Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

Avicins: Triterpenoid saponins from Acacia victoriae (Bentham) induce apoptosis by mitochondrial perturbation

Anticancer agents target various subcellular components and trigger apoptosis in chemosensitive cells. We have recently reported the tumor cell growth inhibitory properties of a mixture of triterpenoid saponins obtained from an Australian desert tree (Leguminosae) Acacia victoriae (Bentham). Here we report the purification of this mixture into two biologically pure components called avicins that contain an acacic acid core with two acyclic monoterpene units connected by a quinovose sugar. We demonstrate that the mixture of triterpenoid saponins and avicins induce apoptosis in the Jurkat human T cell line by affecting the mitochondrial function. Avicin G induced cytochrome c release within 30–120 min in whole cells and within a minute in the cell-free system. Caspase inhibitors DEVD or zVAD-fmk had no effect on cytochrome c release, suggesting the direct action of avicin G on the mitochondria. Activation of caspase-3 and total cleavage of poly(ADP-ribose) polymerase (PARP) occurred between 2 and 6 h posttreatment with avicins by zVAD-fmk. Interestingly, in the treated cells no significant changes in the membrane potential preceded or accompanied cytochrome c release. A small decrease in the generation of reactive oxygen species (ROS) was measured. The study of these evolutionarily ancient compounds may represent an interesting paradigm for the application of chemical ecology and chemical biology to human health.