Concentration-dependent Effects of Nitric Oxide on Mitochondrial Permeability Transition and Cytochrome cRelease

Paul S Brookes,Emmanuel Padilla Salinas,Kenta Darley-Usmar,Jason P Eiserich,Bruce A Freeman,Victor M Darley-Usmar,Peter G Anderson

doi:10.1074/jbc.m001077200

Paul S Brookes, Emmanuel Padilla Salinas + Show 5 more

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https://doi.org/10.1074/jbc.m001077200

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Abstract

The mitochondrial permeability transition pore (PTP) and associated release of cytochrome c are thought to be important in the apoptotic process. Nitric oxide (NO( small middle dot)) has been reported to inhibit apoptosis by acting on a variety of extra-mitochondrial targets. The relationship between cytochrome c release and PTP opening, and the effects of NO( small middle dot) are not clearly established. Nitric oxide, S-nitrosothiols and peroxynitrite are reported to variously inhibit or promote PTP opening. In this study the effects of NO( small middle dot) on the PTP were characterized by exposing isolated rat liver mitochondria to physiological and pathological rates of NO( small middle dot) released from NONOate NO( small middle dot) donors. Nitric oxide reversibly inhibited PTP opening with an IC(50) of 11 nm NO( small middle dot)/s, which can be readily achieved in vivo by NO( small middle dot) synthases. The mechanism involved mitochondrial membrane depolarization and inhibition of Ca(2+) accumulation. At supraphysiological release rates (>2 micrometer/s) NO( small middle dot) accelerated PTP opening. Substantial cytochrome c release occurred with only a 20% change in mitochondrial swelling, was an early event in the PTP, and was also inhibited by NO( small middle dot). Furthermore, NO( small middle dot) exposure resulted in significantly lower cytochrome c release for the same degree of PTP opening. It is proposed that this pathway represents an additional mechanism underlying the antiapoptotic effects of NO( small middle dot).

Highlights

The abbreviations used areNO1⁄7, nitric oxide; DEA NONOate, 2-(N,N-1-diethylamino)-diazenolate-2-oxide; DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N-(ammonio-ethyl)amino]diaze-1-ium-1,2diolate; FCCP, carbonyl-cyanide-p-(trifluoromethoxy)-phenylhydrazine; oxyHb, oxyhemoglobin; PTP, permeability transition pore; tBuOOH, tert-butyl hydroperoxide; MALDI-TOF, matrix-assisted laserpro- and antiapoptotic effects reported depending on both cell type and NO1⁄7 concentration (reviewed in Refs. 1 and 2)
From the Departments of ‡Pathology and **Anesthesiology and the §Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294
The release of cytochrome c and apoptosis-inducing factor from mitochondria leads to activation of caspases 9 and 3 [29], and it was recently reported that caspase 3 can activate caspase 8, which can in turn trigger cytochrome c release [32]. These findings suggest an amplification role for mitochondria in apoptotic signaling and support the concept that the control of cytochrome c release from the mitochondrion is likely to impact the progression of apoptosis

Summary

The abbreviations used are

NO1⁄7, nitric oxide; DEA NONOate, 2-(N,N-1-diethylamino)-diazenolate-2-oxide; DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N-(ammonio-ethyl)amino]diaze-1-ium-1,2diolate; FCCP, carbonyl-cyanide-p-(trifluoromethoxy)-phenylhydrazine; oxyHb, oxyhemoglobin; PTP, permeability transition pore; tBuOOH, tert-butyl hydroperoxide; MALDI-TOF, matrix-assisted laserpro- and antiapoptotic effects reported depending on both cell type and NO1⁄7 concentration (reviewed in Refs. 1 and 2). Mitochondrial cytochrome c release is regulated by the Bcl-2 family of proteins [33], which are targeted at the mitochondrial permeability transition pore (PTP) This multisubunit protein complex includes the mitochondrial voltage-dependent anion channel, the adenine nucleotide translocase, and cyclophilin D [33, 34]. Inhibition of the putative mitochondrial NOS facilitates Ca2ϩ accumulation, consistent with either NO1⁄7-dependent prevention of Ca2ϩ uptake or enhanced release [31] These observations are significant in the context of control of cytochrome c release, since the PTP is initiated by an increase in the intramitochondrial Ca2ϩ concentration, and predict that NO1⁄7 should inhibit PTP opening [34]. This hypothesis was tested by exposing isolated rat liver mitochondria to NO1⁄7 released from NONOate compounds at rates encompassing both the physiological and pathological ranges, and the effects on PTP opening and cytochrome c release were determined

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DISCUSSION