Cytochemical demonstration of increased phospholipid content in cell membranes in chlorphentermine-induced phospholipidosis

We recently introduced a novel cytochemical approach to high-resolution cytochemistry of phospholipids in biological tissues. The technique consists of adsorption of bee venom phospholipase A₂ to colloidal gold particles (PLA₂-gold complex) and subsequent application of this complex for localization of the enzyme substrate, i.e., glycerophospholipids. In the present study, this technique was applied at the post-embedding level, in both light (LM) and transmission electron microscopy (TEM), to investigate drug-induced phospholipids, an experimental disorder in which the lysosomal catabolism of phospholipids is inhibited. Rats received one week of daily treatment (40 mg IP/kg) with chlorphentermine (CP), a cationic amphiphilic drug known to induce phospholipidosis in several tissues. Glutaraldehyde- and osmium-fixed lung and kidney tissues from both treated and control animals, were embedded in Epon and sections processed for labeling by PLA₂-gold. In CP-treated specimens the presence of large osmiophilic inclusions in several cell types of lung parenchyma and kidney cortex confirmed the onset of phospholipidosis. These inclusions were densely labeled by PLA₂-gold at both LM and TEM levels. Two general types of abnormal inclusions were distinguished on the basis of their ultrastructure and labeling pattern by PLA₂-gold, suggesting different content or configuration of phospholipids. Moreover, quantitative evaluation of labeling density over various membrane compartments in lung alveolar cells evidenced significantly increased phospholipid content after CP treatment. In type II pneumocytes, such increases were measured in membranes of the RER, Golgi complex, outer and inner nuclear envelope, and the basolateral and apical domains of the plasma membrane. In capillary endothelial cells, the basal and luminal domains of the plasma membrane also showed an increase in labeling density. These results further demonstrate the potential usefulness of the PLA₂-gold technique for in situ ultrastructural localization of phospholipids in normal and pathological tissues.