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S Mårdh

Publications and source records attributed to S Mårdh.

At least 55 records · Page 3Linked to original sources
PubMed1990

A continuous-flow technique for analysis of stoichiometry and transport kinetics of gastric H,K-ATPase.

A continuous-flow method was developed for determining the stoichiometry of the gastric proton pump H,K-ATPase (EC 3.6.1.36) in its hydrolysis of ATP and translocation of H+ and the K+ congener 86Rb+. H,K-ATPase-containing vesicles which had been isolated from pig gastric mucosa were incubated at 37 degrees C for 2 h in 150 mM 86RbCl, 0.5 mM ethylenebis(oxyethylenenitrilo)tetra-acetic acid and 3 mM 2-(N-morpholino)ethane sulphonic acid (Mes) adjusted to pH 6.1 with Tris, and then applied onto a 0.45 micron pore size cellulose acetate filter. The immobilized vesicles were superfused with 0.15 mM Mes/Tris buffer, pH 6.1, containing 150 mM choline chloride and 0.2 mM MgCl2. After changing to a medium containing 0.1 mM ATP, the amounts and rates of H+ uptake, 86Rb+ efflux and ATP hydrolysis were measured. The initial ratio of Rb+ transported to ATP hydrolysed gave values of 0.96 +/- 0.26 (mean +/- SD, n = 28). The initial ratio of ATP-dependent Rb+ efflux to H+ uptake gave values of 0.92 +/- 0.28 (mean +/- SD, n = 28). The Mg-ATPase activity was measured in vesicles which had been incubated with choline chloride instead of RbCl. This activity was 15.8 +/- 8.7% (mean +/- SD) of the total ATPase activity in the initial fractions used for calculation of the stoichiometry. It is argued that this Mg-ATPase may be an intrinsic activity of the H,K-ATPase and that the relation between these activities is dependent on the amount of K+ (or Rb+) present in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1989

Parietal cell antibodies in pernicious anemia inhibit H+, K+-adenosine triphosphatase, the proton pump of the stomach.

Antibodies to a membrane-bound antigen, localized to the canalicular structures of the parietal cell, are found in most sera of patients with chronic atrophic gastritis and pernicious anemia. In the present study immunoglobulins containing parietal cell antibodies were found to inhibit the activity of H+,K+-adenosine triphosphatase (EC 3.6.1.36) in a tubulovesicular membrane preparation from porcine gastric mucosa. The degree of inhibition correlated to the titer of parietal cell antibodies as assessed by an enzyme-linked immunosorbent assay. The specificity of the enzymatic inhibition was confirmed by the lack of effect of parietal cell antibodies on membrane-bound esterase. A possible interaction of parietal cell antibodies with gastrin binding at the receptor level was investigated in a radioreceptor assay employing 125I-gastrin 1 and gastric mucosal cell suspension from the guinea pig. No blocking capacity was found with immunoglobulins from patients with pernicious anemia as compared with immunoglobulins from healthy controls. The results thus demonstrate a direct inhibitory effect of parietal cell antibodies on the acid producing H+,K+-adenosine triphosphatase of the parietal cell, but also a lack of interaction with the gastrin receptor, and indicate that in the development of hypo/achylia H+,K+-adenosine triphosphatase autoantibodies could have a major pathogenic role.

Adenosine Triphosphatases↗
PubMed1989

Failure to deduce a peptide inhibitor of Na,K-ATPase from the gene coding for the catalytic alpha-subunit of Na,K-ATPase.

It has been proposed (Bost et al. 1985, Blalock & Bost 1986, Smith et al. 1987) that the DNA strand opposite the one coding, for example, for a hormone or a neurotransmitter codes for its receptor protein. This proposal was tested with regard to the possibility of deducing a peptide inhibitor of Na,K-ATPase from the known gene of the catalytic alpha-subunit of the enzyme. Three such peptides, deduced from presumed extracellular domains of the catalytic alpha-subunit, were tested in vitro and in vivo with regard to their effects on Na,K-ATPase activity. No effect on the enzyme could be demonstrated.

4-Nitrophenylphosphatase↗
PubMed1989

The effects of various gastrins on intracellular free Ca2+ in isolated pig parietal cells.

Gastrin 17 (G17) is a potent stimulant of gastric acid secretion in vivo. In this study, the effects of G17 and some related peptides on intracellular free Ca2+ in isolated pig parietal cells were studied. Both G17 and the synthetic peptide pentagastrin increased intracellular free Ca2+ in a dose-dependent manner over the concentration range 10(-9) to 10(-6) M, suggesting a specific action. The EC50 values were 3 X 10(-8) M for G17 and 8 X 10(-8) M for pentagastrin. The N-terminal tridecapeptide of G17 [(1-13)G17] did not have any effect on intracellular free Ca2+, nor was it able to inhibit the action of G17. A glycine-extended gastrin [(5-17)G17-Gly)] elicited a small but significant increase in intracellular free Ca2+ although only at 10(-6) M. This increase was approximately 20% of that obtained with a similar concentration of G17. Sequential incubations with (5-17)G17-Gly and G17 showed that both peptides increased the intracellular free Ca2+ through the same mechanisms.

Animals↗
PubMed1989

Characterization of antigenic structures in auto-immune atrophic gastritis with pernicious anaemia. The parietal cell H,K-ATPase and the chief cell pepsinogen are the two major antigens.

Using isolated cells and subcellular fractions from pig gastric mucosa, antigenic structures with specific binding of IgG from sera of patients with auto-immune atrophic gastritis were characterized by means of immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting experiments using mucosal cells as the antigen source, two dominating bands of 94 and 41 kDa were found. The two major antigens were identified as the H,K-ATPase (94 kDa), which constitutes the parietal cell acid pump, and pepsinogen (41 kDa) located in the chief cells. There was also a small but significant binding of antibodies to a preparation of Na,K-ATPase, an enzyme which is about 60% homologous to H,K-ATPase. Commercial preparations of hog gastric pepsinogen and pepsin bound pernicious anaemia IgG with equal efficacy. When sera from seven patients with the diagnosis pernicious anaemia were tested, all were found to contain auto-antibodies against H,K-ATPase as well as pepsinogen. In intact, isolated H,K-ATPase-containing vesicles the cytosolic part of the ATPase molecule is facing the outside of the vesicles. Both intact and trypsinized vesicles were incubated with patient sera and with a monoclonal antibody against H,K-ATPase. Pernicious anaemia IgG was found to bind to a cytosolic, trypsin-resistant structure, but the binding of the monoclonal antibody was lost upon trypsinization. The present results indicate that intracellular structures of the gastric mucosa, due to cell damage, may be exposed to immune-competent cells, which do not recognize these structures as 'self'.

Adenosine Triphosphatases↗
PubMed1989

The occurrence of gastric and duodenal auto-antibodies in peptic ulcer disease.

The possible relationship between peptic ulcer and the occurrence of auto-antibodies was investigated by means of an enzyme-linked immunosorbent assay (ELISA). Sera from 24 patients with active duodenal ulcer were analysed using cells and subcellular fractions from pig gastric acid duodenal mucosa for binding of immunoglobulins. Four sera (17%) reacted with a homogenate from duodenal mucosa. Nine sera (38%) were found to contain auto-antibodies against gastric mucosal cells. The cell-reactive auto-antibodies were shown to bind preferentially to parietal cells and chief cells. In these cells the antigens were identified as H, K-ATPase and pepsinogen respectively. Six sera were positive against purified H,K-ATPase; 12 sera were positive against pepsinogen, and only one of these sera reacted with both H,K-ATPase and pepsinogen. The results show that auto-antibodies are formed in a large fraction of patients (18/24; 75%) with peptic ulcer disease. The present study further demonstrates that enrichment of antigenic structures is required for obtaining a satisfactory sensitivity in the assay.

Adenosine Triphosphatases↗
PubMed1989

The effect of arachidonic acid and its metabolites on acid production in isolated human parietal cells.

The effect of arachidonic acid and its metabolites on the histamine-stimulated acid production in human isolated parietal cells provenient from endoscopic biopsies was examined. 14C-aminopyrine (14C-AP) accumulation in the parietal cells was used for evaluation of acid production. Histamine dose-dependently increased AP uptake. Histamine stimulation (taken as 100% at 10(-5) M) was significantly inhibited by prostaglandin (PG) E2 to 66 +/- 7% at 10(-8) M, 42 +/- 8% at 10(-6) M, and 13 +/- 10% at 10(-4) M (mean +/- SEM, n = 10). PGF2 alpha, PGD2, and PGI2 showed significant inhibitory effects only at very high concentrations (10(-5)-10(-4) M). Leukotriene (LT) B4 and LTC4 were without effect. The basal acid production (taken as 0%) was lowered significantly by 10(-6) M arachidonic acid to -20 +/- 7.4% (p less than 0.02, n = 10), and the histamine-stimulated (10(-6) M) acid production from 100% to 64 +/- 7.2% (p less than 0.001, n = 10). Aspirin (10(-3) M) increased basal (45 +/- 9.6%, p less than 0.001, n = 10) and histamine-stimulated (10(-6) M) acid production (164 +/- 16.3%, p less than 0.001). It is concluded that PGE2, the major product from arachidonic acid metabolism in the human gastric mucosa, is a significant inhibitor of the histamine-stimulated human parietal cell and may, in humans, play a role as a local physiologic inhibitor of acid secretion.

Adult↗
PubMed1988

Inhibition of H,K-ATPase and Na,K-ATPase by DIDS, a disulphonic stilbene derivative.

Disulphonic stilbenes are effective inhibitors of an anion exchanger which is present in the plasma membranes of many cells (Cabantchik et al. 1978). In the present study, the effects of 4,4'-diisothiocyano-2,2'-disulphonic stilbene acid (DIDS) on the transport activity of the hydrochloric acid pump isolated from pig stomach (H,K-ATPase, EC 3.6.1.36) were tested. Half-maximal inhibition of proton transport carried out by the H,K-ATPase in the isolated vesicles was observed at micromolar concentrations of DIDS. The effects of DIDS on the adenosine-trisphosphatase and p-nitrophenyl-phosphatase activities of isolated H,K-ATPase were also studied and compared with those of the kinetically and structurally related Na,K-ATPase (EC 3.6.1.37). Half-maximal inhibition of the enzymatic activities of both enzymes were observed in the micromolar range of DIDS. The lipid bilayer of the gastric vesicle membrane is highly asymmetric and the original cytosolic side is facing the outside of the vesicle. Since DIDS does not readily cross the membrane, it is most likely that DIDS exerts its inhibitory effects by modifying the transport ATPases on their cytosolic sides.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Major parietal cell antigen in autoimmune gastritis with pernicious anemia is the acid-producing H+,K+-adenosine triphosphatase of the stomach.

In autoimmune gastritis antibodies against a membrane-bound parietal cell antigen of previously unknown function are present in the sera of patients. In this study, a vesicular membrane preparation of porcine gastric mucosa cells was found to be a potent antigenic source. This preparation blocked greater than 90% of antibody binding to a lysate of gastric mucosa cells. The membrane fraction contained H+,K+-ATPase (EC 3.6.1.36) as the major protein, which in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migrated with a weight of 92 kD. After reduction and alkylation, this component was resolved into two bands of similar staining intensity (92 and 88 kD). Immunoblotting analysis showed that sera of patients recognized antigen with pattern identical to the major protein of the vesicular membranes. Protein A-Sepharose beads preincubated with immunoglobulins of five individual patient (but not control) sera were all found to reduce both the H+,K+-ATPase activity and the amount of parietal cell antigen of a preparation of vesicular membranes solubilized in n-octylglucoside. Taken together, the results of this study indicate that the major parietal cell antigen is identical to the acid-producing enzyme, H+,K+-ATPase, of the parietal cell.

Adenosine Triphosphatases↗
PubMed1988

Effects of some antisecretory drugs on acid production, intracellular free Ca2+, and cyclic AMP production in isolated pig parietal cells.

The effects of some inhibitors of acid secretion were tested on isolated, purified pig parietal cells. The cells were stimulated with 10(-4) M histamine, 10(-5) M carbachol, or 10(-7) M pentagastrin. The H,K-ATPase inhibitors SCH 28080 and omeprazole inhibited both the basal and secretagogue-stimulated acid production, as measured by aminopyrine accumulation, irrespective of the type of stimulator used. The IC50 value was 3-5 x 10(-9) M for SCH 28080 and 1-3 x 10(-8) M for omeprazole. Ranitidine inhibited the histamine-stimulated but not the basal acid production. The IC50 value was 2 x 10(-5) M. Stimulation of acid production with carbachol was blocked by pirenzepine, with an IC50 of 6 x 10(-7) M. Pirenzepine (10(-5) M) specifically blocked the carbachol-stimulated increase in cytosolic free Ca2+ in fura-2-loaded cells but not the increase in cytosolic free Ca2+ induced by histamine or pentagastrin. Ranitidine (10(-4) M) prevented the histamine-induced increase in Ca2+ and was also the only one of the four inhibitors which prevented the histamine-stimulated cAMP formation. SCH 28080 (10(-5) M) significantly potentiated the histamine-stimulated increase in cytosolic free Ca2+ and the formation of cAMP, whereas omeprazole (10(-5) M) was without effect.

Aminopyrine↗
PubMed1988

Adrenaline stimulates acid production in isolated pig and human parietal cells.

To investigate the mechanisms of adrenergic stimulation of the parietal cell and to study the possible relationship between the stress hormone adrenaline and duodenal ulcer, the effects of adrenaline and various adrenoceptor agonists and antagonists were investigated in parietal cells isolated from pig stomachs and from endoscopic biopsy specimens taken from the gastric mucosa of patients. Parietal cell acid production was assayed by the aminopyrine accumulation technique. Adrenaline as the sole drug showed poor or no stimulatory effect but potentiated histamine-stimulated acid production. In the presence of histamine, beta-adrenoceptor agonists caused a stimulation of acid formation with the potency order isoproterenol greater than adrenaline greater than noradrenaline. The beta-2-selective antagonist ICI118551 was a more potent inhibitor of acid production than both the beta-1 antagonist practolol and the H2-receptor antagonist cimetidine. Studies of (3H)-dihydroalprenolol (DHA) binding to purified parietal cell membranes showed a protein-concentration-dependent and specific binding of 2.2 +/- 0.6 pmol DHA/microgram. Adrenaline increased gastric acid production in both pig and human parietal cells, most likely through a beta-2 receptor on the parietal cell. The adrenaline stimulatory effect in cells obtained from patients with peptic ulcer was more pronounced than in cells from non-ulcer patients, which indicates a possible role of adrenaline in some types of ulcer disease.

Adrenergic beta-Agonists↗
PubMed1987

Evidence for the existence of an endogenous inhibitor of Na, K-ATPase in plasma from cats and rats.

Plasma samples from cats and rats were filtered through molecular filters to obtain a concentrate of the plasma molecules with a mass between 500 and 10,000. This concentrate was placed on a gel filtration column and eluted with a Tris buffer. Fractions corresponding to a molecular mass of 1500-2000 exhibited a marked Na,K-ATPase inhibitory 'activity', which was not influenced by heating to 95 degrees C for 10 min or by enzymatic degradation by pronase or trypsin.

Animals↗
PubMed1987

Mechanisms of stimulation of acid production in parietal cells isolated from the pig gastric mucosa.

Sequential incubations with pronase and collagenase of pig gastric mucosa resulted in single cell preparations containing 10-20% parietal cells, which could be enriched further to 85-95% purity by density-gradient centrifugation followed by elutriation. Acid production of the isolated cells was measured by means of aminopyrine accumulation in their acid compartments. When small pieces of the mucosa were pretreated for 1 h in the presence of either histamine, pentagastrin or carbachol before preparation of cells, the ability of the subsequently isolated cells to produce acid was increased. In parietal cells isolated from resting (not pretreated) mucosa pentagastrin, carbachol and also adrenaline increased the histamine-stimulated aminopyrine accumulation (50-90% increase). Adrenaline alone had no significant effect on the aminopyrine accumulation. In the presence of 10(-4) M histamine the apparent EC50 for adrenaline was 5 X 10(-7) M. Adrenaline, histamine, forskolin and isobutylmethylxanthin (IBMX) increased the formation of cAMP in purified parietal cells. The three 'classical' secretagogues histamine, pentagastrin and carbachol, but also IBMX and forskolin, increased the cytosolic free Ca2+ from approximately 1.5 X 10(-7) M to 2.2-3.5 X 10(-7) M but adrenaline and dibutyryl cyclic AMP did not. Thus the present results indicate that there are - in addition to histaminergic H2 receptors - specific cholinergic, gastrinergic and adrenergic receptors on the plasma membrane and that there are separate cAMP and Ca2+-dependent stimulatory pathways in the parietal cell.

8,11,14-Eicosatrienoic Acid↗
PubMed1987

Enzyme-linked immunosorbent assay of H+,K+-ATPase, the parietal cell antigen.

Vesicular membranes, purified from porcine gastric mucosa and rich in H+,K+-ATPase, were used to establish an enzyme-linked immunosorbent assay (ELISA) for determinations of parietal cell autoantibodies. Results obtained with the ELISA correlated well with standard immunofluorescence determinations of parietal cell antibodies based on frozen sections of rat stomach. The ELISA however was about 10-fold more sensitive than the immunofluorescence method and had high specificity. Intra- and interassay coefficients of variation, determined with a patient sera of average positivity, were 5.5% and 18%, respectively. The ELISA detected antibody binding in 23 out of 26 sera from patients with known autoimmune atrophic gastritis, in five of 25 sera with autoimmune thyroiditis, in five of 20 sera from patients with Graves' disease, in three out of 20 sera from patients with atoxic nodular goitre, in six of 20 sera of patients with primary biliary cirrhosis, in two out of 20 sera of patients with active duodenal ulcer, in two out of 20 sera with detectable antinuclear antibodies, and in one out of 20 sera with detectable rheumatoid factor. Data determined by an ELISA based on a gastric vesicular membrane preparation of human origin correlated well (r = 0.79, P less than 0.001) to those obtained by the standard ELISA based on porcine membrane material. The assay should be well suited for routine determinations of parietal cell antibodies in investigations of autoimmune gastritis and multiple organ autoimmune endocrinopathies.

Adenosine Triphosphatases↗
PubMed1986

Stimulation of acid formation by histamine, carbachol and pentagastrin in isolated pig parietal cells.

Free cells were obtained by sequential incubations of pig gastric mucosa with pronase and collagenase. Approximately 10-15% of the cell population represented parietal cells. Accumulation of aminopyrine (AP) in the acid compartments of parietal cells was used as an index of their acid production. Histamine, carbachol and pentagastrin each independently stimulated aminopyrine accumulation. The initial rate of aminopyrine accumulation, observed after addition of 10(-4) M carbachol or 10(-6) M pentagastrin, were 32% and 10%, respectively, of that observed with 10(-4) M histamine. Steady-state aminopyrine accumulation in the presence of 10(-4) M histamine, 10(-4) M carbachol or 10(-6) M pentagastrin were 6.2 +/- 3.3, 2.6 +/- 0.6 and 3.0 +/- 1.5 pmol AP per 10(4) parietal cells, respectively (mean +/- SD, n = 5). The EC50 value for histamine was 3.4 +/- 1.4 X 10(-7) M, and for pentagastrin 5.9 +/- 4.2 X 10(-8) M (mean +/- SD, n = 5). The dose-response curve for carbachol was biphasic. A plateau was reached at 10(-5)-10(-4) M carbachol, and for this phase an apparent EC50 of 2.1 +/- 1.4 X 10(-6) M carbachol was calculated (mean +/- SD, n = 5). A further increase to 10(-3) M carbachol increased the aminopyrine accumulation. Atropine (10(-6) M) inhibited the response to concentrations up to 10(-4) M carbachol, but was without effect on the histamine- and pentagastrin-stimulation. The H2-receptor antagonist, cimetidine, right-shifted the dose--response curve for histamine. Also, the pentagastrin-stimulated aminopyrine accumulation was inhibited by cimetidine.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1985

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.

Adenosine Triphosphatases↗
PubMed1985

Kinetics of the acid pump in the stomach. Proton transport and hydrolysis of ATP and p-nitrophenyl phosphate by the gastric H,K-ATPase.

Hydrolysis of adenosine 5'-triphosphate (ATP) and p-nitrophenyl phosphate by the hydrogen ion-transporting potassium-stimulated adenosine triphosphatase (H,K-ATPase) was investigated. Hydrolysis of ATP was studied at pH 7.4 in vesicles treated with the ionophore nigericin. The kinetic analysis showed negative cooperativity with one high affinity (Km1 = 3 microM) and one low affinity (Km2 = 208 microM) site for ATP. The rate of hydrolysis decreased at 2000 microM ATP indicating a third site for ATP. When the pH was decreased to 6.5 the experimental results followed Michaelis-Menten enzyme kinetics with one low affinity site (Km = 116 microM). Higher concentrations than 750 microM ATP were inhibitory. Proton transport was measured as accumulation of acridine orange in vesicles equilibrated with 150 mM KCl. The transport at various concentrations of ATP in the pH interval from 6.0 to 8.0 correlated well with the Hill equation with a Hill coefficient between 1.5-1.9. The concentration of ATP resulting in half-maximal transport rate (S0.5) increased from 5 microM at pH 6.0 to 420 microM at pH 8.0. At acidic pH the rate of proton transport decreased at 1000 microM ATP. The K+-stimulated p-nitrophenylphosphatase (pNPPase) activity resulted in a Hill coefficient close to 2 indicating cooperative binding of substrate. The pNPPase was noncompetitively inhibited by ATP and ADP; half-maximal inhibition was obtained at 2 and 100 microM, respectively. Phospholipase C-treated vesicles lost 80% of the pNPPase activity, but the Hill coefficient did not change. These kinetic results are used for a further development of the reaction scheme of the H,K-ATPase.

Adenosine Triphosphatases↗