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M J Lewin

Publications and source records attributed to M J Lewin.

At least 91 records · Page 5Linked to original sources
PubMed1986

H+ transport by reconstituted gastric (H+ + K+)-ATPase.

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).

Adenosine Triphosphatases↗
PubMed1986

Highly histamine-responsive clones from the human gastric adenocarcinoma cell line HGT-1.

The human gastric epithelial cell line HGT-1 possesses adenylate cyclase-coupled histamine H2 receptors. To test the cellular homogeneity or heterogeneity with respect to these receptors, we have isolated 7 clones from the HGT-1 line and studied their basal and histamine-stimulated adenylate cyclase activities. Basal adenylate cyclase activities of the clones did not differ significantly, nor did 10 mM NaF- nor 0.1 mM Gpp(NH)p-stimulated activities. However, histamine stimulation of adenylate cyclase varied among clones from 1.9 fold to 5.4 fold basal activity. The EC50 values, determined in 3 clones, were not significantly different. These findings support the heterogeneity of histamine responsiveness of the human gastric cell line HGT-1. In addition, they suggest that highly histamine-responsive clones may be useful models to study the gastric histamine H2-receptor and its specific antagonists in the human.

Adenocarcinoma↗
PubMed1986

Growth hormone-releasing factor (somatocrinin) stimulates epithelial cell proliferation in the rat digestive tract.

The possible influence of growth hormone-releasing factor (GHRF) on epithelial cell proliferation in the digestive tract was investigated. Fasted young rats received five hourly subcutaneous injections of either GHRF or saline. They were killed 6, 12, or 18 h after the initial injection and 45 min after [3H]thymidine pulse labeling. At the time of death, blood was taken to determine circulating growth hormone and gastrin levels. After radioautography, DNA synthetic and mitotic activities were estimated in the fundic, antral, duodenal, jejunal, and colonic mucosae. Growth hormone-releasing factor significantly increased labeling indices 6, 12, and 18 h after the initial injection in fundic mucosa, and 6 and 18 h after injection in antral and duodenal mucosae. Furthermore, GHRF significantly increased mitotic indices at 12 h in fundic mucosa and at 12 and 18 h in jejunal mucosa. No effect was seen in the colon. At the three checkpoint times, circulating growth hormone showed no change, but plasma gastrin was increased in the rats treated with GHRF as compared with controls. However, whether the reported stimulatory effect of the GHRF on target cells is direct or indirect remains to be determined.

Animals↗
PubMed1986

Somatocrinin stimulates adenylate cyclase-Ns regulatory subunit in a GH3 cell-line: comparison with VIP.

We used a GH3 cell-line to compare the effects of rat GRF (rGRF) and VIP on the adenylate cyclase activity and to determine on what subunit the site of action of these two peptides is. In the GH3 cell-line, VIP was more potent than rGRF to stimulate adenylate cyclase activity. The stimulatory effects of rGRF and forskolin were additive. Cholera toxin decreased the apparent potency of these peptides and pertussis toxin reversed the inhibition by somatostatin of their adenylate cyclase stimulation. We conclude that rGRF acts on the regulatory subunit Ns, different from the regulatory subunit Ni on which somatostatin is suggested to be acting and that, in the GH3 cells, rGRF stimulates adenylate cyclase through VIP-preferring sites.

Adenylyl Cyclases↗
PubMed1986

Growth hormone releasing hormone stimulates the release of gastrin in rat.

The inhibitory effect of somatostatin on gastrin release is well known. Could the growth hormone releasing hormone (GHRH), an antagonist of somatostatin on growth hormone (GH) release, have a gastrin-releasing effect on gastrin? To answer this question, two types of experiments were conducted: (1) The study of the effect of GHRH on gastrin in rats, which showed a significant and dose related release for the three doses studied: 0.12, 0.6 and 3.0 micrograms/rat. (2) The study on the recurrence of this gastrin releasing effect which has been found to be significant (1 to 5 injections at 1 hour intervals). Our data suggest that GHRH is an hypothalamic releasing factor for gastrin release. Accordingly, gastrin may mediate the observed proliferative effect of GHRH in the upper digestive tract.

Animals↗
PubMed1986

Gastric (H+,K+)-ATPase.

Gastric acid secretion results from the activity of a specific ATPase, the (H+,K+)-ATPase. This enzyme, discovered in 1973, exchanges H+ for K+. It has two ATP binding sites, both involved in enzyme activity, whose affinities vary as a function of the H+ and K+ concentrations. Hydrolysis of ATP at the highest affinity site leads to the synthesis of a covalent aspartyl phosphate which accumulates in the absence of K+. The presence of this cation accelerates dephosphorylation resulting in the stimulation of ATPase (and PNPPase) activity. The structure of membranous (H+,K+)-ATPase is poorly defined. n-Octylglucoside solubilizes an active enzyme of 390-420 kDa which can be partly depolymerized using cholate. The monomer, characterized in SDS has a 95 kDa molecular mass and is inactive. In the presence of magnesium, (H+,K+)-ATPase catalyzes the active and neutral exchange of H+ for K+ at the expense of ATP. In the absence of ATP, (H+,K+)-ATPase acts as a passive transporter exchanging K+ for K+ at maximal rate and H+ for K+ at a 20 times slower rate.

Adenosine Triphosphatases↗
PubMed1986

Somatostatin receptors.

More insight into the biochemical structure and operation of the somatostatin receptor(s) has been gained in recent years from several approaches. The minimal active structure of the receptor(s) has been identified, and active minisomatostatins have been synthesized. High-affinity binding sites (KDS ranging from 0.1 to 1 nM) have been demonstrated in brain and peripheral organs. In pancreas, stomach, and intestine additional low-affinity sites (or states) have been also suggested Furthermore, cytosolic receptors might be present. Binding affinities of synthetic minisomatostatins, somatostatin-14 and somatostatin-28, show different tissue specificities, suggesting the existence of different receptor subtypes. Two possible interactions of somatostatin with stimulus-secretion coupling in secretory cells have been suggested: a direct activation of the GTP-dependent inhibitory subunit of adenylate cyclase and a distal activation of cytosolic phosphoprotein phosphatases.

Adenylyl Cyclases↗
PubMed1985

Gastric microsomal NADH-cytochrome b5 reductase: characterization and solubilization.

NADH-cytochrome b5 reductase from hog gastric microsomes was studied with respect to substrate dependence, optimum pH, thermal denaturation as well as anti-cytochrome b5 antibodies and different ions. The reduction of potassium ferricyanide by the enzyme was specific for NADH. Using potassium ferricyanide or trypsin-solubilized liver cytochrome b5 (Tb5) as substrates, enzyme activity was inhibited by ADP and to a lesser extent by ATP. Tb5- (but not ferricyanide-) reductase was activated by ionic strength up to 0.05 ion equivalent per liter and inhibited at higher strengths whatever the ion used (Cl-, Na+, Ca2+, Mg2+). Enzyme solubilization occurred with Triton X100. The solubilization increased the Tb5- (but not the ferricyanide-) reductase activity up to a Triton:protein ratio of 15. We therefore suggest that gastric microsomes contain a Triton soluble membrane-bound NADH cytochrome b5 reductase which is in many respects similar to the liver and red cell enzymes.

Animals↗
PubMed1984

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase.

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.

Adenosine Triphosphatases↗
PubMed1984

Inhibition of the histamine-stimulated adenylate cyclase activity of guinea pig gastric cells by the H2-receptor antagonists cimetidine, oxmetidine and SKF 93479.

The effect of cimetidine and two new histamine H2-receptor antagonists, oxmetidine and SKF 93479, on histamine-stimulated adenylate cyclase activity was studied in guinea pig gastric mucosal cells. Histamine stimulated the enzyme activity in concentration-dependent fashion. The concentration-response curve of histamine was progressively shifted to the right in the presence of increasing concentrations of each antagonist. The Schild plot gave a straight line for all three compounds, with a slope not significantly different from unity and this suggested a competitive antagonism. The calculated pA2 values were 8.45 +/- 0.20, 7.73 +/- 0.21 and 6.81 +/- 0.15 for SKF 93479, oxmetidine and cimetidine, respectively. These results are in accordance with the pharmacological potencies of the antagonists reported on isolated heart preparation and on gastric secretion in vivo. Therefore, the inhibition of histamine-sensitive adenylate cyclase of gastric cells may represent an additional tool for the in vitro evaluation of the H2-receptor antagonists.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Solubilization of active (H+ + K+)-ATPase from gastric membrane.

(H+ + K+)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg2+-ATPase and p-nitrophenylphosphatase activities (68 +/- 9 mumol Pi and 2.9 +/- 0.6 mumol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K+ (up to 0.69 +/- 0.09 nmol Pi incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1-2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 X g X 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K+-stimulated ATPase and p-nitrophenylphosphatase activities and K+-sensitive phosphorylation: Mg2+-ATPase (mumol Pi/mg protein per h) 32 +/- 9 (basal) and 86 +/- 20 (K+-stimulated); Mg2+-p-nitrophenylphosphatase (mumol p-nitrophenol/mg protein per h) 2.6 +/- 0.5 (basal) and 22.2 +/- 3.2 (K+-stimulated); Mg2+-phosphorylation (nmol Pi/mg protein) 0.214 +/- 0.041 (basal) and 0.057 +/- 0.004 (in the presence of K+). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H+ + K+)-ATPase in a still active structure.

Adenosine Triphosphatases↗
PubMed1983

Cytochrome b5-dependent delta 9 desaturation of fatty acids in gastric microsomes.

Stearyl-CoA was shown to stimulate the reoxidation rate of cytochrome b5 of gastric microsomes and to decrease the reduction rate of trypsin-purified hog liver cytochrome b5 by the NADH-cytochrome b5 reductase of these microsomes. This latter effect was (1) proportional to microsome concentration and to stearyl-CoA concentration with an apparent Km of 3.3 . 10(-6) M and a Vmax of 71 nmol per min and per mg microsomal protein, (2) insensitive to ATP and inhibited by 1.4 mM KCN, (3) mimicked by palmityl-CoA but not by stearic nor palmitic acid. Direct assays carried out using [14C]stearyl- and [14C]palmityl-CoA as substrates showed a production of 0.12 nmol of oleic and palmitoleic acid, respectively, per min per mg of microsomal protein. In the presence of Tb5 antibodies the reaction was inhibited by 40%. These results support the occurrence of cytochrome b5-dependent fatty acid delta 9 desaturation in gastric microsomes.

Acyl Coenzyme A↗
PubMed1983

Somatocrinin receptor coupled with cAMP-dependent protein kinase on anterior pituitary granules.

The molecular mechanism of growth hormone release by synthetic somatocrinin was investigated on purified hog anterior pituitary secretory granules; the granules were found to contain a cAMP-dependent protein kinase that catalyzed [gamma-32P]-ATP histone phosphorylation with maximal rates ranging from 1 to 5 nmol of Pi incorporated per mg of protein per 20 min. The activity of this enzyme was further stimulated by somatocrinin. Stimulation was observed at concentrations as low as 0.3 pM, and the half-maximal effect was obtained with 35 +/- 8 pM (n = 4). Michaelis-Menten analysis of phosphorylation kinetics suggested that the peptide did not change significantly the reaction's Vmax, but produced a dramatic increase in enzyme affinity for cAMP: the apparent Km for the nucleotide decreased from 400 X 10(-9) M under unstimulated conditions to 15 X 10(-9) M in the presence of 100 pM somatocrinin. Furthermore, a Hill plot of concentration-dependence curve indicated the existence of negative cooperativity. At the concentration of 35 pM, the less potent analogs of somatocrinin [designated hpGRF-44 to indicate source (human pancreas, hp), activity (growth hormone-releasing factor, GRF), and amino acid composition], hpGRF-(1-37) and [Phe1]hpGRF-(1-40) had 20% and 7%, respectively, of the effect of somatocrinin. The biologically inactive analog hpGRF-(2-40) had no evident effect at concentrations up to 0.1 microM. Therefore, we suggest that somatocrinin stimulation of growth hormone release involves activation of exocytosis through a phosphorylation mechanism mediated by a granular receptor coupled with a cAMP-dependent protein kinase.

Animals↗
PubMed1983

[Increase by somatocrinin of a cyclic AMP-dependent protein kinase activity in adenohypophyseal, secretory granules in the hog].

This study shows that the growth hormone hypothalamic releasing factor (somatocrinin) stimulates the activity of cyclic AMP-dependent protein kinase associated with a purified fraction of hog anterior pituitary secretory granules. Threshold, half-maximal and maximal concentrations (0.3, 35 pM and 10 nM, respectively, in the presence of 50 nM cyclic AMP) are similar to those observed for in vitro stimulation of growth hormone release by somatocrinin.

Animals↗