Phospholipid organization in H,K-ATPase-containing membranes from pig gastric mucosa

The transverse distribution of the phospholipids in vesicular H+-translocating membranes prepared from pig gastric mucosa was investigated with the aid of phospholipase C, sphingomyelinase, and trinitrobenzenesulfonic acid. The major part (80-90%) of the phosphatidylcholine and the phosphatidylethanolamine, 60% of the phosphatidylserine, and 45% of the sphingomyelin was located on the external, cytoplasmic side of the vesicle membranes. After treatment with phospholipase C the vesicles still behaved as osmometers and appeared as closed vesicles on the electron micrographs. 31P NMR indicated that the phospholipids in untreated vesicles as well as the unhydrolyzed phospholipids in phospholipase C-treated vesicles were arranged in lamellar structures. The 31P NMR spectrum of untreated vesicles to which Pr3+ ions had been added supported the conclusion that the major part of the membrane phospholipids was located on the external surface of the vesicles. A small fraction of the lipids, 3.6 mol %, was found to consist of glycosphingolipids which occurred at a concentration of 52 nmol/mg of protein.     

Phosphorylation of membranes from the rat gastric mucosa

Gastric mucosal membranes derived primarily from parietal cells were found to contain endogenous protein kinase systems as well as several phosphate-accepting substrates. One specific membrane protein with a molecular weight of 88 000 was phosphorylated only in the presence of calcium, while the degree of phosphorylation of three other membrane proteins was similarly increased. The activity of the calcium-dependent protein kinase was found to be totally inhibited in the presence of trifluoperazine, a phenothiazine known to specifically inactivate calmodulin. These results suggest that a calmodulin- and calcium-dependent phosphorylation system may be a component of the parietal cell membrane. Phosphorylation of the membrane proteins was not affected by either cyclic AMP or cyclic GMP. The heat-stable inhibitor protein of cyclic AMP-dependent protein kinase did not inhibit the endogenous protein kinase activity suggesting that the membrane enzyme is not similar to the cytosolic protein kinase. However, the catalytic subunit of the soluble enzyme was capable of phosphorylating a number of membrane proteins indicating that after maximal autophosphorylation of the gastric membranes, phosphate-acceptor sites are still available to the cytosolic cyclic AMP-dependent protein kinase.     

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.     

Characterization of gastric mucosal membranes: lipid composition of purified gastric microsomes from pig, rabbit, and frog

The lipid compositions of the gradient-purified gastric microsomal membranes from the fundic mucosa of pig, rabbit, and frog were determined. The total lipid content varied widely. Compared to the rabbit (21.6 ± 0.6 mg/100 mg protein), the pig had about twice as much and the frog about three times as much lipid. The levels of cholesterol were higher in both mammalian species (about 32% of the lipid) compared to frog (23%). Phospholipids accounted for about 45, 54, and 52% of the total microsomal lipids from pig, rabbit, and frog and the molar ratios of cholesterol to phospholipid in the three species were 1.95, 1.6, and 1.17, respectively. Phosphatidyl choline and phosphatidyl ethanolamine together constituted about 75% of the total phospholipids in pig and frog and 93% in rabbit gastric microsomes. Sphingomyelin comprised 19.3, 3.2, and 1.5% in pig, rabbit, and frog, respectively. Phosphatidyl inositol constituted 5, 2.7, and 23.6% in pig, rabbit, and frog, respectively. The ratios of phosphatidyl ethanolamine to phosphatidyl choline were 1.17, 1.1, and 0.85 in pig, rabbit, and frog, respectively. The saturated fatty acids 16:0 and 18:0 and the unsaturated fatty acid 18:1 and 18:2 were the predominant fatty acids in all phospholipids. The ratios of saturated to unsaturated fatty acids were between 0.8 and 0.9 in phosphatidyl choline and 0.27 and 0.5 in phosphatidyl ethanolamine from all three species. The contributions by saturated fatty acids were much more in phosphatidyl inositol and sphingomyelin than in phosphatidyl choline and phosphatidyl ethanolamine from all species. Position 1 of phosphatidyl choline had 63% saturated and 37% unsaturated fatty acids; while the reverse was true for position 2. Phosphatidyl ethanolamine, however, had 85% saturated fatty acids in position 1 compared to only 25% in position 2. Arachidonic acid (20:4) was present in significant amounts in all species located exclusively at position 2 of both phosphatidyl choline and phosphatidyl ethanolamine.     

Characterization of mucus glycoprotein fatty acyltransferase from gastric mucosa

A fatty acyltransferase activity which catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to gastric mucus glycoprotein has been demonstrated in the rat gastric mucosa. Subcellular fractionation studies revealed that the enzyme activity was present in a Golgi-rich membrane fraction. Optimum enzymatic activity for acylation of mucus glycoprotein was obtained with 0.5% Triton X-100, 25 mM NaF, and 2 mM dithiothreitol at a pH of 7.4. The enzymatic activity increased proportionally, over a given range, with increased concentrations of both substrates and of enzyme. The apparent Km of the enzymes for the undegraded mucus glycoprotein was 4.5 X 10(-7) M and for palmitoyl-CoA, 3.8 X 10(-5) M. The 14C-labeled product of the reaction cochromatographed on Bio-Gel A-50 column and migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gastric mucus glycoprotein. Treatment of this 14C-labeled glycoprotein with mild alkali released hexane-extractable product which was identified as [14C]palmitate. The enzyme was also capable of fatty acylation of the deglycosylated glycoprotein, but did not catalyze the transfer of palmitic acid to the proteolytically degraded mucus glycoprotein. This indicates that the acceptor site for fatty acyltransferase is situated in the protease-susceptible nonglycosylated region of the mucus glycoprotein polymer.     

Characterization of three new fucolipids from hog gastric mucosa.

Three new fucolioids have been isolated from the water-soluble glycolipid fraction of hog gastric mucosa. Fucolipid A and C exhibited blood-group-A activity, whereas Fucolipid B was not active in A-anti-A, B-anti-B and H-anti-H systems. The structures of these glycolipids were identified by partial acid hydrolysis and methylation analysis, as: (see article)     

Characterization of oxyntic glands isolated from the rat gastric mucosa

A simple and reproducible method for isolating oxyntic glands from the rat gastric mucosa was developed. The mucosa was incubated with pronase and EGTA, and then treated mechanically to release glands that were separated from single cells by sedimentation. Parietal cells were identified by immunostaining using a monoclonal antibody against H,K-ATPase. The glandular cells appeared morphologically intact. By careful control of the conditions of gland isolation, long glandular structures comprising hundreds of cells surrounding the lumen were obtained. Intraperitoneal injection of Br-deoxyuridine in the rat 1.5 h before the isolation procedure resulted in glands with a labeling of cells in their neck region. The glands were viable, as demonstrated by their ability to respond to various hormones. Histamine dose-dependently stimulated the acid formation which was measured as the accumulation of [14C]aminopyrine. At 100 microM histamine the accumulation was increased 5-10-fold. At 100 nM, pentagastrin potentiated the histamine stimulated accumulation by approximately 40% but pentagastrin alone did not stimulate. The oxyntic glands obtained by the present procedure appear useful for studies on cell physiology, including regulation of acid secretion, cellular interactions, and possibly also differentiation and proliferation mechanisms since long glandular fragments that contained the proliferative zone could be isolated.     

Characterization of the proton-transporting photocycle of pharaonis halorhodopsin

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.     

Progesterone metabolism in the gastric mucosa microsomes of guinea pig

The microsomes from guinea pig gastric mucosa were found to convert [4-14C]progesterone to two major metabolites in the presence of NADPH. The gastric metabolizing activity was the highest among the gastrointestinal tissues of guinea pig. 5 alpha-Pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one were identified as the major metabolites by thin-layer chromatography and crystallization to constant specific activity, suggesting the presence of steroid 5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase activities in the gastric mucosa microsomes. Furthermore, time course of progesterone metabolism and analysis of 5 alpha-pregnane-3,20-dione metabolites suggest that the gastric progesterone metabolism is initiated by 5 alpha-reductase and followed by 3 beta-hydroxysteroid dehydrogenase. The progesterone-metabolizing activity was strongly inhibited by SKF 525-A and disulfiram. The activity was also inhibited by methyrapone to a somewhat lesser extent than the above inhibitors. From gastric mucosa microsomes, the progesterone-metabolizing activity was successfully solubilized with 2% digitonin using 0.1 M potassium chloride and 1 mM dithiothreitol, 0.4 mM NADPH and 20% glycerol as stabilizers for the solubilized activity. Among these stabilizers, glycerol was found to be most effective for stabilizing the activity of the solubilized microsomes.     

Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa

Sulfated glycoproteins were extracted and purified from porcine stomach mucous scraping. Four sulfated glycoprotein fractions were separated and subsequently purified. These compounds always accompanied the apparent peptic inhibitory activity and consisted of 15–18% (w/w) protein. The carbohydrate portions contained an equimolar ratio of galactose and hexosamine (mainly glucosamine), together with lesser amounts of fucose and sialic acid. The sulfate content of the above fractions was 2–9% (w/w) of the total sulfated glycoprotein.The mode of inhibition of the sulfated glycoproteins to peptic activity was investigated and suggested that there was binding of the sulfated glycoproteins to the substrate of pepsin, making the substrate resistant to peptic activity. The sulfated glycoproteins neither bound pepsin at pH 1.8 nor inhibited the hydrolysis of a synthetic dipeptide substrate of pepsin. Desulfation of the sulfated glycoproteins resulted in the loss of both the inhibitory activity and the precipitate formation. The precipitation curve for sulfated glycoprotein and porcine serum albumin showed that both bound in varying proportions and suggests that both components are multivalent in this precipitate formation.     

The A2B adenosine receptor colocalizes with adenosine deaminase in resting parietal cells from gastric mucosa

The A2B adenosine receptor (A2BR) mediates biological responses to extracellular adenosine in a wide variety of cell types. Adenosine deaminase (ADA) can degrade adenosine and bind extracellularly to adenosine receptors. Adenosine modulates chloride secretion in gastric glands and gastric mucosa parietal cells. A close functional link between surface A2BR and ADA has been found on cells of the immune system, but whether this occurs in the gastrointestinal tract is unknown. The goal of this study was to determine whether A2BR and ADA are coexpressed at the plasma membrane of the acid-secreting gastric mucosa parietal cells. We used isolated gastric parietal cells after purification by centrifugal elutriation. The membrane fraction was obtained by sucrose gradient centrifugation. A2BR mRNA expression was analyzed by RT-PCR. The surface expression of A2BR and ADA proteins was evaluated by Western blotting, flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are expressed in cell membranes isolated from gastric parietal cells. They show a high degree of colocalization that is particularly evident in the surface of contact between parietal cells. The confocal microscopy data together with flow cytometry analysis suggest a tight association between A2BR and ADA that might be specifically linked to glandular secretory function.     

Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa.

Sulfated glycoproteins were extracted and purified from porcine stomach mucous scraping. Four sulfated glycoprotein fractions were separated and subsequently purified. These compounds always accompanied the apparent peptic inhibitory activity and consisted of 15-18% (w/w) protein. The carbohydrate portions contained an equimolar ratio of galactose and hexosamine (mainly glucosamine), together with lesser amounts of fucose and sialic acid. The sulfate content of the above fractions was 2-9% (w/w) of the total sulfated glycoprotein. The mode of inhibition of the sulfated glycoproteins to peptic activity was investigated and suggested that there was binding of the sulfated glycoproteins to the substrate of pepsin, making the substrate resistant to peptic activity. The sulfated glycoproteins neither bound pepsin at pH 1.8 nor inhibited the hydrolysis of a synthetic dipeptide substrate of pepsin. Desulfation of the sulfated glycoproteins resulted in the loss of both the inhibitory activity and the precipitate formation. The precipitation curve for sulfated glycoprotein and porcine serum albumin showed that both bound in varying proportions and suggests that both components are multivalent in this precipitate formation.     

Subcellular distribution of ionic components in gastric mucosa of the guinea pig

Summary The topographical distribution of cations, anions and polyanions in the guinea-pig stomach has been studied by ultrastructural cytochemical methods. After fixation with the pyroantimonate-osmium tetroxide solution, variable-sized precipitates were localized in the basolateral extracellular space bordering parietal cells or chief cells but not in that bordering mucus-secreting cells. The basal lamina of all gastric cells disclosed a continuous layer of heavy antimonate deposits. Parietal cells disclosed uniformly fine deposits also on the apical plasmalemma both at the main lumen and in the intracellular canaliculi, and revealed, as well, coarse precipitates in the mitochondria. Fixation with a silver acetate-osmium tetroxide solution yielded nitric acid-resistant, silver deposits confined to the luminal surface of the apical plasmalemma in the main lumen and intracellular canaliculi, the lateral intercellular space, the outer surface of the basal plasmalemma and the basal lamina of the parietal cell.Staining with dialyzed iron demonstrated a glycocalyx rich in acid mucosubstance on the basolateral plasmalemma but not on the apical plasmalemma of parietal cells. In contrast, acid glycoconjugate was visualized on the apical plasmalemma of isthmus cells, mucous neck cells and the transitional cell between isthmus and mucous neck cells but little or no acidic glycoconjugate was demonstrated on the basolateral plasmalemma of these cells. The entire plasmalemma of gastroendocrine cells, unlike other epithelial cells, stained uniformly for acidic glycoconjugate. The dialyzed iron and high iron diamine methods stained the outer compartment of mitochondria in parietal cells intensely and that in other gastric cells lightly. These reagents stained the basal lamina of all gastric cells as did ruthenium red. The several characteristic cytochemical properties of parietal cells presumably relate to the unique secretory activity of these cells and are consistent with the view of the intracellular canaliculi of the parietal cell as the main route for hydrogen and chloride ion secretion.