Abstract
The present study shows that rat liver and brain mitochondrial nitric oxide synthase (mtNOS) are functionally associated with mitochondrial respiratory chain complex I. When complex I is activated, mtNOS exerts high activity and generates nitric oxide, whereas inactivation of complex I leads mtNOS to abandon its NOS activity. Functional association of mtNOS with complex I is potentially important in regulating mtNOS activity and mitochondrial functions.
Keywords: Mitochondrial nitric oxide synthase, nitric oxide, respiratory chain complex I, mitochondrial respiration
Introduction
Mitochondrial nitric oxide (NO) synthase (mtNOS) regulates various functions of mitochondria in liver [1–5], heart [6–8], brain [2, 9, 10], kidney [11, 12], as well as cells including endothelial [13], kidney [14], neuronal [15], cardiac myocytes [16], and breast cancer [17]. mtNOS-derived NO competes with oxygen and regulates mitochondrial primary functions including respiration and transmembrane potential (ΔΨ) [1,5,6,9]. mtNOS-derived NO also produces oxidative nitrogen oxide species that cause oxidative damage to mitochondria and cells and play a key role in apoptosis [1–3,8,12,15–17]. Despite significance of the regulation by mtNOS of mitochondrial functions, it is not fully understood how mitochondria regulate mtNOS activity. The rationale for the present study was to delineate whether mitochondria exert regulatory function over mtNOS activity. The present study shows that in isolated rat liver and brain mitochondria, mtNOS activity is associated with mitochondrial respiratory chain complex I (RCC-I) and suggest RCC-I regulates mtNOS activity.
Material and Methods
Purification of mitochondria
Liver mitochondria were isolated from Sprague-Dawley rats by differential centrifugation and purified by Percoll purification as described [18]. Brain mitochondria were purified as described [19]. Brains were removed, washed, and homogenized in Medium A (sucrose 320 mM, potassium EDTA 1 mM, 0.1% fatty acid–free bovine serum albumin [FAF-BSA], Tris-HCl 10 mM, pH 7.40). The homogenate was centrifuged at 1,100 x g for 10 min, and the supernatant was re-centrifuged at 19,000 x g for 20 min to obtain crude mitochondrial fraction. The pellet was resuspended in Medium A, laid on 6% Ficoll solution, and centrifuged at 19,000 x g for 30 min. Supernatant and the fluffy layer above the mitochondria pellet were discarded. Each 10 mg of the brown pellet was resuspended in 70 mg of the Purification Medium (2.56 g of 20% [w/w] dextran T500; 1.28 g of 40% [w/w] polyethylene glycol (PEG) 4000; 2.24 g of 1 M sorbitol; 70 mg of 10 mM potassium EDTA; 700 mg of 1% FAF-BSA; 200 mg of 200 mM potassium phosphate pH 7.80; H2O to 7.00 g) and centrifuged at 600 x g for 2 min in a swing-bucket centrifuge. The upper phase was discarded, 300 μl of the Washing Medium (96 mg of 20% [w/w] dextran T500; 480 mg of 40% [w/w] PEG 4000; 960 mg of 1 M sorbitol; 75 mg of 200 mM potassium phosphate pH 7.80; 30 mg of 10 mM potassium EDTA; 300 mg 1% FAF-BSA; H2O to 3.00 g) was added to the lower part, mixed and centrifuged at 600 x g for 2 min. The upper part was discarded and the lower part was mixed with 3 ml of Medium B (sorbitol 320 mM, potassium EDTA 1 mM, 0.1% FAF-BSA, potassium phosphate 5 mM, pH 7.80).
The suspension was centrifuged at 19,000 x g for 20 min, the supernatant was discarded, and the mitochondrial pellet was collected. All steps were carried out at 4 °C. Purity of the isolated mitochondria was assessed by measuring cytochrome a using ε605–630nm 12 mM−1 cm−1 as described [18]. Only mitochondria with less than 5% impurity were used. Broken mitochondria were prepared by 5 times freezing the intact mitochondria in liquid nitrogen followed by thawing as described [1, 17]. Submitochondrial particles (SMP) were prepared from mitoplasts as described [20]. Briefly, the outer membrane was removed by treating the intact mitochondria with digitonin (0.12 mg/mg of mitochondrial protein) for 15 min at 4 °C followed by 2 times wash. Mitoplasts were frozen-thawed as above and the unbroken mitoplasts were removed by 2 times spinning at 9,500 x g for 10 min. The supernatant of the second centrifugation was centrifuged at 100,000 x g for 30 min. The pellet referred to as SMP and supernatant referred as to mitochondrial matrix were collected.
Activation or inactivation of RCCs
The following experimental conditions were used in freshly isolated mitochondria: A) No respiratory substrate (No RS) refers to when respiratory chain was not altered by exogenous substrates; B) RCC-I activation refers to when RCC-I was activated by pyruvate/malate (10/5 mM); C) RCC-I inactivation refers to when RCC-I was inactivated by rotenone (5 μM); D) RCC-II activation refers to when RCC-I was inactivated by rotenone (5 μM) and mitochondria were energized from RCC-II by succinate (2.5 mM). In SMP, RCC-I was activated by NADH (100 μM).
Determination of mtNOS activity
mtNOS activity was determined using the following assays
Oxyhemoglobin assay
mtNOS activity was detected in broken mitochondria at 401–420 nm in the presence of oxyhemoglobin (4 μM) using ε401–420nm 100 mM−1 cm−1 as described [18].
Citrulline radioassay
Mitochondria were supplemented with L-[3H]arginine (30,000 – 50,000 cpm). Radio-labeled L-citrulline was separated from L-arginine using Dowex columns and the radioactivity was measured as described [3, 17].
Chemiluminescence assay
Mitochondria (50 μg) were injected into the purge vessel containing vanadium chloride (0.8 % in 1M HCl) thermostated at 93 °C, and NO was measured using chemiluminescence analyzer (Sievers 280i, General Electric, Boulder, CO) as described [17].
Amperometric assay
mtNOS activity in SMP was measured by NO-sensitive electrode (Apollo 4000; World Precision Instruments, FL) using a 7 μm tip micro-sensor (ISO-NO-007) placed in the chamber containing SMP (200 μg).
Oxygen consumption
Oxygen consumption was measured using a Clark-type oxygen electrode (Harvard Apparatus, Holliston, MA) as described [18, 21].
Intramitochondrial ionized calcium ([Ca2+]m)
The [Ca2+]m was measured by using dual-wavelength excitation fluorometery as described [8, 17]. Mitochondria were loaded with fura-2/AM for 20 min at room temperature followed by twice wash. Fura-2/AM loaded mitochondria were excited at 352–362 nm and emission was collected at 510 nm.
Statistical analysis
Barograms are Mean ± SEM of n = 4–6. Differences were tested by ANOVA and considered significant at p < 0.05. All computations were performed using PlotiT (Scientific Programming).
Results
mtNOS activity
Figs. 1A–F show mtNOS activity of liver and brain mitochondria as determined by three NOS assays. As shown, mtNOS activity was significantly higher when mitochondria were respiring on complex I, compared to when mitochondria were not supplied with exogenous respiratory substrates, RCC-I was inactivated, or RCC-II was activated. As shown, mtNOS activity in all samples was inhibited by mtNOS inhibitor, L-NG-monomethyl arginine (L-NMMA; Ref 3, 18).
Mitochondrial respiration
Figs. 2A, B show respiration of liver and brain mitochondria supported by activation of RCC-I, or when RCC-I was inactivated and respiration was supported from RCC-II. As shown, respiration supported by RCC-I was decreased by loading mitochondria with Ca2+, and that Ca2+-induced decrease of respiration was prevented by L-NMMA. However, when RCC-I was inactivated and respiration was supported from RCC-II, Ca2+ did not decrease the respiration.
Intramitochondrial calcium ([Ca2+]m)
mtNOS is Ca2+-dependent [1,7,9,13] and elevation of [Ca2+]m per se is sufficient to increases mtNOS activity [3,8,9,13,16,17]. Thus, the present study determined whether RCC-I activation increases [Ca2+]m. Fig. 3 shows that [Ca2+]m was not increased when RCC-I was activated compared to when RCC-I was inactivated and RCC-II was activated.
mtNOS activity of submitochondrial particle (SMP)
Figs. 4A, B show mtNOS activity of SMP. As shown, activation of SMP RCC-I by NADH stimulates mtNOS in an L-NMMA-sensitive manner. Increased mtNOS activity was inhibited when RCC-I was inactivated by rotenone. Replacing NADH with NADPH did not support mtNOS activity.
Discussion
The present study provides evidence strongly suggesting that mtNOS activity in isolated rat liver and brain mitochondria is associated with RCC-I. Using 3 well-established NOS detection assays, Fig. 1 shows mtNOS activity as determined by all NOS assays is significantly higher when RCC-I is activated, compared to when mitochondria were not supplied with exogenous respiratory substrates, RCC-I was inactivated, or when mitochondria were energized from RCC-II. mtNOS is Ca2+-sensitive and increased [Ca2+]m stimulates mtNOS activity [1, 3, 7, 8, 13, 17]. mtNOS-derived NO decreases mitochondrial respiration [22] by competing with O2 for the binding site of cytochrome oxidase [23]. Figs. 2A, B confirm findings of Fig. 1 by showing that mitochondrial respiration supported from RCC-I was decreased by loading mitochondria with Ca2+ and that this effect of Ca2+ was prevented when mtNOS was inhibited. However, when RCC-I was inactivated and mitochondrial respiration was supported from RCC-II, Ca2+ did not decrease mitochondrial respiration. Findings presented under Figs. 1 and 2 strongly support the hypothesis that mtNOS activity in brain and liver mitochondria is associated with RCC-I activity.
Intramitochondrial calcium homeostasis is governed by continuous precipitation of [Ca2+]m to non-ionized calcium stores known as matrix electron-dense granules, and release of [Ca2+]m from the granules [8, 24]. Since mtNOS is [Ca2+]m-sensitive [8, 13, 17] and RCC-I activation stimulated mtNOS activity, we tested the effect of RCC-I activation on [Ca2+]m. As shown in Fig. 3, RCC-I activation did not increase [Ca2+]m, even [Ca2+]m was slightly decreased compared to when RCC-I was inactivated and mitochondria were energized from RCC-II. These findings rule out elevation of [Ca2+]m as a mechanism underling stimulation of mtNOS upon RCC-I activation. To further study mechanisms responsible for association of mtNOS activity with RCC-I, we measured mtNOS activity in SMP. mtNOS is located at the inner mitochondrial membrane [1, 3, 8, 10] and determination of mtNOS in SMP allows excluding potential interference of mitochondrial matrix, intermembrane space, outer membrane, or other components of mitochondria with RCC-I-mtNOS association. Figs. 4A, B show that NADH that activates RCC-I significantly increased mtNOS activity in an L-NMMA sensitive manner. NOS isozymes utilize L-arginine to produce NO and L-citrulline. For cytoplasmic NOS isozymes, this reaction utilizes electrons donated by NADPH rendering a redox potential of −280 mV [25]. Figs. 4A, B show that NADPH that serves native electron donor to cytoplasmic NOS isozymes [26, 27] fails to support mtNOS activity of SMP. These figures also show that NADH-increased mtNOS activity was inhibited when RCC-I was inactivated by rotenone. This finding rules out possible direct interaction of NADH with mtNOS. These findings also explain lack of NO formation when SMP were supplied NADPH, as well as inhibition of mtNOS by rotenone.
NOS isozymes are oxidoreductase proteins comprising of an oxygenase and a reductase domain [25]. Each NOS monomer homodimerizes in a configuration that allows the reductase domain of each monomer couples with the oxygenase domain of the coupling monomer. Electrons enter the NADPH-binding site at the reductase domain of a monomer and transverse to the arginine-binding site at the oxygenase domain of the coupling monomer where NO is generated (Diagram 1). Coupling is essential for NOS isozymes to retain their NOS activity and uncoupling NOS isozymes abolishes their NOS activity [25,26]. mtNOS is located at the inner mitochondrial membrane [1,18] where respiratory chain complexes are embedded. Mitochondrial respiratory chain complexes provide a range of redox potentials from −280 mV for RCC-I to +250 mV for cytochrome c oxidase [28]. Electrons derived from oxidation of NADH enter RCC-I and flow through the chain following the redox hierarchy of the respiratory complexes. Our results show that when RCC-I is active, mtNOS produces NO, while inactivation of RCC-I abolishes mtNOS activity. These findings suggest a functional association between mtNOS and RCC-I whereby mtNOS utilizes RCC-I as its source of electrons (Diagram 1), and describe a reciprocal regulation between mtNOS and mitochondrial respiratory chain.
Taken together, the present study demonstrates that liver and brain mtNOS are functionally associated with RCC-I. When RCC-I is activated mtNOS generates NO, whereas RCC-I inactivation leads mtNOS to abandon NO production. Exact molecular mechanism underlying association of mtNOS with RCC-I needs further investigations.
Acknowledgments
Financial support was provided by the National Institute on Aging (award AG023264-02) and American Heart Association (award 0565221B).
Abbreviations used
- RCC-I
respiratory chain complex I
- RCC-II
respiratory chain complex II
- NO
nitric oxide
- mtNOS
mitochondrial nitric oxide synthase
- L-NMMA
NG-monomethyl-L-arginine
- Rot
rotenone, Succ, succinate, Pyr, Pyruvate
- Mal
malate
Footnotes
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