis. The main endocytotic pathway of MPO did not depend on the Rho-associated protein kinase molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade.

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.

Ca(2+)-ATPases in the cochlear duct.

Differing levels of the Ca(2+)-ATPase enzymes that reside on the plasma membrane (PM) and on the endoplasmic reticulum (ER) were identified in individual rat cochlear tissues by the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA). Unlike other studies, a specific antibody to PM Ca(2+)-ATPase was used to detect significantly greater levels (about 2x) of PM Ca(2+)-ATPase in the stria vascularis (SV) than that in the spiral ligament (SL) and organ of Corti (OC) tissues. Similarly, levels of ER Ca(2+)-ATPase were also significantly higher in the SV than in the SL and OC tissues. The presence of ER Ca(2+)-ATPase in the tissues of the SV has not been demonstrated previously. Given the importance of Ca2+ homeostasis in the inner ear, the statistically significantly higher densities of both PM and ER Ca(2+)-ATPase measured in the SV relative to the SL and OC regions would indicate tissue-specific responses to fluctuations in systemic and local Ca2+ concentrations.

Effects of low-sodium, high-potassium dietary intake on cochlear lateral wall Na+,K(+)-ATPase.

The effect of a low Na+, high K+ diet on Na+,K(+)-ATPase levels in cochlear lateral wall tissues was investigated in laboratory rats by using an enzyme-linked immunosorbent assay. The low Na+, high K+ diet induced high aldosterone plasma levels in the animals as well as changes in plasma cation levels. Animals that received a low Na+, high K+ diet demonstrated a statistically significant (97%) increase in Na+,K(+)-ATPase levels in the stria vascularis when compared to animals that received a control diet. This increase in strial Na+,K(+)-ATPase levels was blocked only 70% by administration of the aldosterone antagonist, spironolactone. Findings therefore indicate that strial Na+,K(+)-ATPase may be modulated by both aldosterone and Na+,K+ plasma levels. Na+,K(+)-ATPase levels in the spiral ligament were not affected by the experimental treatment. These findings suggest that spiral ligament Na+,K(+)-ATPase levels may be regulated by factors other than aldosterone and Na+,K+ plasma levels. This study provides further insight into the mechanisms of the beneficial effects of salt restriction and potassium loading in patients with Méniére's disease.

Effects of adenylate cyclase activation on electrical resistance of scala media.

Electrical resistance of scala media at the second turn of the guinea pig cochlea was measured while cochlear adenylate cyclase was activated by perilymphatic perfusion with 2 x 10(-4) M forskolin. The electrical resistance decreased from the pre-forskolin value (6.36 +/- 0.27 k omega to 5.22 +/- 0.33 k omega, n = 5), while the endocochlear potential (EP) increased from the pre-forskolin value (70.2 +/- 2.1 mV to 81.2 +/- 2.8 mV, n = 5). The time course of the electrical resistance change was more rapid than that of the EP change but almost identical to the time course of the change in the Cl- activity of the endolymph. The results suggest that the forskolin-induced decrease in the electrical resistance of the perilymph-endolymph barrier (presumably Reissner's membrane), and the consequent increase in Cl- flux into the endolymph may trigger EP elevation.

Quantitative immunogold localization of Na+,K(+)-ATPase alpha-subunit in the tympanic wall of rat cochlear duct.

Ultrastructural localization of Na+,K(+)-ATPase was quantitatively investigated in the tympanic wall of rat cochlear duct by use of the protein A-gold method, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. A moderate number of gold particles were found on the basolateral membrane of the interdental cells of the spiral limbus. A small number of gold particles were found on the basolateral surfaces of the border cells and Hensen's cells. On the inner and outer sensory hair cells, however, the plasma membranes were rarely labeled by gold particles. The general pattern of labeling densities in cochlear structures determined here and in a previous communication from our laboratory shows good correlation with the distribution of Na+,K(+)-ATPase activity as previously estimated biochemically, cytochemically, and autoradiographically.

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct.

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissner's membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.

Possible functional roles of Na+,K+-ATPase in the inner ear and their relevance to Ménière's disease.

This article reviews the functions of the enzyme Na+,K+-ATPase in epithelial tissues and discusses early and recent biochemical, physiologic and morphologic studies of the enzyme in the inner ear. The purpose of the investigation was to learn whether a relationship between perturbations in activity of the enzyme and Ménière's disease is possible. It is concluded that the preponderance of the evidence indicates that Na+,K+-ATPase plays a role in regulating ion transport into the scala media, but that the significance of the distribution of the enzyme along only one cell type (the marginal) in the functional chains of cells of the outer cochlear wall needs further study. The possible vasoconstrictive effects of ouabain perfusions employed by some investigators must also be taken into account. Recent cytochemical and autoradiographic studies have demonstrated high levels of Na+, K+-ATPase on cochlear nerve fibers, especially near the foramina nervosa and within the organ of Corti. Thus, perturbations in Na+,K+-ATPase activity in the inner ear not only could affect certain aspects of fluid balance, but also could account for the sensory disturbances experienced by patients who have Ménière's disease.

Cellular localization of Na+,K+-ATPase in the mammalian cochlear duct: significance for cochlear fluid balance.

Cytochemical and autoradiographic procedures were employed to determine the cellular distribution of Na+,K+-ATPase in the guinea pig cochlea. The highest activity was associated with the stria vascularis and was restricted almost entirely to the contraluminal extensions of the marginal cells. Elevated levels of activity were also observed in stromal cells of the spiral prominence, external sulcus, and spiral limbus. The pattern of activity in the latter tissues was unusual in being symmetrically distributed along the plasma membranes of the reactive cells. In the organ of Corti, only neural elements showed appreciable activity. Possible functions of the enzyme are discussed in relation to cochlear fluid balance.

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 cochlear 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.