PepT1-mediated epithelial transport of dipeptides and cephalexin is enhanced by luminal leptin in the small intestine.

Dietary proteins are mostly absorbed as di- and tripeptides by the intestinal proton-dependent transporter PepT1. We have examined the effects of leptin on PepT1 function in rat jejunum and in monolayers of the human enterocyte-like 2 cell Caco-2. Leptin is produced by the stomach and secreted in the gut lumen. We show here that PepT1 and leptin receptors are expressed in Caco-2 and rat intestinal mucosal cells. Apical (but not basolateral) leptin increased Caco-2 cell transport of cephalexin (CFX) and glycylsarcosine (Gly-Sar), an effect that was associated with increased Gly-Sar uptake, increased membrane PepT1 protein, decreased intracellular PepT1 content, and no change in PepT1 mRNA levels. The maximal velocity (Vmax) for Gly-Sar transport was significantly increased by leptin, whereas the apparent Michaelis-Menten constant (Km) did not change. Furthermore, leptin-stimulated Gly-Sar transport was completely suppressed by colchicine, which disrupts cellular translocation of proteins to plasma membranes. Intrajejunal leptin also induced a rapid twofold increase in plasma CFX after jejunal perfusion with CFX in the rat, indicating enhanced intestinal absorption of CFX. These data revealed an unexpected action of gastric leptin in controlling ingestion of dietary proteins.

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.

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.

Specific activation of cyclic AMP-dependent protein kinase(s) by H2-histamine agonists in isolated gastric mucosal cells from guinea-pig.

Histamine stimulated cyclic AMP-dependent protein kinase activity in dispersed mucosal cells from guinea-pig gastric fundus (Ka = 5 microM). The H2-agonists dimaprit and impromidine produced similar effects, while the H1-agonist 2-(2-pyridyl) ethylamine had only a weak one. The H2-antagonist cimetidine competitively inhibited 0.1 mM histamine stimulation (Ki = 2 microM). In contrast, the H1-antagonist diphenhydramine had no effect up to 1 mM.

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.

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.

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).

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.

Antibody epitope mapping of the gastric H+/K(+)-ATPase.

Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes. Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells. Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit. It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain. The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region. The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions. This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions. The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments.

Human colon carcinoma cell-line HCT116 transfected by antisense cDNA as a tool to study the Ku86 involvement in cell proliferation.

In this work, we used colon carcinoma cell-line HCT116 to study the involvement of the 86-kDa subunit (Ku86) of DNA-protein kinase (DNA-PK) in human tumoural cell proliferation. We transfected these cells with a 639-bp cDNA encoding a Ku86 portion inserted into pcDNA3.1 vector in the antisense orientation. After selection by neomycin, we obtained more than 300 resistant colonies. In the Y'A5 colony that we chose as total population, we showed by PCR and RT-PCR that pcDNA3/Ku86 antisense was integrated in genomic DNA and that transcript was present. After cloning, we selected two clones, A20 and A23, which contained significatively reduced level of Ku86 protein. These two clones displayed a reduced DNA-PK activity from 44% to 71% and a slower growth than control cells. These results suggest that the HCT116 cell-line is a useful tool to investigate the role of Ku86 in the regulation of human tumoural cell growth.

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.

Detection of gastrin mRNA in human antral mucosa and digestive endocrine tumors by in situ hybridization: a correlative study with immunocytochemistry and electron microscopy.

In gastrinomas, as well as in other endocrine tumors whose hormone overproduction is responsible for clinical syndromes, antibodies against the bioactive form(s) of hormones can fail to detect immunoreactivity. Moreover, tumor secretory granule morphology may fail to allow tumor type identification. The use of anti-pre-pro-gastrin antibodies has been proposed as an alternative to identify gastrinomas. The aim of the present study was to demonstrate that in situ detection of gastrin mRNA may represent another possibility. A 35S-labeled cDNA probe encoding the human gastrin pre-pro-hormone was used to localize gastrin gene transcripts in antral mucosa and digestive endocrine tumors from patients with a Zollinger-Ellison syndrome characterized by high serum gastrin levels. In situ hybridization was combined with light and electron microscopic immunostaining of the bioactive gastrin 17/34 form and morphological study of secretory granules. Gastrin mRNAs were detected in antral gastrin cells and in a variable proportion of tumor cells in all endocrine tumor studied. Transcript expression correlated well with immunohistochemical staining and granule ultrastructure for most of the tumors, and provided crucial evidence for identifying as gastrinomas two tumors with weak immunoreactivity and poorly granulated cells. Our data show that in situ hybridization is a sensitive method for gastrin mRNA detection and represents a valuable tool for the identification of gastrinomas.

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.

Gastric leptin.

The recent discovery of gastric leptin has initiated several investigations on the possible role of leptin in digestive physiology. The following clues are currently suggested: leptin might control meal size in cooperation with Cholecystokinin, help cytoprotection of the gastric mucosa, play a role in gut inflammatory processes, regulate secretion of gastric hormones such as gastrin and somatostatin, and modulate intestinal transport of small peptides. The present review is a brief survey of the most significant advances in these issues.

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.

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.

Endogenous opioid peptides in the control of food intake in cats.

In this report, we investigated the role of exogenous and endogenous enkephalins on food intake in the cat, using, respectively, exogenous [D-Ala2-Met5]-enkephalin (DAME) and acetorphan (Ac) in order to inhibit the degradation of endogenous enkephalins. In addition, the selective peripheral antagonist naltrexone methylbromide (NTxMB) and the nonselective antagonist naloxone (Nx) were used in an attempt to discriminate central and peripheral opioid receptors. In 18-hours food-deprived animals, Ac (5 mg/kg IV) increased milk intake during sham feeding (+18%, p less than 0.05), but did not modify it in feeding conditions. Nx (1 mg/kg SC) reduced milk intake in sham-feeding experiments (-67%, p less than 0.01) more than in milk-feeding conditions (-30%, p less than 0.01). NTxMB (1 mg/kg SC) did not modify milk intake in sham-feeding but decreased it in feeding experiments. In nonfasted animals, Ac did not modify food intake. IV infusion of DAME (50 micrograms/kg) resulted in a reduction of daily food intake (-32%, p less than 0.01). Nx (1 mg/kg SC) decreased the earlier 30 min intake followed by reduction of daily intake (-30%, p less than 0.01). NTxMB (1 and 4 mg/kg SC) increased the 30-min intake dose dependently, without significant change in daily intake. In conclusion, Ac increases food intake in sham-feeding conditions, suggesting that endogenous enkephalins are likely to be involved in the stimulation of food intake. The effects of Nx and NTxMB furthermore suggest both a central activation, and a peripheral inhibition of food intake by opiates when food is allowed to proceed normally through the digestive tract.

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.

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.