Cyclosporin A-insensitive Permeability Transition in Brain Mitochondria

Abstract

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca 2؉ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca 2؉ -induced Ca 2؉ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg 2؉ . Ca 2؉ release was not mediated by the mitochondrial Na ؉ /Ca 2؉ exchanger or by reversal of the uniporter responsible for energy-dependent Ca 2؉ uptake. Loss of mitochondrial Ca 2؉ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca 2؉ -induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A 2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg 2؉ , Ca 2؉ -induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.Mitochondria are temporo-spatial modulators of cytosolic [Ca 2ϩ ] through their ability to both sequester and release Ca 2ϩ in concert with Ca 2ϩ transport across the endoplasmic reticulum (1, 2). Physiological mitochondrial Ca 2ϩ efflux, including that caused by Ca 2ϩ -induced Ca 2ϩ release (mCICR), 1 is attributed to either activation of the permeability transition pore (PTP) (3, 4) or to reversal of the Ca 2ϩ uniporter (5). Opening of the PTP has been implicated as a mediator of apoptotic and necrotic cell death as well as a regulator of normal cell Ca 2ϩ homeostasis (4, 6 -12). The characteristic traits of the PTP include reversibility (13), sensitivity to inhibition by cyclosporin A (14), and mitochondrial swelling associated with loss of matrix solutes (15-17). Cyclosporin A is not always effective, however, at inhibiting Ca 2ϩ -induced mitochondrial swelling and loss of ⌬⌿ (18 -22). In addition, the observation that normal intracellular concentrations of adenine nucleotides and Mg 2ϩ partially or completely block PTP activation (23, 24) casts doubt on a physiological role of the PTP.The extent to which mitochondria swell in response to accumulation of large Ca 2ϩ loads also varies considerably with experimental conditions and with mitochondrial tissue type (25-27). Brain mitochondria are particularly resistant to Ca 2ϩ -induced swelling (25, 28); moreover, they represent many cell populations, which may explain their heterogeneous response to large Ca 2ϩ loads and to inhibitors of the PTP, e.g. cyclosporin A (26,29).In this study we report that 2-APB (30) prevents Ca 2ϩ -induced PTP in non-synaptosomal brain mitochondria in the ...

TRPC1-mediated Inhibition of 1-Methyl-4-phenylpyridinium Ion Neurotoxicity in Human SH-SY5Y Neuroblastoma Cells

“…Whereas the addition of 2APB, a nonspecific TRP channel blocker, showed no protection of SH-SY5Y cells against MPP ϩ , also no significant decrease in the neuroprotective role of TRPC1 was observed in the presence of 2APB. These results are inconsistent with other published studies where the addition of 2-APB prevented Ca 2ϩ -induced permeability transition pores in nonsynaptosomal brain mitochondria (38). Because these studies were performed on isolated mitochondria, 2APB could function differently.…”

Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

“…Furthermore, rat brain mitochondria contain ϳ5 nmol of total NAD/mg protein (C. Chinopoulos, unpublished observations) similar to the amount found in mitochondria from other tissues (3-7 nmol of total NAD/mg mitochondrial protein) (Tischler et al, 1977;Di Lisa et al, 2001). We have previously demonstrated that under the experimental conditions used in this study, rat brain mitochondria did not release more than ϳ15% of their total NAD content (Chinopoulos et al, 2003). Similar amounts of NADH added exogenously did not affect the H 2 O 2 rates detected by the Amplex Red-HRP assay (data not shown).…”

Excitotoxic Calcium Overload in a Subpopulation of Mitochondria Triggers Delayed Death in Hippocampal Neurons

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