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.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

Isolation and functional characterization of crustacean larval salt gland

1985 ◽  
Vol 248 (6) ◽  
pp. R702-R708
Author(s):  
R. J. Lowy ◽  
F. P. Conte
Keyword(s):  
Specific Activity ◽  
Functional Characterization ◽  
Salt Gland ◽  
Artemia Salina ◽  
The Body ◽  
Secretory Cells ◽  
Cell Integrity ◽  
Salt Glands ◽  
And Function ◽  
First Time

A batch method for isolating viable salt glands from the naupliar brine shrimp (Artemia salina) has been developed. This protocol produces a final preparation consisting of approximately 185 isolated salt glands, representing 1 X 10(4) secretory cells/g wet wt nauplii, with a final purity of 88%. Assays of cell integrity and function indicate good retention of in situ characteristics. Vital dye was excluded by 95% of the cells for at least 24 h. The O2 consumption rate was 22.7 nM O2 X min-1 X mg protein-1 and could be altered predictably by compounds known to affect oxidative phosphorylation and ion transport. The specific activity of the Na+-K+-ATPase in the salt gland, measured here for the first time, was 9.1 mM Pi X h-1 X mg protein-1. This is a substantial proportion of the body total, 17%, as expected for an active ion-transporting epithelium.

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.

scholarly journals Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination.

1976 ◽  
Vol 70 (3) ◽  
pp. 660-670 ◽  
Author(s):  
H Cheng ◽  
M G Farquhar
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Liver Homogenate ◽  
Supernatant Fraction ◽  
Golgi Membranes ◽  
Microsomal Membranes

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.

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.

Fine-Structural Localization of Adenosine Triphosphatase in the Rectum of Calliphora

1968 ◽  
Vol 3 (1) ◽  
pp. 17-32
Author(s):  
M. J. BERRIDGE ◽  
B. L. GUPTA
Keyword(s):  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Fluid Transport ◽  
Microsomal Fraction ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Osmotic Gradient ◽  
Ph Optimum ◽  
Structural Localization

Adenosine triphosphatase (ATPase) activity in the rectal papillae of Calliphora has been studied by biochemical and histochemical techniques. The microsomal fraction contained a Mg2+-activated ATPase with a pH optimum of 8.0. The enzyme was not stimulated by the addition of Na+ plus K+ and was insensitive to ouabain. Histochemical studies using modifications of the Wachstein-Meisel method showed that at pH 7.2 this Mg2+-activated ATPase was specifically localized on the intracellular surface of the lateral plasma membranes. A similar though less intense reaction was obtained with adenosine diphosphate and inosine triphosphate, but not with guanosine triphosphate, uridine triphosphate or β-glycerophosphate as substrates. At an acid pH (6.6-6.8), very little reaction occurred on the lateral plasma membrane but some reaction product was present in mitochondria and nuclei. Very little enzyme activity was found in the flattened rectal epithelium. These results are discussed in relation to the available data on transport ATPases and on the structural basis of fluid transport by rectal papillae. It is proposed that the ATPase localized on the stacks of lateral plasma membrane may be involved with ion secretion into the intercellular spaces to create the osmotic gradient necessary to extract water from the lumen.

Primary culture of duck salt gland. I. Morphology of confluent cell layers

1985 ◽  
Vol 249 (1) ◽  
pp. C32-C40 ◽  
Author(s):  
R. J. Lowy ◽  
J. H. Schreiber ◽  
D. C. Dawson ◽  
S. A. Ernst
Keyword(s):  
Electron Microscopy ◽  
Primary Culture ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Salt Gland ◽  
Direct Access ◽  
Secretory Cells ◽  
Collagen Gels ◽  
Lateral Membrane ◽  
Confluent Cell

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.

scholarly journals SUBSURFACE CISTERNS AND THEIR RELATIONSHIP TO THE NEURONAL PLASMA MEMBRANE

1962 ◽  
Vol 13 (3) ◽  
pp. 405-421 ◽  
Author(s):  
Jack Rosenbluth
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Nerve Cell ◽  
Plasma Membranes ◽  
Granular Endoplasmic Reticulum ◽  
Supporting Cells ◽  
Golgi Membranes ◽  
Subsurface Cisterns ◽  
Central Nervous Systems ◽  
Neuronal Plasma Membrane

Subsurface cisterns (SSC's) are large, flattened, membrane-limited vesicles which are very closely apposed to the inner aspect of the plasma membranes of nerve cell bodies and the proximal parts of their processes. They occur in a variety of vertebrate and invertebrate neurons of both the peripheral and central nervous systems, but not in the surrounding supporting cells. SSC's are sheet-like in configuration, having a luminal depth which may be less than 100 A and a breadth which may be as much as several microns. They are separated from the plasmalemma by a light zone of ∼50 to 80 A which sometimes contains a faint intermediate line. Flattened, agranular cisterns resembling SSC's, but structurally distinct from both typical granular endoplasmic reticulum (ER) and from Golgi membranes, also occur deep in the cytoplasm of neurons. It is suggested that membranes which are closely apposed may interact, resulting in alterations in their respective properties. The patches of neuronal plasmalemma associated with subsurface cisterns may, therefore, have special properties because of this association, resulting in a non-uniform neuronal surface. The possible significance of SSC's in relation to neuronal electrophysiology and metabolism is discussed.

Purification of distinct plasma membranes from canine renal medulla

1978 ◽  
Vol 234 (3) ◽  
pp. F247-F254
Author(s):  
R. Iyengar ◽  
D. S. Mailman ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Collecting Duct ◽  
Gradient Centrifugation ◽  
Phospholipid Composition ◽  
Renal Medulla ◽  
Similar Distribution ◽  
Mitochondrial Atpase ◽  
Fold Enrichment

Two types of plasma membrane were purified from canine distal renal medulla by the techniques of differential and zonal density-gradient centrifugation followed by free-flow electrophoresis. One group of plasma membranes was identified as basal-laterally derived based on a 30-fold enrichment of Na-K-ATPase, a 20-fold enrichment of vasopressin-stimulated adenylate cyclase, and a 33-fold enrichment of [3H]vasopressin binding sites. The second type of plasma membrane was free of these markers, but had a cholesterol and phospholipid composition similar to them. Alkaline phosphatase also had a similar distribution in the two fractions. This lighter membrane fraction contained a membrane-bound cyclic AMP-dependent protein kinase as well as substrate for this kinase. In addition there was a 26-fold enrichment of specific activity of an anion (SO32-)-activated ATPase which was insensitive to mitochondrial ATPase inhibitor protein, in contrast to the mitochondrial fraction of the tissue. Based on the relative preponderance of collecting duct tissue in the distal medulla and the yield of membrane protein, these membranes are tentatively identified as containing apical membranes of the collecting duct.

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.

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