Gabriel Péranzi

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.

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.

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 SSTRs mRNAs in human and rat gastric mucosae and in the human gastric tumoral cell-line HGTL. The SSTR1-5s mRNAs were found in both human fundic and antral mucosae as well as in the HGT1 cell and rat antrum. The four SSTR2-5s mRNAs but not SSTR1s 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 SSTRs mRNA subtypes in the stomach.

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.

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.

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

Summry— 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 μm2 (mean ± SD), having a mean diameter of 8.4 nm (n = 2571). The canaliculus bore 8 369 ± 430 particles per μm2, having a mean diameter of 7.7 nm (n = 3259). 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 < 0.0005). Neither the opposite curvatures of tubulovesicle and canaliculus microvillus fractures nor subpopulations populations 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.

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.

The platinum‐carbon replication of a homogenous population of gold particles makes log‐normal distributions of shadow widths and lengths

Gold particles were prepared, dried on grids and shadowed at 45° with a 1.2 nm platinum‐carbon (Pt‐C) film using the shadowing conditions previously described for the freeze‐fracture of gastric parietal cell membranes. The particle diameters and the particle shadow widths and lengths were measured using an image analysis system. Statistical analysis of 2000 diameters, shadow widths and shadow lengths indicated that a homogenous population of particles had a normal frequency distribution of diameters (mean diameter 14.5 ± 1.5 nm) and that the Pt‐C shadowing transformed that normal curve into a log‐normal frequency distribution of shadow widths. The frequency distribution of shadow lengths was log‐normal too. We conclude that a statistical partition of experimental frequency distributions of particle shadow widths and lengths of natural membranes to determine the number and parameters of individual components should involve log‐normal subdistributions rather than normal ones.

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° 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 homogenous 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.