Mechanisms responsible for SCN increase in resistance of in vitro frog gastric mucosa

1983 ◽  
Vol 245 (1) ◽  
pp. G143-G156 ◽  
Author(s):  
W. S. Rehm ◽  
T. C. Chu ◽  
M. Schwartz ◽  
G. Carrasquer
Keyword(s):  
Plasma Membrane ◽  
Gastric Mucosa ◽  
Plasma Membranes ◽  
Concentration Profiles ◽  
Tubular Cells ◽  
Nacl Concentration ◽  
Separate Site ◽  
Hcl Secretion ◽  
High Conductance

Thiocyanate (SCN) inhibits H+ secretion and increases the resistance and potential difference (PD) of the gastric mucosa. These results support our separate-site electrogenic theory of HCl secretion. Recent work shows that an ATP-driven mechanism in the gastric mucosa can produce H+ by a neutral exchange of K+ for H+. The SCN increase in resistance and PD, if due to an inhibition of a high-conductance mechanism(s) in the secretory plasma membrane, is not easily compatible with the neutral mechanism. Therefore, the possibility was examined that SCN induces the increase in resistance by other mechanisms. The HCl and NaCl concentration profiles in the pit and tubular lumina were calculated. The effects of SCN were determined with isotonic, hypotonic, hypertonic, buffered, and high [H+] secretory solutions. The results indicate that SCN produces an increase in resistance of about 130 omega. cm2 of the plasma membranes of the tubular cells. A scheme is proposed that incorporates the neutral K+-H+ mechanism into an electrogenic system.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals The Vitamin D Receptor Is Present in Caveolae-Enriched Plasma Membranes and Binds 1α,25(OH)2-Vitamin D3in Vivo and in Vitro

2004 ◽  
Vol 18 (11) ◽  
pp. 2660-2671 ◽  
Author(s):  
Johanna A. Huhtakangas ◽  
Christopher J. Olivera ◽  
June E. Bishop ◽  
Laura P. Zanello ◽  
Anthony W. Norman
Keyword(s):  
Vitamin D ◽  
Plasma Membrane ◽  
Vitamin D Receptor ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Kidney Tissue ◽  
Mouse Lung ◽  
Nuclear Fraction

Abstract The steroid hormone 1α,25(OH)2-vitamin D3 (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). This study characterized the VDRmem present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [3H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D KD binding dissociation constant = 1–3 nm. Our data collectively support the classical VDR being the VDRmem in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [3H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r2 = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [3H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [3H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

scholarly journals In vitro maintenance of boar sperm viability by a soluble fraction obtained from oviductal apical plasma membrane preparations

Reproduction ◽  
2003 ◽  
pp. 509-517 ◽  
Author(s):  
A Fazeli ◽  
RM Elliott ◽  
AE Duncan ◽  
A Moore ◽  
PF Watson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Current Knowledge ◽  
Biologically Active ◽  
Apical Plasma Membrane ◽  
Sperm Viability ◽  
Boar Sperm ◽  
Membrane Contact ◽  
Vitro Model

Oviductal apical plasma membrane fractions have been successfully used to provide an in vitro model to study the role of direct membrane contact in sperm-oviduct interactions. Apical plasma membrane preparations from pig oviductal tissues show a dose-response in their ability to maintain boar sperm viability in vitro. Membrane preparations obtained from other tissues (lung and duodenum) are incapable of maintaining boar sperm viability to the same extent as oviductal tissue. The present study examined the validity of two hypotheses that arise from current knowledge of sperm-oviduct interactions, namely, that (i) apical plasma membranes prepared from ampullar regions of the oviduct are less effective than those from isthmus regions, and (ii) sperm survival is more effective in apical plasma membrane preparations derived from follicular phase oviducts than those derived from luteal phase oviducts. Both hypotheses were proved false. The nature of the active component(s) in the oviductal apical plasma membrane fractions was further investigated. Heat treatment (100 degrees C for 20 min) diminished the capacity of membranes to support boar sperm viability. Furthermore, a soluble salt-extracted fraction obtained from oviductal apical plasma membrane preparations was biologically active and supported boar sperm viability in vitro. This may indicate that the active factor(s) responsible for the maintenance of boar sperm viability is not an integral part of oviductal membranes and is peripherally bound to these membranes.

scholarly journals Ponticulin is an atypical membrane protein.

1994 ◽  
Vol 126 (6) ◽  
pp. 1421-1431 ◽  
Author(s):  
A L Hitt ◽  
T H Lu ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Metabolic Labeling ◽  
Cortical Actin ◽  
Intact Cells ◽  
Membrane Spanning

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

scholarly journals Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate

1987 ◽  
Vol 244 (1) ◽  
pp. 209-217 ◽  
Author(s):  
C E King ◽  
L R Stephens ◽  
P T Hawkins ◽  
G R Guy ◽  
R H Michell
Keyword(s):  
Plasma Membrane ◽  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Age Distribution ◽  
Specific Radioactivity ◽  
Human Erythrocytes ◽  
Metabolic Compartmentation ◽  
To Come ◽  
Metabolic Heterogeneity

1. A Hepes-based medium has been devised which allows rapid Pi exchange across the plasma membrane of the human erythrocyte. This allows the metabolically labile phosphate pools of human erythrocytes to come to equilibrium with [32P]Pi in the medium after only 5 h in vitro. 2. After 5-7 h incubation with [32P]Pi in this medium, only three phospholipids, phosphatidic acid (PtdOH), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) are radioactively labelled. The concentrations of PtdIns4P and PtdIns4,5P2 remain constant throughout the incubation, so this labelling process is a reflection of the steady-state turnover of their monoester phosphate groups. 3. During such incubations, the specific radioactivities of the monoesterified phosphates of PtdIns4, PtdIns4,5P2 and PtdOH come to a steady value after 5 h that is only 25-30% of the specific radioactivity of the gamma-phosphate of ATP at that time. We suggest that this is a consequence of metabolic heterogeneity. This heterogeneity is not a result of the heterogeneous age distribution of the erythrocytes in human blood. Thus it appears that there is metabolic compartmentation of these lipids within cells, such that within a time-scale of a few hours only 25-30% of these three lipids are actively metabolized. 4. The phosphoinositidase C of intact human erythrocytes, when activated by Ca2+-ionophore treatment, only hydrolyses 50% of the total PtdIns4,5P2 and 50% of 32P-labelled PtdIns4,5P2 present in the cells: this enzyme does not discriminate between the metabolically active and inactive compartments of lipids in the erythrocyte membrane. Hence at least four metabolic pools of PtdIns4P and PtdIns4,5P2 are distinguishable in the human erythrocyte plasma membrane. 5. The mechanisms by which multiple non-mixing metabolic pools of PtdOH, PtdIns4P and PtdIns4,5P2 are sustained over many hours in the plasma membranes of intact erythrocytes are unknown, although some possible explanations are considered.

Protein phosphorylation and the proliferation of chick embryo fibroblasts: analysis by in vitro phosphorylation using isolated plasma membranes and whole cell homogenates

1980 ◽  
Vol 58 (1) ◽  
pp. 30-39 ◽  
Author(s):  
Philip E. Branton
Keyword(s):  
Cell Proliferation ◽  
Plasma Membrane ◽  
Chick Embryo ◽  
Protein Phosphorylation ◽  
Plasma Membranes ◽  
Kinase Activity ◽  
Cell Protein ◽  
Protein Kinase Activity ◽  
Whole Cell

The relationship between plasma membrane and whole cell protein phosphorylation and chick embryo fibroblast proliferation was studied using an in vitro labeling technique employing [γ-32P]ATP and isolated plasma membranes or whole cell homogenates. Cultures containing proliferating cells in log phase or containing cells stimulated to proliferate by the addition of serum were compared with cultures in which proliferation was inhibited due to cell density. Cell proliferation was found to be associated with increased plasma membrane basal protein kinase activity, but decreased membrane and whole cell cyclic AMP-dependent kinase activity. The phosphorylation of a number of membrane and whole cell polypeptides differed between proliferating and density-inhibited cells, suggesting that these phosphoproteins may be involved in the regulatory events associated with cell proliferation. Of interest, however, was the fact that the phosphoprotein differences found with serum-stimulated and sparse, rapidly dividing cells were generally not the same. These observations suggest that the protein phosphorylation events associated with rapid proliferation of sparse, log-phase cells may differ from those involved in serum-activated proliferation of quiescent cells.

scholarly journals Sphingosine kills bacteria by binding to cardiolipin

2020 ◽  
Vol 295 (22) ◽  
pp. 7686-7696 ◽  
Author(s):  
Rabea Verhaegh ◽  
Katrin Anne Becker ◽  
Michael J. Edwards ◽  
Erich Gulbins
Keyword(s):  
Plasma Membrane ◽  
Bactericidal Activity ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Central Element ◽  
Lipid Binding ◽  
Bacterial Cells ◽  
Bacterial Killing

Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively–charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo. We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.

scholarly journals Subcellular localization of transglutaminase. Effect of collagen

1988 ◽  
Vol 250 (2) ◽  
pp. 421-427 ◽  
Author(s):  
M Juprelle-Soret ◽  
S Wattiaux-De Coninck ◽  
R Wattiaux
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Collagen Type I ◽  
Type I ◽  
Nuclear Fraction ◽  
Binding Studies ◽  
Isopycnic Centrifugation

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5′-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.

scholarly journals In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles.

1985 ◽  
Vol 101 (6) ◽  
pp. 2263-2273 ◽  
Author(s):  
J H Crabb ◽  
R C Jackson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Secretory Vesicles ◽  
Cytoplasmic Face ◽  
In Vitro Reconstitution ◽  
Transport Assay ◽  
Complex Preparation ◽  
A Cell ◽  
Vectorial Transport

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.

312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

2007 ◽  
Vol 19 (1) ◽  
pp. 272
Author(s):  
N. Satake ◽  
A. K. Alhaider ◽  
W. V. Holt ◽  
P. F. Watson
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Plasma Membranes ◽  
Fertilization Rate ◽  
Apical Plasma Membrane ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fertilization Rates

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P < 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P < 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

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