Immunolocalization of Na/K–ATPase and Na/K/2Cl cotransporter in the tubular epithelia of sea snake salt glands

The sublingual salt gland is the primary site of salt excretion in sea snakes; however, little is known about the mechanisms mediating ion excretion. Na⁺/K⁺–ATPase (NKA) and Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) are two proteins known to regulate membrane potential and drive salt secretion in most vertebrate secretory cells. We hypothesized that NKA and NKCC would localize to the basolateral membranes of the principal cells comprising the tubular epithelia of sea snake salt glands. Although there is evidence of NKA activity in salt glands from several species of sea snake, the localization of NKA and NKCC and other potential ion transporters remains unstudied. Using histology and immunohistochemistry, we localized NKA and NKCC in salt glands from three species of laticaudine sea snake: Laticauda semifasciata, L. laticaudata, and L. colubrina. Antibody specificity was confirmed using Western blots. The compound tubular glands of all three species were found to be composed of serous secretory epithelia, and NKA and NKCC were abundant in the basolateral membranes. These results are consistent with the morphology of secretory epithelia found in the rectal salt glands of marine elasmobranchs, the nasal glands of marine birds and the gills of teleost fishes, suggesting a similar function in regulating ion secretion.     

Calmodulin blocker inhibits Ca++-ATPase activity in secretory ameloblast of rat incisor

Summary The effects of the calmodulin blocker, trifluoperazine (TEP), on membrane-bound Ca⁺⁺ -ATPase, Na⁺ -K⁺ -ATPase (EC 3.6.1.3.) and the ultrastructure of the enamel organ were investigated in the lower incisors of normal and TFP-injected rats. The rats, of about 100 g body weight, were given either 0.2 ml physiological saline or 100 g TFP dissolved in 0.2 ml physiological saline through a jugular vein and fixed by transcardiac perfusion with a formaldehyde-glutaraldehyde mixture at 1 and 2 h after TFP administration. Non-decalcified sections of the enamel organ less than 50 m in thickness, prepared from dissected lower incisors, were processed for the ultracytochemical demonstration of Ca⁺⁺-ATPase and Na⁺-K⁺ -ATPase by the one-step lead method at alkaline pH. In control saline-injected animals the most intense enzymatic reaction of Ca⁺⁺-ATPase was demonstrated along the plasma membranes of the entire cell surfaces of secretory ameloblasts. Moderate enzymatic reaction was also observed in the plasma membranes of the cells of stratum intermedium and papillary layer. Reaction precipitates of Na⁺-K⁺-ATPase activity were localized clearly along the plasma membranes of only the cells of stratum intermedium and papillary layer. The most drastic effect of TFP was a marked disappearance of enzymatic reaction of Ca⁺⁺-ATPase from the plasma membranes of secretory ameloblasts, except for a weak persistent reaction in the basolateral cell surfaces of the infranuclear region facing the stratum intermedium. The cells of stratum intermedium and papillary layer, however, continued to react for Ca⁺⁺-ATPase even after TFP treatment. Similarly, Na⁺-K⁺-ATPase activity in these cells was not inhibited by TFP administration. Ultrastructural examination of secretory ameloblasts revealed that administration of TFP caused no considerable cytological changes and did not act as a cytotoxic agent. These results suggest that secretory ameloblasts may have an active Ca⁺⁺ transport system, which is modulated by an endogenous calmodulin.     

Plant proton pumps

Chemiosmotic circuits of plant cells are driven by proton (H(+)) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react to their constantly changing environments and at the same time maintain optimal metabolic conditions, the expression, activity and interplay of the pumps generating these H(+) gradients have to be tightly regulated. In this review, we will highlight results on the regulation, localization and physiological roles of these H(+)- pumps, namely the plasma membrane H(+)-ATPase, the vacuolar H(+)-ATPase and the vacuolar H(+)-PPase.     

Regulation of the paracellular Na+ and Cl- conductances by the NaCl-generated osmotic gradient in a manner dependent on the direction of osmotic gradients

In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na⁺ conductance (G_Na) and the Cl⁻ conductance (G_Cl). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the G_Na associated with a small increase in the G_Cl, whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the G_Na and the G_Cl. These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by basolateral application of sucrose without any NaCl gradients had little effects on the Gp. However, this basolateral application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the basolateral side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells.     

Calcium adenosine triphosphatase and secretion of koilin membrane in the gizzard of the fowl.

The localization of calcium adenosine triphosphatase (Ca(2+)-ATPase) was determined histo- and ultracytochemically in the gizzard gland cells of the adult domestic fowl. Surface and chief gland cells exhibited faint and inconstant basolateral activity in contrast to basal cells, whose basolateral cell membrane constantly showed deposition on the external side. Intracellular enzyme activity was localized on the luminal aspect of Golgi membranes in all types of gland cells. Lysosomes also reacted positive for Ca(2+)-ATPase. Neither membranes of secretory vesicles nor cortical cytoplasm of the secretory pole exhibited enzyme activity. From these results it is speculated that calcium is not essentially involved in the secretion of the koilin membrane in terms of storage of the secretory material, transport to the secretory surface and release into the lumen. Ca(2+)-ATPase activity rather seems to be related to differentiation and maturation processes and to intracellular storage of Ca2+.     

Morphological and functional characterization of the thoracic portion of blowfly salivary glands

The abdominal portion of the salivary glands in the blowfly has been studied intensively. Here, we examine the thoracic part of the salivary glands, emphasizing structural and functional aspects. The initial segment downstream of the abdominal portion is secretory and resembles the latter in most structural and functional aspects: the apical membrane is enfolded, forms a canalicular system and contains V-H⁺-ATPase that assembles upon stimulation with the hormone serotonin (5-HT); Na,K-ATPase is localized in the basolateral membrane; septate junctions are not prominent, as deduced from immunofluorescence staining for the marker proteins discs large and fasciclin III. 5-HT elicits, at low concentrations, cytoplasmic [Ca²⁺] oscillations, and, at saturating concentrations, a tonic [Ca²⁺] rise. The following, so-called “re-absorptive” segment loops through the coiled secretory portion of the salivary gland. The apical membrane of the re-absorptive cells is not enfolded, and septate junctions are prominent. V-H⁺-ATPase and Na,K-ATPase reside on the apical and basolateral membranes, respectively. Finally, re-absorptive cells are also sensitive to 5-HT; however, whereas V-ATPase assembly has a 5-HT concentration dependence similar to other segments, the Ca²⁺ response occurs only at higher 5-HT concentrations, and displays a different kinetic pattern.     

Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets

Summary The basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from –23.7 to –46.7 and –4.3 to –5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 G and 25m, respectively. The cells are electrically coupled via low resistance intercellular communications (58 M). Intracellular ion activities for Na⁺, K⁺ and Cl^– averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence ofP _K>P _Cl>P _Na. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na⁺–K)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques.     

Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells

The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14).     

Vasopressin-dependent control of basolateral Na/H-exchange in highly differentiated A6-cell monolayers

We have used a well-differentiated A6-cell preparation (A6-C1) to study cellular location and vasopressin control of Na/H-exchange activity. After cell acidification, cell pH_i (measured by BCECF-fluorescence) only recovered by the addition of Na medium to the basolateral cell surface; this pH_i recovery was inhibited by dimethylamiloride (2 m) consistent with basolateral location of Na/H-exchange activity. Addition of vasopressin produced stimulation of Na/H-exchange activity and increased the affinity of the exchanger for Na⁺. Stimulation of Na/H exchange was mimicked by pharmacological activation of protein kinase A (forskolin, 8-Br-cAMP) and not by pharmacological activation of protein kinase C (TPA). It is concluded that basolaterally located Na/H-exchange in A6-C1 cells is activated by vasopressin.     

Ultrastructural localization of ouabain-sensitive, K-dependent p-nitrophenyl phosphatase activity in monkey eccrine sweat gland

We studied the electron microscopic localization of ouabain-sensitive, potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity of the Na K-ATPase complex in Rhesus monkey eccrine sweat gland by use of the one-step lead citrate method of Mayahara et al. (Histochemistry 1980; 67:125). Reaction product was observed predominantly in the cytoplasmic side of the basolateral membranes of clear (secretory) cells, especially in the interdigitating membrane folds in the basal labryinth, and were completely abolished by 10 mM ouabain or by removal of K+. Little or no enzyme activity was noted in membrane processes in the intercellular canaliculi and in the secretory coil lumen. Basolateral membranes of the dark cells also showed moderate enzyme activity. The myoepithelial cell membrane was devoid of reaction product, except in a few membrane processes arising from the inner aspect of myoepithelial cells. In the coiled duct, K-pNPPase activity was present predominantly in the entire cell membrane of the peripheral ductal cells. The predominantly basolateral distribution of Na-K-ATPase in the eccrine sweat secretory cells is consistent with the concept that a Na-K-Cl co-transport model may be involved in the mechanism of eccrine sweat secretion.     

Immunocytochemical characterization of two types of microvillar cells in rodent olfactory epithelium

Microvillar cells (MCs) have been identified in the olfactory epithelium of various mammalian species from rodents to humans. Studies on properties and functions of MCs to date have yielded partially controversial results, supporting alternatively an epithelial or a neuronal nature of these cells. In the present study, single and double immunolabeling investigations were carried out using antibodies against cytoskeletal and integral membrane proteins in order to further characterize MCs in rat and mouse olfactory epithelium. Application of antibodies against ankyrin (ANK), a protein that links integral membrane proteins to the submembrane cytoskeleton, led to intense labeling of the basolateral membranes of numerous cells with characteristic MC morphology. ANK-immunoreactive (ir) cells bore an apical tuft of -actin-ir microvilli, were filled with cytokeratin 18 (CK18)-ir filamentous network, and extended a basal process that appeared to end above the basal membrane. Immunoreactions for villin, an actin-crosslinking protein particularly prominently expressed in brush cells in the gastrointestinal and respiratory tract epithelia, and for the -subunit of sodium-potassium ATPase (Na⁺, K⁺-ATPase), revealed that ANK-ir MCs fall into two subpopulations. The less frequent type I MCs displayed villin immunoreactivity in their apical microvilli and underneath the basolateral membranes; the more numerous type II MCs were negative for villin but possessed intense basolateral immunoreactivity for Na⁺, K⁺-ATPase. Strong reactivity for the epithelial-type integral membrane protein of adherens junctions, E-Cadherin, was localized in basolateral membranes of both types of MCs. Our results support an epithelial nature of ANK-ir MCs in rat and mouse olfactory epithelium. Type I MCs strongly resemble brush cells in their immunocytochemical characteristics, namely, their ANK reactivity, CK18 reactivity, and villin reactivity. The intense Na⁺, K⁺-ATPase reactivity of type II MCs implicates these cells in transport processes.     

Ultrahistochemical and autoradiographic evidence for epithelial transport in the uterus of the ovoviviparous salamander,Salamandra salamandra (L.) (Amphibia,Urodela)

Summary The uterine epithelium of pregnant females of the terrestrial ovoviviparous Salamandra salamandra is characterized by a considerable enlargement of its basolateral surface. Chloride and cations (among others sodium), preferentially within the intercellular spaces, can be demonstrated ultrahistochemically. There is indirect evidence of Na⁺-K⁺-ATPase activity along the basolateral plasma membranes of the epithelial cells using the Sr-technique for demonstration of a K⁺-NPPase and ³H-ouabain autoradiography. Preliminary measurements reveal a potential difference across the uterine wall of 15–25mV, the lumenal (mucosal) surface being negative with respect to the coelomic (serosal) surface, and a short circuit current of 200–300 A. The possibly electrogenic ion transport is ouabain-sensitive. The results are in agreement with the model of a forward transporting, i.e. absorptive epithelium. An active transport of solute out of the uterine lumen across the epithelium to the subjacent connective tissue and the blood vessels may be involved in the regulation of an intrauterine milieu appropriate for the development of the offspring.I am indebted to Miss Dr. U. Beigel, Zoologisches Institut der Universität Münster, for linguistic help     

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig

Summary Ca⁺⁺-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase) activity, which represents the second dephosphorylative step of the Na⁺-K⁺-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca⁺⁺-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca⁺⁺- and substrate-dependent and showed sensitivity to oligomycin. When Mg⁺⁺-ions were used instead of Ca⁺⁺-ions, a distinct reaction was also found on mitochondrial inner membranes.In contrast to the localization of the Ca⁺⁺ -ATPase activity, the K⁺-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K⁺ from the incubation medium.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): immunolocalization of Na+,K+-ATPase

The involvement of the antennal urinary glands in the ontogeny of osmoregulatory functions was investigated during the development of Astacus leptodactylus by measurements of hemolymph and urine osmolality in juvenile and adult crayfish and by the immunodetection of the enzyme Na⁺,K⁺-ATPase. In stage II juveniles, 1-year-old juveniles, and adults, all of which were maintained in freshwater, urine was significantly hypotonic to hemolymph. In adults, chloride and sodium concentrations were much lower in urine than in hemolymph. During embryonic development, Na⁺,K⁺-ATPase was detected by immunocytochemistry in ionocytes lining the tubule and the bladder, at an eye index (EI) of 220–250 m, and in the labyrinth, at EI 350 m. In all regions, immunofluorescence was mainly located at the basolateral side of the cells. No immunofluorescence was detected at any stage in the coelomosac. In late embryonic stages (EI 410–440 m), in stage I juveniles, and in adults, strong positive immunofluorescence was found from the labyrinth up to and including the bladder. These results show that, as early as hatching, juvenile crayfish are able to produce dilute urine hypotonic to hemolymph. This ability originates from the presence of Na⁺,K⁺-ATPase in ion-transporting cells located in the labyrinth, the tubule, and the bladder of the antennal glands and constitutes one of the main adaptations of crayfish to freshwater.We thank the University of Tarbiat Modarres and Ministry of Science, Research and Technology, Islamic Republic of Iran for financial aid and support. Special thanks are also due to the Société Française dExportation des Ressources Educatives (SFERE) for the scholarship to S.K.     

Ultracytochemical localization of ouabain-sensitive,potassium-dependent p-nitrophenylphosphatase activity in the lacrimal gland of the rat

The electron-microscopic localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na - K-ATPase complex was studied in the exorbital lacrimal gland of the untreated rat with the use of a newly developed one-step lead-citrate method (Mayahara and Ogawa 1980; Mayahara et al. 1980). In the rat lacrimal gland fixed for 15 min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, an electron-dense reaction product was observed on the plasma membrane of the basal infoldings and the lateral interdigitations of the ductal cells. The most intense reaction product - and thus the major site of the Na - K-ATPase activity - was evident on the basolateral membranes of the cells of the large interlobular ducts; a weak reaction was seen on the basolateral, extensively folded plasma membranes of the small intercalated ducts; no reaction product was observed on the plasma membranes of the acinar cells. Addition of 1) 10 mM ouabain, 2) p-chloromercuri-phenyl-sulfonic acid (PCMB-S), 3) elimination of K-ions from the incubation medium, or 4) preheating abolished completely the K-NPPase reaction. The activity was also substrate-dependent. Mg-ATPase-activity was observed not only in the basolateral membranes of all ductal cells but also in the basal part of the acinar cells and on the walls of blood vessels. This reaction was neither inhibited by ouabain nor activated by K-ions. The precipitate of the Mg-ATPase-activity was localized at the extracellular side of the plasma membrane, whereas the K-NPPase-reaction product was restricted to the cytoplasmic side of the plasmalemma. In contrast, non-specific alkaline-phosphatase (ALPase) activity was missing in cells of the large interlobular ducts, but obvious on the apical plasmalemma of cells lining the small intercalated ducts. With respect to its localization and reactivity pattern the activity of the K-NPPase (member of the Na - K-ATase complex) differs markedly from the Mg-ATPase- and ALPase-activity.     

A comparative ultrastructural study of ‘glue’ production and secretion of the salivary glands in different species of theDrosophila melanogaster group

Summary Salivary gland cells of members of theDrosophila melanogaster group (from four different subgroups) were examined electron microscopically and histochemically during the late larval period of development. The secretory product, which is supposed to be utilized as glue at the time of puparium formation, appears, by analogy to Palade and Jamieson's results, to be synthesized partially in the rough endoplasmic reticulum (RER) and partially in the Golgi complex. The latter is also the usual site of the packaging of the product into secretory granules, except in the case of one of the secretory granule components ofD. lucipennis. The phylogenetic relationships among the subgroups, implied by the morphological appearance of the secretory granules, fit well with the existing phylogenetic relationships within the group. The secretory granules of each species have their own morphological features; granules of species of the same subgroup share some of these features. Secretion occurs from the cells via exocytosis during which the morphology of the secretory granules changes. Light microscope examination of PAS (Periodic Acid-Schiff reaction) stained glands shows a strong positive reaction in most species, with the exception of the species of thesuzukii subgroup which show a weak, or a negative reaction (D. rajasekari). Electron histochemical localization of polysaccharides in the secretory granules was possible inD. melanogaster and the species of theananassae subgroup.     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)