Sialic acids expression in newborn rat lungs: implications for pulmonary developmental biology.

Mammalian lung development proceeds during the postnatal period and continues throughout life. Intricate tubular systems of airways and vessels lined by epithelial cells are developed during this process. All cells, and particularly epithelial cells, carry an array of glycans on their surfaces. N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic (Neu5Gc) acids, two most frequently-occurring sialic acid residues, are essential determinants during development and in the homeostasis of cells and organisms. However, systematic data about the presence of cell surface sialic acids in the postnatal lung and their content is still scarce. In the present study, we addressed the histochemical localization of Neu5Ac > Neu5Gc in 0-day-old rat lungs. Furthermore, both residues were separated, identified and quantified in lung membranes isolated from 0-day-old rat lungs using high-performance liquid chromatography (HPLC) methodologies. Finally, we compared these results with those previously reported by us for adult rat lungs. The Neu5Ac > Neu5Gc residues were located on the surface of ciliated and non-ciliated cells and the median values for both residues in the purified lung membranes of newborn rats were 5.365 and 1.935 µg/mg prot., respectively. Comparing these results with those reported for the adults, it was possible to observe a significant difference between the levels of Neu5Ac and Neu5Gc (p < 0.001). A more substantial change was found for the case of Neu5Ac. The preponderance of Neu5Ac and its expressive increase during the postnatal development points towards a more prominent role of this residue. Bearing in mind that sialic acids are negatively charged molecules, the high content of Neu5Ac could contribute to the formation of an anion "shield" and have a role in pulmonary development and physiology.

Presence of N-acetylneuraminic acid in the lung during postnatal development.

Sialic acids, particularly N-acetylneuraminic acid (Neu5Ac), are present as terminal components of rich and complex oligosaccharide chains, which are termed glycans, and are exhibited on the cell surfaces, especially on epithelial cells. Crucial in the 'social behavior' of the cell, sialic acids play vital roles in many physiological and pathological phenomena. The aim of the present study was to separate, identify, and quantify Neu5Ac in purified lung membranes from 4-, 14-, and 21-day-old animals, followed by the statistical analysis of these results with our previously reported data (0-day-old and adult results). Complementary, ultrastructural methodologies were used. The differences in the Neu5Ac values obtained across the examined postnatal-lung development relevant ages studied were found to be statistically significant. A substantial increase in the mean level of this compound was found during the period of 'bulk' alveolarization, which takes place from postnatal day 4 to 14 (P4-P14). The comparison of the mean levels of Neu5Ac, during microvascular maturation (mainly between P12 and P21), reveals that the difference, although statistically significant, is the least significant difference among all the pair-wise differences between the developmental stages. The presence of sub-terminal N-acetylgalactosamine (GalNAc)/Galactose (Gal) residues with terminal sialic acids on the bronchioloalveolar cell surfaces was confirmed using lung ultra-thin sections of adult and 0-day-old animals. These results showed that, although Neu5Ac levels increase throughout postnatal lung development, this sialic acid was substantially added to epithelial cell surfaces during the "bulk" alveolarization period, while its presence was less important during the microvascular maturation period. Bearing in mind that sialic acids are negatively charged and create charge repulsions between adjacent cells, we hypothesized that they can substantially contribute to postnatal alveolar formation and maturation.

Presence of sialic acids in bronchioloalveolar cells and identification and quantification of N-acetylneuraminic and N-glycolylneuraminic acids in the lung.

The lung, in air-breathing vertebrates, is a tree-like structure composed of branching tubes ending in alveoli and lined by diverse and highly specialized epithelial cells. A dense array of complex and diverse glycoconjugates is present on essentially all animal cell surfaces. Sialic acids are widely allocated at the outermost ends of glycan chains, attached to membrane proteins and lipids below. Due to their abundance and their terminal position in glycans, sialic acids are implicated in many physiological and pathological functions. Although the composition of lung epithelial cell-surface glycans has been studied over the years, it is not yet completely understood. In the present work, we aimed to histochemically localize N-acetylneuraminic acid (Neu5Ac)>N-glycolylneuraminic acid (Neu5Gc) residues on rat bronchioloalveolar epithelial cell surfaces using light microscopy (LM) methods. In lung membranes isolated from adult rat lung homogenates, we also separated, identified and quantified Neu5Ac and Neu5Gc by means of high-performance liquid chromatography (HPLC), and systematically described the optimized HPLC methods used. Sialic acid residues were localized on the surface coat of bronchioloalveolar cells, and the mean quantification of Neu5Ac and Neu5Gc in the adult rat lung homogenates was 12,26 and 2,73 µg/mg prot., respectively, revealing a manifest preponderance of Neu5Ac. A coefficient of variation (CV) of 4,98% and 4,40%, respectively was obtained and an optimal dispersion variability expressed by the SD and the CV was also reported, confirming the efficiency of the methodology. To the best of our knowledge, our group was the first to identify, separate and quantify sialic acids in purified lung membranes. The presence of these residues contributes to a strong anionic shield and may provide an hydrating and protective barrier as well as a repulsive structure that is crucial to lung physiology.