Peroxynitrite causes calcium efflux from mitochondria which is prevented by Cyclosporin A

Abstract

Superoxide reacts with nitric oxide to form peroxynitrite, a potent oxidising agent which may contribute to tissue damage in pathological situations such as inflammation and ischaemia/reperfusion. One mechanism by which oxidative stress damages tissues is the induction of a specitic Cyclosporin A-sensitive mitochondrial calcium eflhrx pathway. Here we show that peroxynitrite induces calcium efflux from mammalian mitochondria and that this efflux is blocked by Cyclosporin A. These dam suggest that disruption of mitochondrial calcium eBhtx may contribute to tissue damage when superoxide and nitric oxide are present together in vivo.Key words: Peroxynitrite; Mitochondrial oxidative damage; Calcium efi%rx; Cyclosporin A; Mitochondrial permeability transition IIltroductloIlNitric oxide ('NO) is formed in many mammalian tissues where it acts as a chemical messenger [l] and superoxide (O,'-) is a by product of normal metabolism [2]. The reaction of 02'-with 'NO produces the potent oxidant peroxynitrite (ONOO-) [3,4] which contributes to tissue damage in inflammation and ischaemia/reperfusion where significant amounts of 'NO and Or*-are produced [4,5]. At 37"C, the pK, of ONOO-is 6.8 and the half life of its protonated form, peroxynitrous acid, is less than 1 second [6]. Peroxynitrous acid spontaneously cleaves to produce the powerful oxidants nitrogen dioxide and the hydroxyl radical [4,7]. The ONOO-anion itself oxidises thiols to distides [7] and therefore formation of ONOO-from 'NO and 02'-may lead to oxidative damage in vivo by a number of mechanisms.Mitochondria are an important target for oxidative damage by ONOO-since they produce 02'- [2]. Therefore exposure to 'NO can lead to the formation of ONOO-within mitochondria [6]. Furthermore, it has been shown that 'NO and ONOO-disrupt mitochondrial function [6,8]. In addition to directly inactivating mitochondrial enzymes [9, lo], many oxidants disrupt mitochondrial calcium metabolism by inducing a specific mitochondrial calcium efflux pathway [ 111. This calcium efflux is associated with, or leads to, the opening of a non-specific pore in the mitochondrial inner membrane which depolarises the mitochondrion pathway [l 1,121. While the role of calcium efflux and pore opening is unclear, it may contribute to cell damage following oxidative stress as CsA protects cells from oxidants [13].We have investigated whether or not oxidative damage by ONOO-disrupts mitochondrial calcium transport. To do this we measured calcium efflux and swelling in isolated rat liver mitochondria exposed to ONOO-and found that ONOO-induces mitochondrial calcium efflux and opening of the inner membrane pore, both of which can be prevented by CsA. Materials and methods MaterialsArsenaxo III, superoxide dismutase and catalase were from Sigma. "CaClz was from Amersham. Cyclosporin A was a kind gift from Sandoz Pharma Ltd., Basel, Switzerland. Preparation of mitochondriaLiver mitochondria were prepared from fed female Wistar rats (150-200 g) by homogenisation and differential centrifugatio...

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