Calpain‐mediated breakdown of cytoskeletal proteins contributes to cholecystokinin‐induced damage of rat pancreatic acini

Abstract

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 microM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 microM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins alphaII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, mu- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of alphaII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Increase in motility and invasiveness of MCF7 cancer cells induced by nicotine is abolished by melatonin through inhibition of ERK phosphorylation

“…Indeed, active dephosphorylating processes, especially those sustained by Slingshot phosphatase, also regulate cofilin activity. Coronin, which targets Slingshot at lamellipodia and filopodia, fosters slingshot activity and thus enhances cofilin dephosphorylation at the leading edge . Indeed, we observed that melatonin in nicotine‐treated cells significantly reduces coronin pool.…”

“…Coronin, which targets Slingshot at lamellipodia and filopodia, fosters slingshot activity and thus enhances cofilin dephosphorylation at the leading edge. 66 Indeed, we observed that melatonin in nicotine-treated cells significantly reduces coronin pool. Overall, melatonin may thus decrease activation of cofilin by directly promoting its phosphorylation (through PKCα) and, indirectly, by antagonizing its dephosphorylation (by decreasing coronin), through an ERK-independent mechanism.…”

Calpain- and talin-dependent control of microvascular pericyte contractility and cellular stiffness

“…Talin has also been demonstrated to be the most strongly bound component in the FA protein network (Lele et al 2008). Furthermore, although vinculin has been suggested to contribute to strength of cell adhesion (Gallant, Michael and Garcia 2005) and to act as a target for calpain cleavage (Goll et al 2003; Weber et al 2009), it has also been shown that mechanical stretching of talin unmasks its vinculin binding sites (del Rio et al 2009). Thus, it is likely that vinculin recruitment and activation in FAs (Chen, Choudhury and Craig 2006) depends strongly on presence and conformation of talin.…”

Cardiocirculatory pathophysiological mechanisms in severe acute pancreatitis

“…Moreover, increased oxidative stress causes an antioxidative capacity reduction and increases lipid peroxidation, and both effects could also play an important role in acinar cell damage [30] . Other early events that take place in the onset of AP is cytoskeletal proteins breakdown, probably mediated by cytosolic proteases activation [32] and disruption of acinar cell membranes, which could allow the influx of calcium and the exit of molecules, such as enzymes, from acinar cells [33] . Trypsin activation, which is a consequence of the pro longed elevated cytosolic Ca 2+ and also of the deficient lysosomal degradation [34] , occurs in postexocytotic endo cytic vacuoles [35] .…”