Expression of H⁺,K⁺-ATPase and Glycopattern Analysis in the Gastric Glands of Rana esculenta

Abstract

A multidisciplinary study involving lectin histochemistry, IHC, immuno-lectin blotting, and immunogold was carried out to determine the distribution of sugar residues in the glycoproteins of Rana esculenta oxynticopeptic cells. We considered animals in two experimental conditions, fasting and fed. It is known that, in mammals, the tubulovesicular membranes are rich in proteins with several functions. The proton pump H+,K+-ATPase, a heterodimeric complex with a catalytic α-subunit and a heavily glycosylated β-subunit, responsible for acid secretion, is the most abundant. No data have been published regarding the localization and the structures of H+,K+-ATPase in amphibians. In the water frog, the luminal membrane and tubulovesicular system of oxynticopeptic cells, which differ in morphology according to their functional stage, reacted with the primary gold-conjugated antibody against the H+,K+-ATPase α-subunit. By lectin histochemistry and immunoblotting, in the oxynticopeptic cells of R. esculenta we detected the presence of N-linked glycans having fucosylated (poly)lactosamine chains, which could correspond to the oligosaccharide chains of the β subunit. The latter are somewhat different from those described in mammals, and this is probably because of an adaptation to the different microenvironmental conditions in which the oxynticopeptic cells find themselves, in terms of their different habits and phylogeny.

Histochemical and Immunohistochemical Analysis of the Stomach of Rhinella icterica (Anura, Bufonidae)

“…In anurans, mucins are produced by goblet cells found in the esophagus and the intestines, but also by epithelial cells and mucous neck cells in the stomach [43]. In R. icterica, mucous neck cells of fundic region produce neutral glycoprotein, rich in galactose residues, similar to those of R. esculenta [19], Ceratophrys ornata [44], Ceratophrys chalcides [10], and reptile Natrix natrix [45].…”

“…Yet, our results corroborated the reports by Loo and Wong [3], who described that the lining epithelial cells of the stomach of B. viridis and B. melanostictus produce only neutral mucins. Moreover, lectin histochemical studies revealed that in R. aurora aurora [13], B. viridis [14], and R. esculenta [19] mucous secreting superficial cells produce galactose, but not galactosamine, since in B. viridis [14] and R. aurora aurora [13] gastric secretion is rich in galactose and mannose.…”

“…The mucosubstances, which phylogenetically first appeared in mucous neck cells of the gastric glands of anurans [12], have already been reported for Rana aurora aurora [13], Bufo viridis [14], Rana perezi [15], Bufo melanostictus [3], and Bufo marinus [16], indicating variability among anuran species. Furthermore, different types of carbohydrates were identified in secretory cells in the stomach of Bufo viridis [17,18] and Rana esculenta [19].…”

Functional reconstitution of a rice aquaporin water channel, PIP1;1, by a micro-batchwise methodology

“…Pellets of empty liposomes or 10His-OsPIP1; 1 proteoliposomes obtained by centrifugation at 152,000 g for 1 h at 4 C were fixed in a mixture of 3% paraformaldehyde and 1% glutaraldehyde in 0.1 mol/l PBS at pH 7.4 for 4 h at 4 C. Specimens were postfixed in 1% OsO 4 in PBS for 1 h at 4 C. Fixed specimens were dehydrated in ethanol and then embedded in Epoxy Resin-Araldite (M) CY212 (TAAB, Aldermaston, UK) as previously reported (Mastrodonato et al, 2009). Ultrathin sections were mounted on formwar-coated nickel mesh grids and stained with uranyl acetate and lead citrate (Reynolds, 1963).…”

Glycopattern analysis and structure of the egg extra-cellular matrix in the Apennine yellow-bellied toad, <i>Bombina pachypus (Anura: Bombinatoridae)</i>

“…ransmission electron microscopy. The eggs were fixed in 4% glutaraldehyde and processed for embedding in Epoxy Resin-Araldite (M) CY212 (TAAB, Aldermaston, UK) as previously reported [46]. Semi-thin sections 2 μm thick were stained with Toluidine blue-PAS (PAS-TB).…”