scholarly journals Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

1977 ◽  
Vol 75 (1) ◽  
pp. 74-94 ◽  
Author(s):  
SA Ernst ◽  
JW Mills
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Salt Gland ◽  
Physiological Data ◽  
Secretory Cells ◽  
Ouabain Binding ◽  
Salt Glands ◽  
Cytochemical Localization ◽  
Secretory Epithelium ◽  
Basolateral Plasma Membrane

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.

Biogenesis of Plasma Membranes in Salt Glands of Salt-Stressed Domestic Ducklings: Localization of Acyltransferase Activity

1972 ◽  
Vol 11 (3) ◽  
pp. 855-873
Author(s):  
A. M. LEVINE ◽  
JOAN A. HIGGINS ◽  
R. J. BARRNETT
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Salt Gland ◽  
Secretory Cells ◽  
Salt Glands ◽  
Acyltransferase Activity ◽  
Structural Localization ◽  
Differentiated Cells ◽  
Golgi Membranes

In response to salt water stress there is a marked increase in the plasma membranes of the epithelial secretory cells of the salt glands of domestic ducklings. In the present study, the fine-structural localization of the acyltransferases involved in synthesis of phospholipids has been investigated in this tissue during this increased biogenesis of plasma membranes. The specific activity of the acyltransferases of the salt gland rose in response to salt stress, and this preceded the rapid increase in weight and cellular differentiation. After the weight increase of the gland became established, the specific activity of the acyltransferases declined, but the total activity remained constant. Salt gland tissue fixed in a mixture of glutaraldehyde and formaldehyde retained 35% of the acyltransferase activity of unfixed tissue. Cytochemical studies of the localization of acyltransferase activity in fixed and unfixed salt gland showed reaction product associated only with the lamellar membranes of the Golgi complex. This localization occurred in partially differentiated cells from salt-stressed glands to the greatest extent; and to only a small extent in cells of control tissue from unstressed salt glands. Omission of substrates resulted in absence of reaction product in association with the Golgi membranes. In addition, vesicles having limiting membranes morphologically similar to the plasma membrane occurred between the Golgi region and the plasma membrane in the partially differentiated cells. The phospholipid component of the plasma membrane appears therefore to be synthesized in association with the Golgi membranes and the membrane packaged at this site from which it moves in the form of vesicles to fuse with the pre-existing plasma membrane.

Ouabain binding during plasma membrane biogenesis in duck salt gland

1978 ◽  
Vol 31 (1) ◽  
pp. 179-197
Author(s):  
F.E. Hossler ◽  
M.P. Sarras ◽  
R.J. Barrnett
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Binding Assay ◽  
Specific Activity ◽  
Salt Gland ◽  
Secretory Cells ◽  
Ouabain Binding ◽  
Salt Diet ◽  
Maximal Binding ◽  
Enzyme Molecules

The conditions necessary for optimal ouabain binding in the avian salt gland were examined. Binding was enhanced by ATP and Mg2+ and was decreased by K+, but was unaffected by added Na+. Both maximal binding and complete inhibition of Na, K-ATPase activity were obtained at 1 X 10(−6) M ouabain. Half maximal binding and half maximal inhibition of Na, K-ATPase activity were obtained at 1.7 X 10(−7) M ouabain. Ouabain binding increased in parallel with increasing specific activity of the Na, K-ATPase duringsalt-induced salt gland specialization. The ratio of Na, K-ATPase activity to ouabain-binding sites remained constant during the salt stress as well as after removal of the salt diet. Autoradiography indicated binding to partially and fully differentiated secretory cells of the salt gland. The ouabain binding assay appeared to be a more useful indicator of membrane amplification than Na, K-ATPase activity since it is rapid, essentially irreversible, less sensitive to tissue fixatives, and quantitatively measured the number of enzyme molecules.

Morphology of isolated crustacean larval salt glands

1985 ◽  
Vol 248 (6) ◽  
pp. R709-R716
Author(s):  
R. J. Lowy ◽  
F. P. Conte
Keyword(s):  
Plasma Membrane ◽  
Salt Gland ◽  
Artemia Salina ◽  
Apical Plasma Membrane ◽  
Secretory Cells ◽  
Cell Contact ◽  
Salt Glands ◽  
Transmission Electron

Larval salt glands isolated from the naupliar brine shrimp (Artemia salina) were examined using light microscopy and scanning and transmission electron microscopy. These methods demonstrated that most cellular and subcellular features of the in vitro organ compared favorably with those seen in vivo. This salt gland measures 130 micron in diameter and is comprised of 50-70 secretory cells, which are of a single epithelial cell type. Characteristic ultrastructural features that are well preserved include apical to basal cell polarity, apical plasma membrane projections, and the extent of the basolateral tubular labyrinth and its association with numerous mitochondria. Some features that have been altered are a decrease in cell-cell contact, separation of septate junctions, and expansion of tubular labyrinth lumens and mitochondrial cristae. Use of this preparation has allowed examination of the salt gland cell's hemocoelic surface for the first time and provided information about the ultrastructure of the tufts formed by the apical plasma membrane.

Immunocytochemical studies of the Na-K-Cl cotransporter of shark kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F927-F936 ◽  
Author(s):  
D. Biemesderfer ◽  
J. A. Payne ◽  
C. Y. Lytle ◽  
B. Forbush
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Rectal Gland ◽  
Apical Plasma Membrane ◽  
Secretory Cells ◽  
Weak Staining ◽  
Basolateral Plasma Membrane ◽  
Kidney Functions

The Na-K-Cl cotransporter (NKCC or BSC) has been described in numerous secretory and reabsorptive epithelia and is an important part of the mechanism of NaCl reabsorption in both the mammalian and elasmobranch kidneys. We have recently developed a panel of four monoclonal antibodies (MAbs) raised to the 195-kDa Na-K-Cl cotransport protein of the shark rectal gland (sNKCC1), which is expressed along the basolateral plasma membrane of secretory cells in this tissue (29). Here, we report immunologic studies of the Na-K-Cl cotransporter in the kidney of the dogfish shark Squalus acanthias. Western blot analysis of shark renal microsomes using MAbs J3, J7, and J25 identified proteins of approximately 195 and 150 kDa, whereas MAb J4 was not reactive. To define the cellular and subcellular distribution of the cotransport protein, immunofluorescence and immunoelectron microscopy studies were performed on fixed kidneys. Immunofluorescence microscopy on semithin (0.5-micron) cryosections demonstrated that MAbs J3, J7, and J25 intensely stained the apical plasma membrane of all distal tubule segments. Weak staining was also seen along the basolateral membrane of most distal nephrons. Immunoelectron microscopy confirmed this observation and showed that some of these segments were morphologically similar to diluting segments from other species. MAbs also reacted with the brush border and, to a lesser extent, the basolateral membrane of proximal tubules. This study supports the hypothesis that the lateral bundle zone of the elasmobranch kidney functions as a countercurrent exchanger and is consistent with the presence of multiple isoforms of the Na-K-Cl cotransporter in the shark kidney.

Effect of methacholine on Na+ pump activity and ion content of dispersed avian salt gland cells

1981 ◽  
Vol 241 (1) ◽  
pp. R77-R86
Author(s):  
S. R. Hootman ◽  
S. A. Ernst
Keyword(s):  
Binding Sites ◽  
Gland Cell ◽  
Salt Gland ◽  
Ion Content ◽  
Ouabain Binding ◽  
Gland Cells ◽  
Na Pump ◽  
Pump Activity ◽  
Number Of Binding Sites ◽  
Dissociated Cells

The effects of the cholinergic agonist methacholine chloride (MCh) on cellular ion content and Na+ pump activity of dissociated duck salt gland cells were studied. Dispersed salt gland cells regulate intracellular ion levels in a ouabain-sensitive manner. MCh (0.5 mM) caused no detectable change in cell Na+ levels over the first 10 min of exposure of cells to the agonist but elicited decreases of 23 and 13%, respectively, in intracellular Cl- and K+ content. The rate of turnover of salt gland cell plasmalemmal Na+ pumps, as measured by [3H]ouabain binding to the dissociated cells, was markedly stimulated by 0.5 mM MCh, although the total number of binding sites at equilibrium remained unchanged. Replacement of medium Na+ with choline abolished the MCh-stimulated increase in ouabain binding but had no effect on the rate of glycoside binding in the absence of the agonist. Substitution of Cl- in the medium by NO3-, SO42-, or benzene sulfonate- reduced the stimulated component of Na+ pump turnover by 85-90%. Addition of 1 mM furosemide to the medium abolished the increase in ouabain binding and ouabain-sensitive oxygen consumption observed after exposure of salt gland cells to MCh. These data are consistent with the hypothesis that cholinergic stimulation of salt gland cells triggers a Cl--dependent uptake of Na+, which elicits a compensatory increase in Na+ pump turnover. In addition, the decrease in cellular Cl- content caused by MCh suggests that the agonist either directly or indirectly mediates an efflux of Cl- from the cells.

Salt stress increases abundance and glycosylation of CFTR localized at apical surfaces of salt gland secretory cells

1994 ◽  
Vol 267 (4) ◽  
pp. C990-C1001 ◽  
Author(s):  
S. A. Ernst ◽  
K. M. Crawford ◽  
M. A. Post ◽  
J. A. Cohn
Keyword(s):  
Salt Stress ◽  
Single Cells ◽  
Salt Gland ◽  
Cell Protein ◽  
Secretory Cells ◽  
Salt Glands ◽  
Cl Secretion ◽  
Peptide Antibody ◽  
Nacl Secretion ◽  
Apical Membranes

Osmotic stress elicits hypertonic NaCl secretion and promotes structural and biochemical differentiation in avian salt glands. In addition to cholinergic control, Cl- secretion is stimulated by vasoactive intestinal peptide (VIP), suggesting that the cystic fibrosis transmembrane conductance regulator (CFTR) may be present and that its expression may be regulated by chronic salt stress. Anion efflux, assayed by 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence changes in single cells, was stimulated by VIP or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Immunoblots with a COOH-terminal peptide antibody to human CFTR revealed approximately 170- and approximately 180-kDa bands in lysates from control and salt-stressed glands, respectively. Both variants reduced to approximately 140 kDa after N-glycanase digestion and gave identical tryptic phosphopeptide maps after immunoprecipitation and phosphorylation by protein kinase A. CFTR was localized to apical membranes by immunofluorescence and, additionally, to subapical vesicles by immunoelectron microscopy. Salt stress induced an approximately twofold increase in CFTR abundance/cell protein (approximately 5-fold/cell) and intensified apical membrane immunofluorescence. For comparison, Na+ pump expression increased approximately fourfold per cell protein with little change in actin. Thus differentiation induced by salt stress is accompanied by alteration in CFTR abundance and glycosylation. Upregulation of CFTR likely contributes to increased efficiency of Cl- secretion.

Plasma Membrane Biogenesis

1974 ◽  
Vol 32 ◽  
pp. 312-313
Author(s):  
Russell J. Barrnett
Keyword(s):  
Plasma Membrane ◽  
Trigeminal Nerve ◽  
Salt Gland ◽  
Ultrastructural Localization ◽  
Secretory Cells ◽  
Membrane Biogenesis ◽  
Cell Plasma ◽  
Microsome Fraction ◽  
Myelin Fraction

This report presents two examples of plasma membrane biogenesis in which the synthesis and assembly of components, phospholipid and enzyme proteins, were studies by a combination of biochemistry, cytochemistry and electron microscopy. These were: the proliferation of Schwann cell plasma membrane during the process of myelination of the trigeminal nerve in neonatal rats and amplification of the plasma membrane at the lateral and basal borders of secretory cells of the ducklings' salt gland as a result of salt stress.In the study concerning myelination a method for the ultrastructural localization of acyltransferase activities (the first two steps in phospholipid synthesis) was applied to the developing rat trigeminal nerve. Determination of acyltransferase levels in the nerve indicated that a peak of activity occurs at the 8th day after birth with gradual declines of activity up to 15 days. This peak coincided with the peak of a-glycerophosphate incorporation into phospholipids in the microsome fraction of the nerve: wheras, no incorporation was noted in the myelin fraction.

scholarly journals PRESERVATION OF Na-K-ACTIVATED AND Mg-ACTIVATED ADENOSINE TRIPHOSPHATASE ACTIVITIES OF AVIAN SALT GLAND AND TELEOST GILL WITH FORMALDEHYDE AS FIXATIVE

1970 ◽  
Vol 18 (4) ◽  
pp. 251-263 ◽  
Author(s):  
STEPHEN A. ERNST ◽  
CHARLES W. PHILPOTT
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Electron Microscopic Examination ◽  
Salt Gland ◽  
Chloride Cells ◽  
Secretory Cells ◽  
Salt Glands ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Gill Filaments

The effect of glutaraldehyde and formaldehyde fixation on the level of biochemically demonstrable Na-K-adenosine triphosphatase (Na-K-ATPase) and Mg-ATPase of avian salt glands and teleost gill filaments was studied. Sections, 100-200 µ, prepared with the Smith-Farquhar tissue chopper, were fixed for varying periods, homogenized and assayed for ATPase activity. Fixation of salt gland tissue with 0.5% glutaraldehyde for 40-60 min completely inhibited the Na-K-ATPase activity and reduced the level of Mg-ATPase by 85%. In contrast, fixation with 2 or 3% formaldehyde, prepared from paraformaldehyde, for 60-90 min resulted in a loss of only 30% of the Na-K-ATPase activity and 65% of the Mg-ATPase activity. Similar results were obtained with gill filaments. In addition, Na-K-ATPase of formaldehyde-fixed tissue retained an obligatory requirement for Na+ and K+ and was fully sensitive to ouabain. Electron microscopic examination of formaldehyde-fixed tissue, sectioned with either the tissue chopper or in the cryostat and incubated in the Wachstein-Meisel medium, showed excellent morphologic preservation. Reaction product deposition (presumably due to Mg-ATPase) was associated with the extracellular side of the plasma membrane in the secretory cells of the salt gland and over the mitochondrial matrix of chloride cells present in the gill epithelium.

scholarly journals Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes

1990 ◽  
Vol 269 (3) ◽  
pp. 757-766 ◽  
Author(s):  
G Schmalzing ◽  
S Kröner
Keyword(s):  
Binding Sites ◽  
Rank Order ◽  
Secretory Cells ◽  
Free Calcium ◽  
Membrane Insertion ◽  
Xenopus Laevis Oocytes ◽  
Ouabain Binding ◽  
Permeabilized Cells ◽  
Sodium Pumps ◽  
Maximal Stimulation

As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca2+ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K0.5 0.5 microM-Ca2+, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca2+ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg2+ or ATP completely abolished the Ca2+ effect. Half-maximal stimulation by Ca2+ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5′-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). Pi, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5′-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5′-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca2+ effect by some 50%. Inhibitors of chymotrypsin and the Ca2+ proteinase calpain had no effect. Ca2+ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca2+ response entirely. Neomycin also abolished a Ca2(+)-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca2(+)-regulated pathway for the plasma membrane insertion of sodium pumps.

scholarly journals Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous β1 subunits of the sodium pump

1991 ◽  
Vol 279 (2) ◽  
pp. 329-336 ◽  
Author(s):  
G Schmalzing ◽  
S Gloor ◽  
H Omay ◽  
S Kröner ◽  
H Appelhans ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Binding Sites ◽  
Sodium Pump ◽  
Binding Capacity ◽  
Polyclonal Antiserum ◽  
Scatchard Analysis ◽  
Xenopus Laevis Oocytes ◽  
Ouabain Binding ◽  
Na Pump

Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.

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