Bidirectional action of nitric oxide on mitochondrial respiration and permeability transition pore induced by calcium and palmitoylcarnitine

The role of mitochondrial calcium-dependent NO synthase in the control of respiration and mitochondrial permeability transition pore (MPTP) opening, as well as possible involvement of mitochondrial NO synthase/guanylate cyclase/kinase G-signaling system (mtNOS-SS) in the regulation of these processes are not sufficiently studied. In this work, using rat liver mitochondria, we applied specific inhibitors of the enzymes of this signaling system to evaluate its role in the control of respiration and MPTP. The respiration was supported by pyruvate and glutamate or succinate in the presence of hexokinase, glucose and ADP. The results indicate that L-arginine and NO donors SNP and SNAP produce bidirectional concentration-dependent effects on the respiration and MPTP opening evoked by calcium ions or D,L-palmitoylcarnitine. Maximal activation of respiration was observed at 20 µM of L-arginine or SNP. At low concentrations, L-arginine (to 500 µM) and NO donors (to 50 µM) increased the threshold concentrations of calcium and D,L-palmitoylcarnitine required for the dissipation of the mitochondrial membrane potential and pore opening. The application of the inhibitors of NO synthase, guanylate cyclase, and kinase G eliminated both effects. These data indicate the involvement of mtNOS-SS in the activation of respiration and deceleration of MPTP opening. At high concentrations, L-arginine and NO donors inhibited the respiration and promoted pore opening, indicating that the inhibition induced by NO excess dominates over the protection caused by mtNOS-SS. These results demonstrate that the functioning of mtNOS-SS might provide a feedforward activation of respiration and a lowering of MPTP sensitivity to calcium and palmitoylcarnitine overload.


Introduction
Numerous studies clearly demonstrate that exogenous nitric oxide (NO) suppresses mitochondrial respiration by inhibiting cytochrome c oxidase (COX) and complexes I and II of the electron transport chain [1][2][3][4][5]. The activation of calcium-dependent mitochondrial NO synthase (mtNOS) by its substrate L-arginine [6,7] or by Ca 2+ plus L-arginine [8] also causes a sharp rise in the production of mitochondrial NO, which is followed by the inhibition of oxygen consumption [7].
In contrast to unidirectional action of NO on mitochondrial respiration, contradictory effects of NO on mitochondrial calcium retention capacity (CRC), mitochondrial permeability transition pore (MPTP) opening, and mitochondrial cytochrome c (CytC) release have been demonstrated over the last two decades. As early as 1999, it was shown that the activation of . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/443986 doi: bioRxiv preprint 3 mtNOS by Ca 2+ and L-arginine induced CytC release, while the inhibition of mtNOS diminished it, raised mitochondrial potential (ΔΨm) and CRC [9]. Later it was demonstrated that NO evoked concentration-dependent effects on pore opening and CytC release [10]. It was shown that, being added at very low or high concentrations, the NO donor SpermineNONOate promoted mitochondrial swelling, CytC release, and MPTP opening induced by calcium, whereas in intermediate concentrations this compound caused protective effects. The protective and adverse effects of NO donors were attributed to possible action of S-nitrosothiols and peroxynitrite, correspondingly [9,10].
However, opposite to these results, ensuing studies indicated that the inhibitors of mtNOS promoted, while NO donors prevented the dissipation of Δ Ψ m and mitochondrial swelling induced by Ca 2+ in isolated mitochondria [11]. Wherein, accumulated S-nitrosothiols were considered as final mediators providing the prevention of MPTP opening.
The concentration-dependent effects of NO donors were also demonstrated on permeabilized cells. It was shown that NO donors dose-dependently diminished mitochondrial Ca 2+ uptake and, being applied in high doses, promoted MPTP opening [12,13]. It was assumed that the inhibition of Ca 2+ uptake by intramitochondrial NO may represent negative feedback, which could prevent Ca 2+ overload and MPTP opening [12]. According to another point of view, the inhibition of mitochondrial Ca 2+ accumulation was explained by mitochondrial membrane depolarization and fall of Δ Ψ m induced by NO [14].
Modern experiments also demonstrate that moderate doses of nitroglycerine increase CRC and prevent Ca 2+ -dependent MPTP opening. NO and reactive nitrogen species are considered as the mediators, which may improve mitochondrial calcium handling and suppress pore opening [15].
Diverse effects of NO donors and L-arginine on mitochondrial respiration, Δ Ψ m, and MPTP are generally explained by the mechanisms based on the redox regulation of mitochondrial processes with the involvement of S-nitrosylation [16][17][18] and S-glutationylation [19][20][21][22] of numerous proteins.
However, potential intramitochondrial mechanisms of protection may also include some signaling chains involved in calcium and NO interplay. Recently Seya and coauthors discovered that cardiac mitochondrial protein fraction possesses PKG activity [23] and provides cGMP synthesis triggered by NO donors [24]. Besides, the hydrolysis of cGMP by mitochondrial cyclic nucleotide phosphodiesterase PDE2A was demonstrated in brain and liver mitochondria in independent experiments [25]. All these data indicate that the elements Ca 2+ -dependent mtNOS/ GC/PKG-signaling system (mtNOS-SS) may operate in mitochondria. However, according to . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/443986 doi: bioRxiv preprint 4 the results presented by Seya and coauthors [23], SNAP or 8-Bromo-cGMP induced calciumdependent CytC release and apoptosis, while the inhibitors of NOS, GC, and PKG prevented these effects. These results contradict to the generally admitted pro-survival action of cytosolic NOS/GC/PKG1-SS directed at the prevention of MPTP opening and cell death [26][27][28][29].
MPTP is considered as a common pathway leading to the development of apoptosis and necrosis, which are observed in myocardial ischaemia-reperfusion (I/R) [28][29][30][31][32][33][34], acute steatohepatits and oxidative stress [31, 35,36], and under the action of various drugs and toxins [31,37]. Calcium and reactive oxygen species (ROS) are recognized as key mediators involved in MPTP opening [31,32]. However, I/R and some other pathologic processes are characterized not only by a steep rise of Ca 2+ and ROS but also by the accumulation of long-chain fatty acids [38][39][40] and their carnitine derivatives [38], which are often considered as triggering agents evoking mitochondrial calcium overload and oxidative stress [31, 40,41]. Long-chain fatty acids and acylcarnitines are oxidized in mitochondria as long-chain acyl CoA's, which inhibit various enzymes including NAD(P)H-dependent dehydrogenases [42][43][44]. Our preliminary results indicate that D,L-palmitoylcarnitine (PC) excess induces CsA-dependent dissipation of Δ Ψ m, which may be prevented by SNAP [45].
In this work, we investigated the involvement of mitochondrial Ca 2+  The data show that the application of 10-50 µM SNP markedly activate steady state respiration rate. The maximal increase in VO2ss by 39% was observed at 20 µM SNP ( Fig. 2A). In a like manner, VO2ss increased by 32% at low concentration of L-arginine (Fig. 2B, 20µM). which may be based on competitive inhibition of COX by the excess of NO [1,6].

Involvement of mtNOS-SS in the activation of mitochondrial respiration
Detected activation of the respiration by low concentrations of SNP, SNAP, and L-arginine contradicts the known inhibition of mitochondrial respiration by these substances [1][2][3][4][5][6][7]. We suggested that mtNOS-SS may be involved in the activation of mitochondrial respiration by SNP, SNAP, and L-arginine. In the next experiments we applied selective inhibitors of mtNOS-SS to evaluate the influence of this signaling system on the respiration. The results presented on  Figure 2A shows that ODQ and KT prevented the activating effect of 20 µM SNP by lowering VO 2 ss by 27% and 34%, respectively. Also, the activation of respiration . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/443986 doi: bioRxiv preprint 6 by 20 µM L-arginine was not observed after the incubation of mitochondria with 7-NI, ODQ, and KT (Fig. 2B).

Dissipation of the mitochondrial potential and inhibition of respiration by PC. Protection provided by low concentrations of L-arginine
Previously we have shown that high concentrations of PC (above 50 µM) induced a steep

Impact of mtNOS-SS on mitochondrial calcium retention capacity
Calcium retention capacity (CRC) was used as second parameter, which may characterize the involvement of mtNOS-SS in MPTP control. The values of CRC were measured by the standard procedure of sequential loading the medium with 20 µM Ca 2+ (CaCl 2 ). Like to the experiments presented above (Fig. 4), PC (20 µM) was added to reinforce the effect of calcium on MPTP.
The inhibitor of PKG KT (2 µM) diminished CRC in comparison with control value by 30 % (Fig. 5A). The diagrams presented on Figs. 5B,C demonstrate the involvement of mtNOS-SS in The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/443986 doi: bioRxiv preprint 9 by increasing the values of two regulatory parameters (CRC and PC*) characterizing MPTP sensitivity to calcium and PC overload.
The application of the inhibitors of NOS, GC and PKG eliminated both effects, indicating the involvement of mtNOS-SS in the activation of respiration (Fig. 2) and deceleration of MPTP opening (Figs. 4, 5). Mitochondrial PKG may act as a final mediator involved in the activation of respiration and MPTP control. It is known that mitochondrial protein kinase A activates the respiration by phosphorylating the enzymes of respiratory chain [46][47][48][49]. A similar mechanism of action can also be inherent in PKG. It is worth to note that mtNOS-SS cannot be considered as a redundant element in the multi-level control of oxidative phosphorylation and MPTP. Being calcium-dependent, this signaling system may be involved in the functioning of several feedback and feedforward regulatory loops, two of which are directed toward the activation of mitochondrial respiration and MPTP control. In this case, mitochondrial PKG, along with cytosolic PKG1, may be considered as a final mediator of protection involved in MPTP control.

Calcium
It is well known that a number of kinases and phosphatases are transported into mitochondria and have here, as the targets, multiple proteins, including the components of MPTP complex [27,[49][50][51][52]. Special mechanisms provide the transport of these proteins [50]. We might suppose that GC and PKG are imported into mitochondria using like mechanisms. However, direct experimental data confirming the localization of PKG and GC inside of mitochondria are currently lacking. This issue requires further investigations.
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Materials and methods
All animal procedures were fulfilled in accordance with the EU directive 86/609/EEC and approved by the Ethics Committee at the Institute of Theoretical and Experimental Biophysics, RAS, Russia. Male (six-to eight-week-old) Wistar rats were kept under the same conditions in air-conditioned and ventilated rooms at 20-22°C with a 12 h/12 h light-dark cycle. All experiments were performed at 26°C. Liver mitochondria were isolated using standard techniques of differential centrifugation in the medium containing 300 mM sucrose, 1 mM EGTA, and 10 mM Tris-HCl (pH 7.4). Mitochondrial preparations were washed twice with the release medium containing no EGTA, resuspended in the medium of the same composition, and stored on ice as described earlier [45]. Mitochondria incubation medium contained: 125 mM The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/443986 doi: bioRxiv preprint 11 opening was induced by the sequential loading of the incubation medium with 20 µM Ca 2+ (CaCl 2 ) or D,L-palmitoylcarnitine (PC). In this way, the values of mitochondrial CRC and the threshold concentration of PC (PC*) were determined as total concentrations of added Ca 2+ and PC required for pore opening. To investigate the impact of mtNOS-SS on MPTP opening, we selected these values of CRC and PC* as regulatory parameters characterizing the sensitivity of MPTP to Ca 2+ and PC overload. Besides, to evaluate the involvement of mtNOS-SS in the regulation of mitochondrial respiration, we determined the rates of steady-state respirationVO2ss.
The activation of mtNOS-SS was produced by the application of L-arginine, NO donors, and Ca 2+ ; 7-NI, ODQ, and KT5823 were used to inhibit mtNOS, GC and PKG, correspondingly.    The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/443986 doi: bioRxiv preprint 18 performed on mitochondria preincubated with SNAP (blue and violet columns), SNAP + ODQ and SNAP + KT (red and green columns); (C) The influence of 500 µM L-arginine on PC* value (violet) and elimination of its effect by the inhibitors of NOS, GC and PKG (7-NI, ODQ and KT; brown, red and green columns, respectively). All concentrations are given in µM. L-arg = Larginine.Data represent mean ± S.E.M. n = 4. The compared pairs of PC* values are marked by horizontal lines placed above columns. Symbol * indicate p < 0.05.  The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/443986 doi: bioRxiv preprint