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

1989 ◽  
Vol 37 (3) ◽  
pp. 353-363 ◽  
Author(s):  
T Iwano ◽  
A Yamamoto ◽  
K Omori ◽  
M Akayama ◽  
T Kumazawa ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Stria Vascularis ◽  
Basolateral Membrane ◽  
Lateral Wall ◽  
Alpha Subunit ◽  
Membrane Domain ◽  
Cochlear Duct ◽  
Gold Particles ◽  
Marginal Cells

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.

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

1990 ◽  
Vol 38 (2) ◽  
pp. 225-232 ◽  
Author(s):  
T Iwano ◽  
A Yamamoto ◽  
K Omori ◽  
K Kawasaki ◽  
T Kumazawa ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
General Pattern ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Alpha Subunit ◽  
Cochlear Duct ◽  
Gold Particles ◽  
Sensory Hair Cells

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.

Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 453-463 ◽  
Author(s):  
K.P. Steel ◽  
C. Barkway
Keyword(s):  
Neural Crest ◽  
Basal Cell ◽  
Stria Vascularis ◽  
Normal Adult ◽  
Basal Cells ◽  
Cochlear Duct ◽  
Cell Layers ◽  
Marginal Cells ◽  
Intermediate Cells ◽  
And Function

The stria vascularis of the mammalian cochlea is composed primarily of three types of cells. Marginal cells line the lumen of the cochlear duct and are of epithelial origin. Basal cells also form a continuous layer and they may be mesodermal or derived from the neural crest. Intermediate cells are melanocyte-like cells, presumably derived from the neural crest, and are scattered between the marginal and basal cell layers. The marginal cells form extensive interdigitations with the basal and intermediate cells in the normal adult stria. The stria also contains a rich supply of blood vessels. We investigated the role of melanocytes in the stria vascularis by studying its development in a mouse mutant, viable dominant spotting, which is known to have a primary neural crest defect leading to an absence of recognisable melanocytes in the skin. Melanocytes were not found in the stria of most of the mutants examined, and from about 6 days of age onwards a reduced amount of interdigitation amongst the cells of the stria was observed. These ultrastructural anomalies were associated with strial dysfunction. In the normal adult mammal, the stria produces an endocochlear potential (EP), a resting dc potential in the endolymph in the cochlear duct, which in mice is normally about +100 mV. In our control mice, EP rose to adult levels between 6 and 16 days after birth. In most of the mutants we studied, EP was close to zero at all ages from 6 to 20 days. Melanocyte-like cells appear to be vital for normal stria vascularis development and function. They may be necessary to facilitate the normal process of interdigitation between marginal and basal cell processes at a particular stage during development, and the lack of adequate interdigitation in the mutants may be the cause of their strial dysfunction. Alternatively, melanocytes may have some direct, essential role in the production of an EP by the stria. Melanocytes may be important both for normal strial development and for the production of the EP. We believe this is the clearest demonstration yet of a role for migratory melanocytes other than their role in pigmentation.

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

2006 ◽  
Vol 291 (5) ◽  
pp. C1038-C1048 ◽  
Author(s):  
Toshiaki Shibata ◽  
Hiroshi Hibino ◽  
Katsumi Doi ◽  
Toshihiro Suzuki ◽  
Yasuo Hisa ◽  
...  
Keyword(s):  
Stria Vascularis ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Lateral Wall ◽  
Endocochlear Potential ◽  
Pcr Analysis ◽  
Spiral Ligament ◽  
Gastric Type ◽  
Marginal Cells ◽  
Strong Immunoreactivity

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 α1-, but not colonic α2-, and β-subunits of H+,K+-ATPase. Immunolabeling of an antibody specific to the α1 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.

scholarly journals Localization of Carbonic Anhydrase XII to the Basolateral Membrane of H+-secreting Cells of Mouse and Rat Kidney

2003 ◽  
Vol 51 (9) ◽  
pp. 1217-1224 ◽  
Author(s):  
Matti S. Kyllönen ◽  
Seppo Parkkila ◽  
Hannu Rajaniemi ◽  
Abdul Waheed ◽  
Jeffrey H. Grubb ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Western Blotting ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Type A ◽  
Immunofluorescence Staining ◽  
Intercalated Cells ◽  
Basolateral Plasma Membrane ◽  
Proximal Tubule Segments

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO3− absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40–45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H+-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO3− absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.

Forskolin stimulates phosphorylation and membrane accumulation of UT-A3

2007 ◽  
Vol 293 (4) ◽  
pp. F1308-F1313 ◽  
Author(s):  
Mitsi A. Blount ◽  
Janet D. Klein ◽  
Christopher F. Martin ◽  
Dmitry Tchapyjnikov ◽  
Jeff M. Sands
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Membrane Staining ◽  
Protein Protein Interaction ◽  
Medullary Collecting Duct

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

scholarly journals Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH.

1987 ◽  
Vol 35 (5) ◽  
pp. 601-611 ◽  
Author(s):  
T Kobayashi ◽  
T Okada ◽  
H Seguchi
Keyword(s):  
Plasma Membranes ◽  
Stria Vascularis ◽  
Enzymatic Reaction ◽  
Salt Gland ◽  
Enzyme Concentration ◽  
Secretory Cells ◽  
Cerium Chloride ◽  
Reaction Products ◽  
Cytochemical Method ◽  
Marginal Cells

We have developed a new cytochemical method for detecting the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (K-NPPase) activity of the sodium-potassium-activated adenosine triphosphatase (Na-K ATPase) complex. The incubation medium contains p-nitrophenylphosphate (p-NPP) as substrate, cerium chloride as capture agent, Tricine buffer, MgCl2, and KCl. Tricine buffer protected against the medium turbidity caused by non-enzymatic reaction at pH 7.5. Biochemically, the accumulation of p-nitrophenol and phosphate in the reaction precipitate was proportionally related to the enzyme concentration. Ultracytochemically, the reaction products of the K-NPPase activity were localized as fine and uniform electron-dense deposits in the cytoplasmic side of specialized basolateral plasma membranes of cells of kidney distal convoluted tubules, secretory cells of salt gland, and marginal cells of stria vascularis. This method has the advantage of being useful at physiological pH.

Distribution and significance of endothelin 1 in guinea pig cochlear lateral wall

2007 ◽  
Vol 121 (8) ◽  
pp. 721-724 ◽  
Author(s):  
D-Y Xu ◽  
Y-D Tang ◽  
S-X Liu ◽  
J Liu
Keyword(s):  
Guinea Pig ◽  
Stria Vascularis ◽  
Lateral Wall ◽  
Complex Method ◽  
Spiral Ligament ◽  
Basal Cells ◽  
Endothelin 1 ◽  
Marginal Cells ◽  
Spiral Ligament Fibrocytes ◽  
Avidin Biotin Complex

AbstractEndothelin 1 is a vasoconstrictive peptide with many biological functions. To investigate the distribution of endothelin 1 in guinea pig cochlear lateral wall and the significance of endothelin 1 in maintaining cochlear homeostasis, the immunohistochemistry avidin biotin complex method was applied by using rabbit anti-endothelin 1 polyclonal antibody as primary antibody. Endothelin-1-like activities were detected in the marginal cells, spiral prominence epithelial cells, outer sulcus cells, stria vascularis capillaries, basal cells and spiral ligament fibrocytes.These results suggest that endothelin 1 may play an important role in maintaining cochlear homeostasis.

Cholesterol distribution in cells of the stria vascularis of the mammalian cochlea and some effects of ototoxic diuretics

1985 ◽  
Vol 79 (1) ◽  
pp. 181-197
Author(s):  
A. Forge
Keyword(s):  
Cell Membranes ◽  
Plasma Membranes ◽  
Early Stage ◽  
Stria Vascularis ◽  
Freeze Fracture ◽  
Ultrastructural Level ◽  
Body Region ◽  
Intermediate Cell ◽  
Marginal Cells ◽  
In Cells

The distribution of cholesterol in cells of the stria vascularis of guinea pigs and gerbils has been investigated at the ultrastructural level by incubation of tissue in filipin, followed by freeze-fracture. Verification of results has been sought by using tomatin. It is shown that in the cell body region of the marginal cells, the apical and lateral membranes reacted intensely with both agents, but the membranes of the basal processes of the marginal cells did not respond significantly to either filipin or tomatin. On basal cell membranes, filipin-cholesterol complexes were present at a high density, even within the strands of the tight-junctional network of these cells and occasionally within the gap-junctional areas also. Complexes were present on intermediate cell membranes at a lower density than on other plasma membranes that showed a positive response. Tissue from animals that had received an ototoxic diuretic, either ethacrynic acid or furosemide, was characterized by the appearance of membrane regions with closely clustered filipin complexes, suggesting some change in cell membrane structure. At an early stage following diuretic administration, such clusters were particularly noticeable on the membranes of intermediate cells. As intercellular spaces enlarged in response to the effects of diuretics, vesicles released into the extracellular spaces appeared to be cholesterol-enriched. The results are discussed in relation to known features of the structure and function of cells in the normal stria vascularis and of the changes that follow from acute diuretic ototoxicity.

scholarly journals Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland.

1987 ◽  
Vol 35 (8) ◽  
pp. 871-879 ◽  
Author(s):  
T Iwano ◽  
M Akayama ◽  
A Yamamoto ◽  
K Omori ◽  
T Kumazawa ◽  
...  
Keyword(s):  
Parotid Gland ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Protein A ◽  
Acinar Cells ◽  
Gold Particles ◽  
Duct Cells ◽  
Rat Parotid Gland ◽  
Basal Plasma ◽  
Rat Parotid

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.

Localization of ecto-ATPase in rat kidney and isolated renal cortical membrane vesicles

1992 ◽  
Vol 262 (2) ◽  
pp. F217-F228 ◽  
Author(s):  
I. Sabolic ◽  
O. Culic ◽  
S. H. Lin ◽  
D. Brown
Keyword(s):  
Atpase Activity ◽  
Western Blotting ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Regional Distribution ◽  
Membrane Vesicles ◽  
Specific Staining ◽  
Peritubular Capillaries ◽  
Long Form

Brush-border (BBMV) and basolateral membrane vesicles (BLMV) from rat renal cortex exhibit an ecto-ATPase activity that is distinct from other ATPases. We have examined the cellular and regional distribution of this enzyme in rat kidney using antibodies against rat liver ecto-ATPase. In isolated vesicles, the distribution shown by biochemical assays of ATPase activity was confirmed by immunocytochemistry and Western blotting. Indirect immunofluorescence and immunogold labeling showed that brush borders of the S1 and S3 segments of the proximal tubule (PT) were stained, but the S2 segment was negative. Staining was most intense in the S3 segment. The luminal membrane of the initial part of the thin descending limb of Henle also showed a marked staining. Surprisingly, basolateral plasma membranes of PT had no detectable staining. However, the plasma membrane of endothelial cells was heavily stained, both in larger vessels and in peritubular capillaries. Using an antibody against rat thrombomodulin, a marker for endothelial cell plasma membranes, we showed that preparations of BBMV, BLMV, and endocytic vesicles are all contaminated with these membranes. This may explain, at least partially, the biochemically measured ecto-ATPase activity in renal cortical membrane vesicles. Finally, no specific staining in the kidney was found using polyclonal antipeptide antibodies against the “long form” of liver ecto-ATPase, either by immunocytochemistry or by Western blotting. This indicates either that there is no long isoform of the ecto-ATPase in the kidney or that the intracellular domains of the long form are different in the two tissues.

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