Molecular characterization of a purified human gastric somatostatin receptor.
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Humans↗
Biomedical subjects
M J Lewin
Publications and source records attributed to M J Lewin.
[Plasma pharmacokinetics of roxatidine in the healthy man: correlation with gastric antisecretory effect].
Inhibitory effects of roxatidine acetate upon human gastric secretion have been shown to be dose-related after oral administration. In order to correlate this effect with drug pharmacokinetics, blood concentration of the active metabolite, roxatidine, was assessed in 10 healthy volunteers, each receiving in random order 75, 150, 300, 600 mg of roxatidine acetate and placebo. Blood was drawn for roxatidine measurement at T 0 (time of drug intake) T 75, 90, 120, 150, 210, 240 and 300 min. Meal-induced gastric secretion was studied by intragastric titration (IGT) at 15 min intervals, from T 150 to T 240. Mean peak of blood concentration occurred at T 150 with significant correlation with the administered dosage. Gastric secretion inhibition over the 90 min of IGT correlated with peak concentration exclusively for the subpopulation with peak occurrence at T 150. Beyond the peak, decrease in roxatidine concentrations was slow, as expected from a known 6-h half-life. However, overall results showed that mean blood concentration was correlated with gastric secretion inhibition from T 180 on. The last IGT sample also correlated with the peak. Interpretation of these results would be different whether roxatidine concentration variations are appreciated as exhibiting a significant decrease or not. Infusion studies of roxatidine would be a valuable technique for further analysis and comparison of blood-concentration/efficacy relationships as compared with other H2-antagonists.
Adult↗
A monoclonal antibody which inhibits H+/K(+)-ATPase activity but not chloride conductance.
A mouse monoclonal antibody was raised against hog gastric membranes. This antibody (95-111 mAb) has a very high affinity for the 95 kDalton band of H+/K(+)-ATPase-enriched membranes, and does not react with Na+/K(+)-ATPase. The epitope is located on the tubulovesicles and canaliculi of the parietal cells. The 95-111 mAb also inhibits the ATP hydrolytic activity, decreases the steady-state phosphorylation level and inhibits the phosphatase activity of H+/K(+)-ATPase, strongly suggesting that the epitope is on the catalytic subunit of H+/K(+)-ATPase. The 95-111 mAb also recognizes rat, rabbit and human gastric H+/K(+)-ATPase. This mAb differs from the H+/K(+)-ATPase-inhibiting mAb previously described (Asano et al. (1987) J. Biol. Chem. 262, 13263-13268), in that it does not inhibit the chloride conductance opened by Cu-o-phenanthroline in gastric vesicles.
Adenosine Triphosphatases↗
Solubilization and immunopurification of a somatostatin receptor from the human gastric tumoral cell line HGT-1.
The human gastric tumoral cell line HGT-1 was previously shown to contain a membrane somatostatin receptor negatively coupled to adenylate cyclase through a pertussis toxin-sensitive inhibitory GTP-binding regulatory protein (Gi) (Reyl-Desmars, F., Laboisse, C., and Lewin, M. J. M. (1986) Regul. Pept. 16, 207-215). In this study, we have solubilized this receptor in a free unoccupied form using Triton X-100 as detergent and [125I-Tyr11]somatostatin-14 to monitor specific binding. Furthermore, we have prepared a monoclonal antibody against a chromatographically enriched soluble receptor fraction and used this antibody (30F3) to immunopurify the receptor in conjunction with Sepharose-somatostatin-14 immunopurification and steric exclusion high pressure liquid chromatography (HPLC). The purified fraction showed 18,600-fold enrichment in terms of specific binding (i.e. from 0.6 +/- 0.05 to 11,300 +/- 830 pmol/mg of protein) and a single dissociation constant (kappa D) of 76 +/- 8 nM. On HPLC, it migrated as a single and symmetric 90-kDa peak. Moreover, after 125I-protein labeling, it gave a single 90-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. On the other hand, the 30F3 monoclonal antibody immunoblotted with a single 90-kDa band contained in the HGT-1 cell membrane. We therefore suggest that this antibody is specific to the HGT-1 membrane somatostatin receptor, that this receptor has a molecular mass of 90 kDa, and that we have obtained a homogeneous preparation of nondenatured receptor suitable for further cloning studies.
Antibodies, Monoclonal↗
Gastric endocrine cell behavior in Zollinger-Ellison patients upon long-term potent antisecretory treatment.
Serum gastrin and gastric endocrine cell numerical densities were examined in 22 patients with long-standing Zollinger-Ellison syndrome who were receiving either ranitidine, omeprazole, or other antisecretory drugs (SMS 201-995 or pirenzepine with or without ranitidine) for long periods of time. Fifteen patients had iterative biopsies. Twenty-one subjects with normal endoscopy, serum gastrin, and acid secretion served as controls. Individual fundic argyrophil cell density was above the highest control value in 77% of the patients, whatever the treatment. Argyrophil cell densities tended to be higher in women than in men. During the survey, fundic carcinoids developed in one ranitidine- and in one omeprazole-treated patient. Fundic argyrophil cell densities were correlated with serum gastrin levels (r' = 0.730, p less than 0.001). Antral somatostatin cell density was not modified in any patients as compared with controls, nor was antral gastrin cell density except in omeprazole-treated patients. In these patients, gastrin cell density and gastrin to somatostatin cell ratio were significantly higher than in all other patients or controls. Such increases may indicate true gastrin cell hyperplasia in relation to drug-induced profound acid inhibition.
Adult↗
Cell isolation techniques: use of enzymes and chelators.
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Animals↗
Histamine release in the isolated vascularly perfused stomach of the rat: regulation by autoreceptors.
1. In the isolated vascularly-perfused stomach of the rat, gastrin 1-17 (520 pmol 1(-1)) increased acid output from basal values of 13.7 +/- 2.7 to 92.5 +/- 11.4 mumol h-1 and venous histamine output from 10.1 +/- 2.3 to 54.7 +/- 7.9 nmol h-1 (mean +/- s.e.mean). 2. The H1 receptor agonist 2-methylhistamine (10 mumol 1(-1)) increased acid output to 21.6 +/- 2.9 mumol h-1 (P less than 0.05) and reduced basal histamine output to 4.0 +/- 0.8 nmol h-1 (P less than 0.05). Gastrin-stimulated acid secretion and vascular histamine output was not significantly affected by 2-methylhistamine (10 mumol 1(-1)). 3. The H2 receptor agonist, impromidine, dose-dependently increased basal acid secretion, reaching a maximal value of 145.5 +/- 11.7 mumol h-1 with impromidine (10 mumol 1(-1)), and maximal gastrin-stimulated acid secretion to 167.4 +/- 15.1 mumol h-1 with impromidine (10 mumol 1(-1)). Impromidine dose-dependently inhibited basal and gastrin-stimulated vascular histamine output. 4. The H3 receptor agonist R-a-methylhistamine, (1 and 10 mumol 1(-1)) minimally increased basal acid secretion. R-a-methylhistamine (10 mumol 1(-1)) did not significantly affect maximal gastrin-stimulated acid secretion. Basal and gastrin-stimulated vascular histamine outputs decreased to 4.0 +/- 0.8 (P less than 0.05) and 24.7 +/- 4.7 nmol h-1 (P = 0.05) with R-a-methylhistamine (10 mumol 1(-1)). 5. The H2 receptor antagonist ranitidine (2 mumol 1(-1)) did not inhibit basal acid secretion, but acid outputs with gastrin and all histamine agonists were reduced. Ranitidine did not affect histamine release in the basal state, with gastrin or with any histamine agonist tested. 6 We conclude that gastric histamine release in the rat is regulated via a histamine H2 receptor sensitive to the histamine agonists tested, but not to ranitidine. It is unlikely that the inhibition of histamine release is secondary to increased gastric acidity.
Animals↗
Effects of bombesin on food intake and gastric acid secretion in cats.
The brain and gut peptide bombesin has been reported both to stimulate gastric secretion and to induce satiety. To understand how the peripheral administration of bombesin affects food intake and whether gastric mechanisms are involved, a comparative study of the doses of bombesin active on gastric secretion, gastric emptying, and food intake was undertaken in cats provided with a gastric fistula and a denervated Heidenhain pouch. The smallest dose of intravenous bombesin that stimulated significantly basal acid secretion (20 pmol.kg-1.h-1) by the gastric fistula also enhanced meal-stimulated acid secretion by the Heidenhain pouch (+138%, P less than 0.01), delayed gastric emptying of a liquid protein meal (-30%, P less than 0.01), and suppressed food intake when the test meal was allowed to reach the stomach (-15%, P less than 0.01). Conversely, in sham-feeding experiments, the same dose of bombesin increased food intake (+35%, P less than 0.01). In full-day experiments conducted in nonfasted cats, bombesin decreased both the food intake in the 4-h period after the infusion and the daily food intake, whereas octapeptide cholecystokinin induced a transient satiety but did not decrease daily food intake. These results indicate that in cats the interaction of bombesin with "pregastric" mechanisms is not sufficient to induce satiety and that a relation could exist between the effects of bombesin on gastric secretion, emptying, and food intake. A single class of receptors might be involved in these peripheral effects of bombesin.
Animals↗
Gastrin processing in primary culture of gastrinoma cells.
There is a general agreement on the cell specificity of gastrin processing. In order to investigate this processing in Zollinger-Ellison (ZE) patients, we have studied in two primary gastrinoma cultures (one from a pancreatic tumor, the other from a liver metastasis) the proportion of progastrin fragments using immunochemical and immunohistological methods. In tumor extracts as well as in sera, the predominant gastrin form differed between the two patients (i.e. being G17 and G34, respectively). In the two gastrinoma cultures, RIA determinations and electron microscopic observations indicated that the proportion of progastrin increased with time while that of G17 and G34 decreased. On the other hand, as the culture time extended, an increasing proportion of nonimmunostained secretory granules was observed suggesting the presence of other gastrin precursors (e.g. Gly-extended progastrin). From these findings, we suggest that gastrinoma culture cells could be a valuable tool in the biochemical approach to gastrin processing in ZE tumors.
Cells, Cultured↗
Immunohistochemical characterization of gastrinomas with antibodies specific to different fragments of progastrin.
The characterization of the tumors and their metastasis in patients with the Zollinger-Ellison syndrome is currently based on the immunohistochemical identification of gastrin cells. However, sometimes tumoral cells fail to react with common C-terminal gastrin antibodies. In order to clarify this failure, we carried out morphologic, morphometric and immunocytochemical analyses performed on light and electron microscope levels of 6 pancreatic and 1 metastatic gastrinomas, using antibodies raised against various sequences of human progastrin. On the basis, in light microscopy, of qualitative analysis of immunostaining within cells and of immunostained cell numbers, gastrin 34 residue seemed to be the prominent form in 2 of the tumor tissues, G-17 in 1 tumor which was not responsive with C terminus progastrin and N terminus G-34 antisera, and progastrin in the metastatic tissue that did not contain typical gastrin (G-like) cells. Two tumors failed to react with all antisera used. At the electron microscope level, immunogold staining revealed that progastrin was present only in the progranules and gastrin 34 in both progranules and intermediate granules. Quantitative studies performed on 3 tumors showed that, within a given tumoral cell, about 25 percent of progranules contained progastrin while 75 percent contained gastrin 34. We concluded that different forms of gastrin can be immunodetected in a gastrinoma tissue, depending on the regions, and that the distribution of progastrin fragments is variable from tumor to tumor. So, specific antibodies to different fragments of progastrin may help to the characterization of gastrinomas.
Antibodies↗
[Purification to apparent homogeneity of the somatostatin receptor of the human cell line HGT-1 of gastric origin].
This communication reports the isolation and the purification of the gastric somatostatin receptor from the human cell line HGT-1. The receptor has been extracted from the cell membrane by Triton X 100, and a monoclonal antibody to this was prepared. A series of affinity chromatographies (Sepharose-antibody and Sepharose-somatostatin-14) and a final purification by steric exclusion on high performance liquid chromatography columns (HPLC) allowed us to obtain a fraction enriched 20,000 fold in 125I-Tyrll-somatostatin-14 specific binding (apparent dissociation constant: 7.6 x 10(-8) M). This fraction corresponded to a molecular mass of about 90 kDa (in presence of detergent) and to a maximal binding capacity of more than 10,000 pmol/protein. It therefore has a theoretical homogeneity close to 100%.
Cell Membrane↗
[Relation of the kinetics of the antisecretory effect and kinetics of blood concentration in man. Comparing cimetidine and ranitidine].
The time course of antisecretory effects of ranitidine and cimetidine and their relationship to plasma concentration were studied in 4 healthy volunteers. Placebo, cimetidine (266 mg) or ranitidine (100 mg) were infused during one hour preceding the 90 minute assessment of food-stimulated gastric acid secretion (intragastric titration); cimetidine and ranitidine blood levels and gastric acid outputs were measured at 15 min intervals. For each test a significant (p less than or equal to 0.01) correlation was found between concentration and effect. The half-life effect of ranitidine (t1/2 E) was significantly (p less than or equal to 0.05) greater than t1/2 E of cimetidine whereas their plasma half-lives (t1/2) were similar. The ratio t1/2 E/t1/2 was significantly (p less than or equal to 0.05) lower with cimetidine than with ranitidine. Explanations for this discrepancy are analyzed according to various hypotheses. The determination of t1/2 E seems to be a good approach to the duration of action of H2-antagonists. Our results show that the ratio t1/2 E/t1/2 and the t1/2 E might be important pharmacokinetic parameters to compare various H2-receptor antagonists.
Adult↗
Cholecystokinin suppresses food intake in cats: structure-activity characterization.
Our experimental models in this study were cats fitted with gastric fistulae. Intravenous infusion of sulfated CCK 8 and nonsulfated Boc CCK 7 inhibited both sham-feeding and feeding in fasted cats. The threshold dose (1.2 pmol/kg.hr) required for inhibition of sham-feeding was identical to that required to inhibit feeding in the same animals. However, the gastric secretory studies indicated that this dose was 90 times lower than the threshold dose stimulating gastric acid secretion (109 pmol/kg.hr). In nonfasted animals, sulfated CCK 8 and nonsulfated Boc CCK 7 (219 and 875 pmol/kg.hr) are both capable of decreasing the food intake at different intervals following the infusion with no significant effect on daily food intake. Our findings clearly show that there is no difference in the sensitivity of CCK's ability to inhibit sham-feeding and feeding, suggesting that CCK's suppressive effect on food intake does not solely involve gastric distension mechanisms. In contrast to gastric acid secretion, the sulfate group is not a "restrictive" factor for peripherally-induced CCK satiety.
Animals↗
The effects of intravenously administered bombesin on pentagastrin-stimulated acid secretion in cats.
The effects of bombesin (BBS) infusion or BBS injection on the plateau gastric secretion stimulated by pentagastrin (Pg) were compared in cats fitted with gastric fistula (GF) and Heidenhain pouch (HP). Injection of 81 pmol/kg of BBS inhibited Pg-stimulated acid secretion in both GF and HP by 47 +/- 5% and 37 +/- 5% (P less than 0.01), respectively. Infusion of 324 pmol/kg.h of BBS did not significantly modify acid secretion, but as soon as the infusion stopped, an inhibition appeared which lasted 1 h (37 +/- 5% in GF and 53 +/- 4% in HP P less than 0.01). The inhibition was reversed in GF by infusion of BBS 324 pmol/kg.h. In HP, reversion of inhibition required the addition in the Pg infusion of subthreshold dose of carbachol. We suggest that under non-steady state conditions (i.e. injection or after the end of the infusion) a concentration gradient of BBS is created which favors the response of D-cells over that of G-cells, whereas under steady-state conditions (i.e. during infusion) the effects of BBS on G- and D-cells are balanced. This finding argues for a physiological role of BBS in the regulation of gastric acid secretion.
Animals↗
Gastrin secretion by gastrinoma cells in long-term culture.
Gastrinoma cells from surgical specimens of a primary pancreatic tumor and an hepatic metastasis in two patients with a Zollinger-Ellison syndrome were grown and subcultured for 7 mo. Cultured cells displayed a strong reactivity to heptadecapeptide gastrin antibody and maintained an ultrastructural appearance resembling that of the original tumor cells with the presence of secretory granules of variable size and electron density. Cultured cells also showed the ability to secrete immunoreactive gastrin, and this secretion was further concentration-dependently stimulated by secretin (10(-10)-10(-6) M), carbachol (10(-6) M), and bombesin (10(-10)-10(-6) M). The latter peptide was the more potent stimulant with a maximal effect at 10(-9) M (460 +/- 20% of basal release; P less than 0.05). This stimulation occurred in the absence of extracellular Ca2+ and was potentiated by the addition of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; 10(-3) M) into the culture medium. The somatostatin analogue, somatostatin-(201-995), did not alter basal gastrin release but inhibited secretin, carbachol, and bombesin stimulation. Moreover, DBcAMP (10(-3) M) and Ca2+ (1-3 mM) stimulated gastrin release; Ca2+ ionophore A23187 (6 micrograms/ml) enhanced gastrin response to Ca2+ in the early time intervals of incubation. Furthermore the phorbol ester derivative, 12-O-tetradecanoyl phorbol-13-acetate, dramatically stimulated gastrin release (10 times the basal value). We conclude that gastrinoma cells can be cultured over an extended period with maintenance of their capacity to secrete gastrin in response to various hormones and mediators.(ABSTRACT TRUNCATED AT 250 WORDS)
Bombesin↗
A somatostatin receptor negatively coupled to adenylate cyclase in the human gastric cell line HGT-1.
Somatostatin receptors are demonstrated in the human derived gastric cell line HGT-1. Using 125I-Tyr11-somatostatin as ligand, two classes of sites were characterized with apparent dissociation constants KD1 = 0.9 X 10(-10) M and KD2 = 4 X 10(-9) M and maximum binding capacities of N1 = 20 and N2 = 556 fmol per mg protein, respectively. These values are close to those previously reported in freshly isolated parietal cells (Reyl, F., Silve, C. and Lewin, M.J.M., Somatostatin receptors on isolated gastric cells. In S. Bonfils et al. (Eds.), Hormone Receptors in Digestion and Nutrition, Elsevier/North-Holland, Amsterdam, 1979, pp. 391-400). Somatostatin binding to the high affinity sites was partially inhibited by the non-hydrolysable guanyl nucleotide analog Gpp(NH)p and by pretreating the cells with islet activating protein (IAP). Furthermore, IAP counteracted the inhibitory effect of somatostatin on histamine stimulation of adenylate cyclase. These findings are interpreted in terms of somatostatin interaction with the 41,000 Da adenylate cyclase GTP-dependent inhibitory subunit, Ni.
Adenylate Cyclase Toxin↗
Thiophosphorylation of hog gastric (H+ + K+)-ATPase membranes by endogenous protein kinases.
(H+ + K+)-ATPase-enriched membranes from hog stomachs were tested for their capacity to autophosphorylate using [gamma-32P]ATP or [gamma-35S]ATP[S] as phosphate donors. The radioactive polypeptides were characterized by SDS-PAGE. In the presence of Mg2+ and 5 microM [gamma-32P]ATP, rapid and transient incorporation of 32P occurred at 0 degrees C. Radioactivity was essentially found in the major polypeptide of the material, the 95 kDa subunit of (H+ + K+)-ATPase. Under the same experimental conditions, thiophosphorylation was slower and reached a plateau within 1 h. Incorporation levels were higher with manganese than with magnesium. After one hour at 0 degrees C, and in the presence of 10 mM manganese and 5 microM ATP[S], 0.58 +/- 0.06 nmoles of thiophosphate were incorporated per mg of protein. Twenty seven percent of the thiophosphorylated amino acids were acylphosphates i.e. likely to be the ATPase thiophosphointermediate. The remaining thiophosphorylated amino acids (73%) were thought to be produced by protein kinases. This was supported by the autoradiographies of membrane SDS-PAGE which indicated that, in addition to the 95 kDa ATPase subunit, other polypeptides were thiophosphorylated especially at 108, 58, 47, 45 and 36-40 kDa. A previous study had provided strong evidence that chloride transport in gastric microsomes, is modulated by a protein kinase-dependent phosphorylation (Soumarmon, A., Abastado, M., Bonfils, S. and Lewin M.J.M. (1980) J. Biol. Chem. 255, 11682-11687). In the present work, we demonstrate that the peptidic inhibitor of cAMP-dependent protein kinases decreased thiophosphorylation of a 45 kDa polypeptide. We suggest that this polypeptide could be regarded as a candidate for the role of chloride transporter or chloride transport regulator.
Animals↗
Depolymerization of solubilized gastric (H+ + K+)-ATPase by n-octylglucoside or cholate.
We have previously shown that an active (H+ + K+)-ATPase can be extracted from gastric apical membranes using n-octylglucoside (Soumarmon, A., Grelac, F. and Lewin, M.J.M. (1983) Biochim. Biophys. Acta 732, 579-585). This extract contained an holomeric enzyme of 390-420 kDa and contained 68% of the K+-stimulated ATPase specific activity originally present. We demonstrate here that inactivation, induced during a more classically designed protocol, is associated with the appearance of smaller, polymorphic structures with molecular mass of 330-360 and 240-250 kDa estimated using molecular sieve chromatography and glycerol gradients. This suggests that (H+ + K+)-ATPase solubilization by n-octylglucoside is a complex process involving first extraction of the enzyme as an active polymer, with subsequent depolymerication and inactivation of this polymer. Depolymerization was specifically studied by treating the large holomeric n-octylglucoside-extracted (H+ + K+)-ATPase with increasing concentrations of either n-octylglucoside or cholate. Detergent-induced changes were characterized by centrifugation on glycerol gradients. Progressive displacement of ATPase activity into three different peaks at 32%, 26% and 20% glycerol was found with increasing detergent concentrations. n-Octylglucoside inhibited enzyme activities and was more deleterious for phosphatase than for ATPase activity. Moreover, it induced the dissociation of phosphatase and ATPase distribution profiles. At concentrations of 0.2 to 1.15%, cholate induced the displacement of the glycerol gradient profiles but no loss of activities and no dissociation of phosphatase and ATPase profiles. Higher concentrations of this detergent (2.5%) also inactivated the ATPase concomitantly with the appearance of a protein peak with no related activity at 16-18% glycerol. From this study we suggest that solubilization of gastric (H+ + K+)-ATPase can be achieved through the extraction of a polymer by n-octylglucoside and through subsequent depolymerization using cholate. We suggest that the different sizes correspond to monomers, dimers, trimers and perhaps tetramers. The monomers were apparently inactive under present test conditions.
4-Nitrophenylphosphatase↗