Characterization of a functional NTPDase in the endoplasmic reticulum of rat submandibular salivary gland.

Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca(2+) pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with a NTPDase identity. The presence of a NTPDase was demonstrated by immunohistochemistry in rat SMG. Immunoreactivity was stronger in ductal cells than in mucous and serous acini. Although this enzyme was observed in the plasma membrane, colocalization with the ER marker calnexin revealed a specific subcellular localization in this organelle of all three types of cell. The putative function of this NTPDase activity in salivary glands is discussed.

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands.

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.

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.

Expression and regulation of leptin receptor proteins in afferent and efferent neurons of the vagus nerve.

Leptin, the product of the ob gene, plays a key role in the regulation of food intake via a cross-talk between hypothalamic leptin receptors and neuropeptides that affect feeding behaviour. Recent studies have shown a synergistic interaction between leptin and cholecystokinin (CCK) leading to suppression of food intake, which involves CCK-1 receptors and capsaicin-sensitive vagal fibres. In this study, we have investigated the presence of leptin receptors in afferent and efferent neurons of the vagus nerve. By using reverse transcription-polymerase chain reaction, mRNAs encoding long (Ob-Rb) and short (Ob-Ra) leptin receptor isoforms were detected in the rat nodose ganglion, which contains the cell bodies of the vagal afferent neurons. Western blot analysis confirmed the presence of leptin receptor-immunoreactive proteins in extracts from the vagal trunk. Immunohistochemistry showed the presence of leptin receptors and the leptin-induced transcription factor STAT3 in the cytoplasm of nodose ganglion cells. In cervical vagal segments, levels of leptin receptor protein displayed physiological regulation, with decreased amounts after feeding and increased levels after food restriction. In addition, leptin receptor and STAT3 immunoreactivities were detected in neurons of the nucleus of tractus solitarius (NTS) and the dorsal motor nucleus of the vagus nerve (DMNX) by immunofluorescence histochemistry. Furthermore, direct double-labelling demonstrated colocalization of Ob-Rb and STAT3 immunoreactivities in cholinergic vagal efferent cell bodies of the DMNX. It is speculated that vagal leptin receptors, apart from being activated by adipocyte-derived leptin, may also be influenced by leptin produced by the stomach. This may explain the synergistic action of leptin and CCK on neuronal activity in the NTS and on food intake.

Structural and functional study of reconstituted peripheral benzodiazepine receptor.

Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His. Bind metal chelation resin. Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein. The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol.

Is aggregation of beta-amyloid peptides a mis-functioning of a current interaction process?

In a previous study, Hughes et al. [Proc. Natl. Acad. Sci. USA 93 (1996) 2065-2070] demonstrated that the amyloid peptide is able to interact with itself in a two-hybrid system and that interaction is specific. They further supported that the method could be used to define the sequences that might be important in nucleation-dependent aggregation. The sequence of the amyloid peptide can be split into four clusters, two hydrophilic (1-16 and 22-28) and two hydrophobic (17-21 and 29-42). We designed by molecular modeling and tested by the two-hybrid approach, series of mutations spread all over the sequence and changing the distribution of hydrophobicity and/or the spatial hindrance. In the two-hybrid assay, interaction of native Abeta is reproduced. Screening of mutations demonstrates that the C-domain (residues 29-40 (42)), the median domain (residues 17-22) and the N-domain (1-16) are all crucial for interaction. This demonstrates that almost all fragments of the amyloid peptide but a loop (residues 23-28) and the C-term amino acid are important for the native interaction. We support that the folded three-dimensional (3D) structure is the Abeta-Abeta interacting species, that the whole sequence is involved in that 3D fold which has a low secondary structure propensity and a high susceptibility to mutations and thus should have a low stability. The native fold of Abeta could be stabilized in Abeta-Abeta complexes which could in other circumstances facilitate the nucleation event of aggregation that leads to the formation of stable senile plaques.

Prediction of epitopes and production of monoclonal antibodies against gastric H,K-ATPase.

Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.

Messenger RNA expression of somatostatin receptor subtypes in human and rat gastric mucosae.

In several tissues including gastric mucosa, somatostatin displays various biological effects. Five seven-transmembrane-domain somatostatin receptor subtypes (SSTR1-5) have been recently cloned and only SSTR1 has been shown to be present in the human stomach. We used the polymerase chain reaction on reverse transcripts (RT-PCR) to characterize further the SSTR's mRNAs in human and rat gastric mucosae and in the human gastric tumoral cell-line HGTL. The SSTR1-5's mRNAs were found in both human fundic and antral mucosae as well as in the HGT1 cell and rat antrum. The four SSTR2-5's mRNA's but not SSTR1's were detected in the rat fundic mucosa. Furthermore, the use of rat isolated and purified fundic mucosal cells allowed us to localize SSTR2-5 in the parietal cell-enriched fraction, whereas SSTR2 and SSTR5 were the only subtypes found in the endocrine cell-enriched fraction. These results are the first to demonstrate the presence of five SSTR's mRNA subtypes in the stomach.

Statistical evidence for two major proteins in freeze-fractured gastric parietal cell tubulovesicles and canaliculus.

In a previous work, resting and acid-secreting rabbit gastric mucosa were freeze-fractured and shadowed at 45 degrees with Pt-C. The shadow widths of proteic particles of tubulovesicle and canaliculus membranes were measured and compared. It was concluded that the frequency distributions of widths are significantly different in resting and secreting membranes and that each distribution accounts for several subpopulations of homogeneous particles. In the present study, an attempt is made to describe the experimental distributions as a mixture of those of two major proteins, say A and B and their aggregates (AA, AB and BB). The modelling, although simple, gave a very satisfactory statistical fit between observed and computed distributions. The comparison of parameters calculated from histamine and ranitidine experimental data further improves the fits and finally, component A accounts for 69% of the particles. Most replica of A particles are heart-shaped and the median shadow widths are 6.1 and 6.8 nm in canaliculus and tubulovesicles respectively. The component B accounts for 31% of the particles. They mainly appear as small barrels and the median shadow widths are 8.8 and 10.3 nm in canaliculus and tubulovesicles respectively. According to calculated parameters and observed particle replica, the onset of secretion does not change the relative ratio of proteins but changes their shapes. Component A should be the (H+,K+)-ATPase whereas debate on the identity of B is wide open.

Gastric acid secretion in the chicken: effect of histamine H2 antagonists and H+/K(+)-ATPase inhibitors on gastro-intestinal pH and of sexual maturity calcium carbonate level and particle size on proventricular H+/K+ ATPase activity.

1. Cimetidine was more potent 4 hr after a single injection of 25 or 100 mg/kg body wt in increasing gastric pH than other H2 receptor antagonists, ranitidine and famotidine but was less efficient than H+/K(+)-ATPase inhibitors. Omeprazole rose proventricular and gizzard pH at a lower dose than SCH 28080 and Ro 18-5364 (30, 50 and 200 mg/kg body wt, respectively). 2. Proventricular and gizzard pH values were maximal 1 and 4 hr after a single injection of 7.5 mumol/kg body wt omeprazole. Inhibition of acid secretion was maintained for 24 hr after an injection of 100 mumol/kg. 3. H+/K(+)-ATPase activity in vitro was 10 mumol Pi/hr/mg protein in the microsomal fractions of the proventriculus. It was doubled by nigericine and inhibited by SCH 28080. However, western blots by high specific H+/K(+)-ATPase monoclonal antibody 95-A3 and 95-111 recognized a 42 kDa band but hardly exhibited the specific 95 kDa band recognition. 4. Chickens and immature pullets showed a higher H+/K(+)-ATPase activity than laying hens. Calcium level of the diet did not affect the enzyme activity but coarse particles of calcium fed to pullets or laying hens enhanced the H+/K(+)-ATPase activity when compared with ground particles.

Immunopurification of gastric parietal cell tubulovesicles.

1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.

Analysis of human mitochondrial transcripts using electron microscopic in situ hybridization.

Human mitochondrial transcripts have been examined at the ultrastructural level. After contact with ultrathin sections of a human lymphoid cell line (CEM) embedded in Lowicryl K4M, biotinylated mitochondrial probes yield specific hybrids identified by a colloidal gold immunocytochemistry marker that visualizes rRNA and mRNA coding for respiratory chain polypeptides CO II, CO III and ATPase-6. The mitochondrial transcripts are preferentially located close to the inner membrane, particularly the cristae, suggesting that intra-organelle protein synthesis is intimately associated with the mitochondrial membrane system. Quantitative analysis indicates that the mitochondria concentrate the labeling with intensities that vary with the type of RNA and that the nucleus induces a light hybridization signal with each mitochondrial probe. The visualization of human mitochondrial DNA expression in correlation with the fine anatomy of the mitochondria constitutes a new approach for fundamental research on the organelle and for analyzing its behaviour in human mitochondrial diseases.

The intramembranous particles of resting and secreting gastric (H+,K+)-ATPase membranes.

The fundic mucosa of resting and acid-secreting rabbit stomachs were freeze-fractured and replicated to compare the intramembranous particles on the parietal cell tubulovesicles (rest) and canaliculus (secretion). The particles were counted and their shadow diameters were measured using an image analysis program. The tubulovesicles bore 9,726 +/- 400 particles per microns2 (mean +/- SD), having a mean diameter of 8.4 nm (n = 2,571). The canaliculus bore 8,369 +/- 430 particles per microns2, having a mean diameter of 7.7 nm (n = 3,259). The data were reproducible: three fractures of tubulovesicles and canaliculus gave essentially the same distributions of particle diameters. By contrast, the frequency distributions of tubulovesicle and of canaliculus particle diameters were significantly different (P less than 0.0005). Neither the opposite curvatures of tubulovesicle and canaliculus microvillus fractures nor subpopulations of tubulovesicles with different particle diameters, were the cause of the difference, since there was only one population of tubulovesicles. We therefore postulate that the diameters of intramembranous particles of tubulovesicles and canaliculus are different and suggest, as a working hypothesis, that the difference could be due to a conformational change of the major intramembranous protein, the (H+,K+)-ATPase.

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.

The ontogeny of rat gastric H+/K+-ATPase.

The ontogeny of rat H+/K+-ATPase was studied between foetal day 18 and neonatal day 18, using a specific monoclonal antibody (95-111 mAb). The H+/K+-ATPase content of gastric subcellular membranes was assayed and the ATPase subunits were characterized by Western blot. The epithelium density in parietal cells was measured by immunohistochemistry. H+/K+-ATPase was present in the 18-day-old foetuses and parietal cells were detected on foetal day 19. The H+/K+-ATPase concentration remained stable from foetal day 18 to neonatal day 1, while the parietal cell density increased 2.5-fold. The H+/K+-ATPase concentration increased by 2.5-fold on day 6, then remained constant up to day 18. The parietal cell density remained unchanged during this period, suggesting that the concentration increase on day 6 was due to an increase in parietal cell ATPase content. The 95-111 mAb recognized a 95 kDa single band on foetal day 18 and a doublet at all the other stages of development. Previous studies had demonstrated that acid secretion drops critically at day 12 post partum in the rat and that H+/K+-ATPase activity is lost. The present study demonstrates that the H+/K+-ATPase is, however, present on day 12.

Endocrine cell populations in the colon and rectum of cat, dog, and monkey: fine structure, immunocytochemistry, and distribution.

Comparative and quantitative ultrastructural studies of endocrine cells from the large bowel of European cat, beagle dog, and the monkey Callitrix jacchus were performed. The cat and monkey exhibited a roughly similar distribution of colonic endocrine cells with a frequency increasing toward the distal. On the contrary, the highest endocrine cell frequency in the dog colon was in the cecum. In the dog and monkey, enterochromaffin (EC) cells were predominant in all segments. In the cat, non-EC cells were predominant in the proximal colon. For each colonic segment, relative percentages of EC and non-EC cells appeared on the whole to be roughly stable between individuals of the same species. Three subtypes of EC cells were distinguished in each species. Non-EC cells were characterized by large variation in size and electron densities of their granules: Mean granule size per cell extended from 210 to 850 nm in cat, 310 to 770 nm in dog, and 130 to 470 nm in monkey. In each species, statistical analyses indicated that the non-EC cell population was composed of two or more subpopulations. Some similarities were found between colonic endocrine cells of the monkey and man, whereas obvious differences appeared between the two carnivorous mammals. Immunocytochemical studies demonstrated the presence of cells containing enteroglucagon, somatostatin, or a pancreatic polypeptidelike substance in the colon of the monkey and the rectum of the three mammals. Correlative immunocytochemical and ultrastructural studies showed that the three kinds of immunostained endocrine non-EC cells in each species had rather round granules, with great electron densities. Some subpopulations, morphologically distinguished, did not react to any of the antisera used. This suggests either the existence of secretory cycle in some endocrine cells or, perhaps, the presence of peptides still unknown in this part of the gut.

Correlative immunocytochemical and electron microscopic studies: identification of (entero)glucagon- somatostatin- and pancreatic polypeptide-like-containing cells in the human colon.

Correlative immunocytochemical and electron microscopic studies, using the semi thin-thin technic, were performed to identify the (entero) glucagon, somatostatin and pancreatic polypeptide-like immunoreactive cells of the human colonic mucosa. Mean granule diameter for each cell type was estimated according to two methods and histograms showing the granule size distribution were constructed. A total of 139 immunostained cells identified at the ultrastructural level were analyzed. Mean granule diameter for (entero)glucagon-containing cells was 318 +/- 11 nm but a reduction of granule size with age was noteworthy: granules were larger in the fetus (mean diameter 350 +/- 15) than in adults (mean diameter 310 +/- 10 nm). Somatostatin-containing cells, very rare in adults, were present in the fetal distal colon. Their general mean granule diameter was 354 +/- 18 nm but many cells had a mean granule diameter of more than 400 nm. A pancreatic polypeptide-like immunoreactivity was found only in (entero)glucagon-containing cells, pointing out the possible occurrence of both peptides (or of similar sequences) in the same cells. Previous ultrastructural studies dealing with a tentative classification of the human colonic endocrine cells were compared with the present data.