Role and function of the Ca2+-dependent protease calpain-1 associated to membrane lipid rafts

The Ca2+-dependent protease calpain-1 has been found constitutively present in lipid raft/caveolin-1 membrane microdomains isolated from endothelial bEnd5 cells. In this membrane localization calpain-1 is associated with some of its substrates, such as endothelial Nitric Oxide Synthase (eNOS) and the chaperone Heat Shock Protein 90 (HSP90), whereas the calpain-2 and the inhibitor calpastatin have not been detected. Alteration in intracellular Ca2+ homeostasis, induced by either treatment with Ca2+-ionophore A23187 or prolonged cell exposure to high glucose, leads to a significant decrease in the level of eNOS associated to lipid rafts, followed by a recruitment of HSP90 at this site. The decrease of eNOS is due not only to its Ca2+-dependent release from the caveolin-1 microdomains, but also to its digestion mediated by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2). However, the modifications observed in lipid rafts protein composition make bEnd5 cells more resistant against cell death caused by Ca2+ overload. In this context, calpain-1 seems to play a protective role against NO overproduction. Alterations in eNOS, calpain-1 and HSP90 levels have been also detected in aorta of Zucker Diabetic Rats (ZDR). The digestion of HSP90 indicates an aberrant activation of calpain, due to a chronic alteration in Ca2+ homeostasis occurring in these animals, and thereby the transition from a physiological to a pathological cell condition. These changes cannot be detected in brain cortex, suggesting a tissue-specificity of the alteration in Ca2+ homeostasis related to diabetes. A similar protein organization of lipid rafts has been also observed in neuroblastoma SK-N-BE cells, where calpain-1 is associated with NMDAR, HSP90 and neuronal NOS (nNOS), as well as in brain cortex of normotensive rats. Preliminary results have demonstrated that the increase in [Ca2+]i, that characterizes the nervous tissue of hypertensive rats, leads to a protein reorganization of the lipid rafts. The results presented in this thesis provide new information on a selective localization and role of calpain-1. In endothelial cells the protease and its associated substrates seem to have a crucial role in responding to fluctuations of [Ca2+]i. Further investigations will be necessary to analyse the role of the protease associated to lipid rafts in nervous tissue both in physiological and pathological conditions.