Othon B. Kotoulas

cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria.

We showed recently that actin assembly by phagosomal membranes facilitates fusion with late endocytic organelles in macrophages. Moreover, lipids that induced phagosomal actin also stimulated this fusion process. In macrophages infected with pathogenic mycobacteria actin-stimulatory lipids led to an increase in pathogen destruction, whereas inhibitors facilitated their growth. A model was proposed whereby phagosomal membrane actin assembly provides tracks for lysosomes to move towards phagosomes, thereby facilitating fusion. Here, we investigated how cAMP affected phagosomal actin assembly in vitro, and phagosomal actin, acidification and late fusion events in J774 macrophages. Latex bead phagosomes are shown to possess adenylyl cyclase activity, which synthesizes cAMP, and phosphodiesterase activity, which degrades cAMP. The system is regulated by protein kinase A (PKA). Increasing cAMP levels inhibited, whereas decreasing cAMP levels stimulated, actin assembly in vitro and within cells. Increasing cAMP levels also inhibited phagosome-lysosome fusion and acidification in cells, whereas reducing cAMP had the opposite effect. High cAMP levels induced an increase in intraphagosomal growth in macrophages of both the non-pathogenic Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis, whereas low cAMP levels or inhibition of PKA correlated with increased bacterial destruction. We argue that the phagosome cAMP-PKA system behaves as a molecular switch that regulates phagosome actin and maturation in macrophages.

An electron microscopic and biochemical study of the effects of propranolol on the glycogen autophagy in newborn rat hepatocytes

The effects of propranolol on the glycogen autophagy in newborn rat hepatocytes were studied by using biochemical determinations, electron microscopy and morphometric analysis. Propranolol lowered the liver cyclic AMP and cyclic AMP-dependent protein kinase activity. It also decreased the formyl-methionyl-leucyl-phenylalanine (FMLP)-inhibitable Ca2+-ATPase activity including lysosomal calcium uptake pump. The normal postnatal increase in the volume of autophagic vacuoles and the activity of acid glycogen-hydrolyzing alpha glucosidase were inhibited. Also, the degradation of glycogen inside the autophagic vacuoles was apparently inhibited. The activity of acid mannose 6-phosphatase was increased. These findings indicate that propranolol influences several steps in the sequence of events leading to the breakdown of glycogen in the autophagic vacuoles of newborn rat hepatocytes. This supports our previous studies suggesting that cyclic AMP regulates glycogen autophagy.