Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Polarized calcium and calmodulin signaling in secretory epithelia

This review examines polarized calcium and calmodulin signaling in exocrine epithelial cells. The calcium ion is a simple, evolutionarily ancient, and universal second messenger. In exocrine epithelial cells, it regulates essential functions such as exocytosis, fluid secretion, and gene expression. Exocrine cells are structurally polarized, with the apical region usually dedicated to secretion. Recent advances in technology, in particular the development of videoimaging and confocal microscopy, have led to the discovery of polarized, subcellular calcium signals in these cell types. The properties of a rich variety of local and global calcium signals have now been described in secretory epithelial cells. Secretagogues stimulate apical-to-basal waves of calcium in many exocrine cell types, but there are some interesting exceptions to this rule. The shapes of intracellular calcium signals are determined by the distribution of calcium-releasing channels and mechanisms that limit calcium elevation. Polarized distribution of calcium-handling mechanisms also leads to transcellular calcium transport in exocrine epithelial cells. This transport can deliver considerable amounts of calcium into secreted fluids. Multicellular polarized calcium signals can coordinate the activity of many individual cells in epithelial secretory tissue. Certain particularly sensitive cells serve as pacemakers for initiation of intercellular calcium waves. Many calcium signaling pathways involve activation of calmodulin. This ubiquitous protein regulates secretion in exocrine cells and also activates interesting feedback interactions with calcium channels and transporters. Very recently it became possible to directly study polarized calcium-calmodulin reactions and to visualize the process of hormone-induced redistribution of calmodulin in live cells. The structural and functional polarity of secretory epithelia alongside the polarity of its calcium and calmodulin signaling present an interesting lesson in tissue organization.</jats:p

The role of Ca 2+

Acute pancreatitis is a human disease in which the pancreatic pro-enzymes, packaged into the zymogen granules of the acinar cells, become activated and cause auto-digestion. The main causes of pancreatitis are alcohol abuse and biliary disease. A considerable body of evidence indicates that the primary event initiating the disease process is excessive release of Ca2+ from intracellular stores, followed by excessive entry of Ca2+ from the interstitial fluid. However, Ca2+ release and subsequent entry are also precisely the processes that control physiological secretion of the digestive enzymes in response to stimulation via the vagal nerve or the hormone cholecystokinin. The spatial and temporal Ca2+ signal patterns in physiology and pathology, as well as the contributions from different organelles in the different situations, are therefore critical issues. There has recently been significant progress in our understanding of both physiological stimulus-secretion coupling and the pathophysiology of acute pancreatitis. Very recently, a promising potential therapeutic development has occurred with the demonstration that blockade of Ca2+ release-activated Ca2+ currents in pancreatic acinar cells offers remarkable protection against Ca2+ overload, intracellular protease activation and necrosis evoked by a combination of alcohol and fatty acids, which is a major trigger of acute pancreatitis