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The International Journal of Biochemistry & Cell Biology 49 (2014) 26–31

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

jo u r n al homep ag e: www.elsev ier .com/ locate /b ioce l

7 nicotinic acetylcholine receptors control cytochrome c releaserom isolated mitochondria through kinase-mediated pathways

alyna Gergalova1, Olena Lykhmus1, Sergiy Komisarenko, Maryna Skok ∗

alladin Institute of Biochemistry, 9, Leontovicha Str., Kyiv 01601, Ukraine

r t i c l e i n f o

rticle history:eceived 18 September 2013eceived in revised form1 December 2013ccepted 2 January 2014vailable online 9 January 2014

eywords:icotinic acetylcholine receptoritochondria

ytochrome c

a b s t r a c t

Nicotinic acetylcholine receptors are ligand-gated ion channels found in the plasma membrane of bothexcitable and non-excitable cells. Previously we reported that nicotinic receptors containing �7 sub-units were present in the outer membranes of mitochondria to regulate the early apoptotic events likecytochrome c release. Here we show that signaling of mitochondrial �7 nicotinic receptors affects intrami-tochondrial protein kinases. Agonist of �7 nicotinic receptors PNU 282987 (30 nM) prevented the effectof phosphatidyl inositol-3-kinase inhibitor wortmannin, which stimulated cytochrome c release in iso-lated mouse liver mitochondria, and restored the Akt (Ser 473) phosphorylation state decreased by either90 �M Ca2+ or wortmannin. The effect of PNU 282987 was similar to inhibition of calcium-calmodulin-dependent kinase II (upon 90 �M Ca2+) or of Src kinase(s) (upon 0.5 mM H2O2) and of protein kinase C.Cytochrome c release from mitochondria could be also attenuated by �7 nicotinic receptor antagonist

uperoxiderotein kinases

methyllicaconitine or �7-specific antibodies. Allosteric modulator PNU 120526 (1 �M) did not improvethe effect of agonist PNU 282987. Acetylcholine (1 �M) and methyllicaconitine (10 nM) inhibited super-oxide release from mitochondria measured according to alkalization of Ca2+-containing medium. It isconcluded that �7 nicotinic receptors regulate mitochondrial permeability transition pore formationthrough ion-independent mechanism involving activation of intramitochondrial PI3K/Akt pathway andinhibition of calcium-calmodulin-dependent or Src-kinase-dependent signaling pathways.

. Introduction

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ionhannels initially discovered in the neuromuscular junctions andsh electric organs and further found in the central and autonomicervous system, as well as in many non-excitable cells (Changeux,012). The growing evidence suggests a universal role of acetyl-holine (ACh), an evolutionary ancient mediator regulating vitalellular processes like proliferation, survival, adhesion and motilityWessler and Kirkpatrick, 2008). Consequently, receptors to ACh of

oth nicotinic and muscarinic type were found in representatives oflant and animal kingdoms (Kawashima and Fujii, 2008; Kummer

Abbreviations: ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; cyt, cytochrome c; MLA, methyllicaconitine; MPTP, mitochondria permeability tran-ition pore; ROS, reactive oxygen species; CaMKII, calcium-calmodulin-dependentinase; PKC, protein kinase C; PI3K, phosphatidylinositol-3-kinase.∗ Corresponding author at: Laboratory of Immunology of Cellular Receptors,epartment of Molecular Immunology, Palladin Institute of Biochemistry, 9, Leon-

ovicha Str., Kyiv 01601, Ukraine. Tel.: +380 44 234 33 54;ax: +380 44 279 63 65.

E-mail address: skok@biochem.kiev.ua (M. Skok).1 These two authors contributed equally to the paper.

357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.biocel.2014.01.001

© 2014 Elsevier Ltd. All rights reserved.

et al., 2008; Sugiyama and Tezuka, 2011), and their prototypes havebeen discovered in bacteria (Bocquet et al., 2007).

Structurally, the nAChRs are homo- or heteropentamers com-posed of various combinations of homologous subunits. Muscularreceptors are composed of (�1)2�1��(�) subunits and are simi-lar in all parts of the body. Neuronal type nAChRs, which are alsoexpressed in non-excitable cells, are much more heterogeneous:they consist of �2–�10 and �2–�4 subunits combined either ashomomers (�7, �8, �9) or heteromers (�3�2, �3(�5)�4, �4�2,etc.; reviewed in Kalamida et al. (2007), Albuquerque et al. (2009).Homomeric �7 nAChRs are considered to be the most evolutionaryancient (Ortells and Lunt, 1995); they are found in both neuronsand non-excitable cells to control the cell viability (Parada et al.,2010; Resende and Adhikari, 2009); motility (Chernyavsky et al.,2004), activation (Koval et al., 2011), as well as angiogenesis (Ariaset al., 2009) and inflammation (De Jonge and Ulloa, 2007).

Previously we reported the presence of nAChRs composedof �7 subunits in mouse liver mitochondria and in mitochon-dria of several cell lines. This novel and unexpected findingwas confirmed by various experimental approaches includ-

ing electron and confocal fluorescent microscopy, the bindingof �7-specific antibodies and toxins, as well as the use ofmitochondria from �7−/− animals lacking such binding (Gergalovaet al., 2012; Kalashnyk et al., 2012). The nAChRs were found in

l of Biochemistry & Cell Biology 49 (2014) 26–31 27

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Fig. 1. The effects of kinase inhibitors and PNU 282987 on cyt c released in responseto 90 �M Ca2+ (A) or 0.5 mM H2O2 (B). Ctrl – mitochondria without Ca2+ or H2O2; Noinh – no kinase inhibitor added; Kn – KN62 (1 �M, CaMKII inhibitor); SB – SB 202190(10 �M, p38 inhibitor); Bis – bisindolylmaleimide (50 nM, PKC inhibitor); PP2 (1 �M,

G. Gergalova et al. / The International Journa

he outer membrane of mitochondria and were shown to influencea2+ accumulation and early pro-apoptotic events like cytochrome

(cyt c) release. However, the mechanism of �7 nAChR signaling initochondria remained unclear. In the present work we addressed

his question by studying the effects of various �7 nAChR ligandsn cyt c release and related intramitochondrial signaling pathways.

. Materials and methods

.1. Animals

We used male C57BL/6J mice, 3–5 months of age, and Chinchillaabbits (to produce cyt c-specific antibodies). Animals were kept inhe animal facility of Palladin Institute of Biochemistry, housed in auiet, temperature-controlled room (22–23 ◦C) and were providedith water and dry food pellets ad libitum. Before removing the liverice were sacrificed by cervical dislocation. All procedures con-

ormed to the guidelines of the Animal Care and Use Committee ofalladin Institute of Biochemistry. Before starting the experiments,he protocols were approved by the IACUC (Protocol 1/7-421).

.2. Reagents

All reagents were of chemical grade and were purchased fromigma–Aldrich unless specially indicated. Antibodies against totalkt (Akt1/2/3 (H-136)) and phosphorylated Akt (p-Akt1/2/3 (Ser73)) were from Santa-Cruz Biotechnology. Antibodies against7(1–208) and �7(179–190) nAChR fragments were obtained andharacterized by us previously (Skok et al., 1999; Lykhmus et al.,010). To obtain cyt c-specific antibodies a rabbit was immunizedwice with 0.3 mg of bovine cyt c (the first time with complete andhe second one with incomplete Freund’s adjuvant, with the inter-al of 3 weeks). A week after the second immunization, the bloodas collected from the ear vein and immunoglobulins were puri-ed from the serum using Protein A-conjugated Sepharose 4B. Theurified immunoglobulins were biotinylated according to standardrocedure (Harlow and Lane, 1988).

The following �7 nAChR ligands were used: acetylcholinehloride, PNU 282987, PNU 120596, methyllicaconitine (all fromigma–Aldrich).

The following protein kinase inhibitors were used: KN62calcium/calmodulin-dependent kinase inhibitor); SB 202190 (p38nhibitor); wortmannin (phosphatidylinositol-3-kinase inhibitor);P2 (Src kinase inhibitor), all from Sigma–Aldrich; bisindolyl-aleimide (protein kinase C inhibitor), from Cell Signaling

echnology.

.3. Mitochondria purification and fractionation

Mitochondria isolation from the mouse liver and purification ofheir outer membranes were performed by differential ultracen-rifugation according to standard procedure (Sottocasa et al., 1967)nd as described previously (Gergalova et al., 2012). To prepareetergent lysates, the membranes were freezed at −70 ◦C, thawednd treated with the lysing buffer (0.01 M Tris–HCl, pH 8.0; 0.14 MaCl; 0.025% NaN3; 1% Tween 20 and protease inhibitors cock-

ail) for 2 h on ice upon intensive stirring. The resulting lysateas cleared by centrifugation (20 min at 25,000 × g) and dialysed

gainst PBS containing 0.025% NaN3 and protease inhibitors. Therotein concentration was established by BCA assay (Thermo Sci-ntific).

.4. Cyt c release studies

The purified mitochondria (120 �g of protein per ml) werencubated with either 90 �M CaCl2 or 0.5 mM H2O2 for 2 min at

Src kinase inhibitor). The inhibitors were added to mitochondria 10 min prior toCa2+; PNU 282987 (30 nM) was applied just before Ca2+. Each column correspondsto mean ± S.E. (n = 3).

room temperature and were immediately pelleted by centrifuga-tion (10 min, 7000 × g) at 4 ◦C. The incubation medium contained10 mM HEPES, 125 mM KCl, 25 mM NaCl, 5 mM sodium succinateand 0.1 mM Pi(K), pH 7.4. The nAChR ligands (agonists, antago-nist, allosteric modulator or antibodies) were applied 2–3 min priorto apoptogenic agents. The kinase inhibitors were added either3–10 min before or simultaneously with PNU 282987 (as specifiedin the figures). The mitochondria supernatants were collected andtested for the presence of cytochrome c (cyt c) by sandwich assay asdescribed previously (Gergalova et al., 2012). Experimental valuesof optical density (OD 490 nm) shown in Figs. 1 and 3 were withinthe linear part of the calibration curve built with bovine cyt c.

2.5. Superoxide release studies

The purified mitochondria were resuspended in RPMI 1640medium and were incubated in the presence or absence ofacetylcholine (ACh, 1 mM), methyllicaconitine (MLA, 10 nM),�7(179–190) antibody (6.0 �g/ml) or their combinations for10 min. Similar medium samples without mitochondria were pre-pared as negative controls. The light absorbance (optical density,� = 545 nm) of phenol red within the medium was read usingStatFax-2100 Microplate reader (Awareness Technology, USA). ThepH values were calculated according to calibration curve built withRPMI 1640 medium of pH established with conventional pH-meter(Orion 501, USA).

2.6. Study of Akt phosphorylation

Mitochondria were purified by usual procedure. The suspension

obtained from one liver was distributed into five equal samples(100 �l) in the separation medium. The samples were treated withCa2+ (90 �M); Ca2+ + PNU 282987 (30 nM); wortmannin (1 �M);wortmannin + PNU 282987 or PNU 282987 alone, respectively, for

2 l of Biochemistry & Cell Biology 49 (2014) 26–31

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Fig. 2. The effect of PNU 282987 on cyt c released in response to either 90 �M Ca2+

or 1 �M wortmannin (Wor, PI3K inhibitor) (A) and on the level of mitochondrial Aktphosphorylation influenced by either Ca2+ or wortmannin (B). In (B) PNU 282987

8 G. Gergalova et al. / The International Journa

0 min at room temperature. Then the samples were centrifugedor 10 min at 7,000 × g. The pellets were resuspended in 150 �l ofysing buffer (0.01 M Tris–HCl, pH 8.0; 0.14 M NaCl; 0.025% NaN3;% Tween 20, protease inhibitors cocktail, 1 mM NaF and 1 mMa3VO4), freezed at −70 ◦C and lysed during 1 h on ice. The resulting

ysates were again centrifuged at 25,000 × g for 30 min, the pel-ets were withdrawn, while the supernatants were used for furtherxamination. The protein content in the supernatants was estab-ished using BCA assay and the level of Akt phosphorylation wasurther studied by sandwich ELISA.

The immunoplates were coated with the Akt-specific antibody60 �g/ml) for 2 h at 37 ◦C and were subsequently blocked with% BSA. The mitochondria lysates were applied in triplicates at00 �g/ml (the optimal concentrations were established in prelim-

nary experiments) for 2 h at 37 ◦C. The plates were washed withater and the second biotinylated (phospho Ser 473) Akt-specific

ntibody (60 �g/ml) was applied for additional 2 h at 37 ◦C. Afterubsequent washing, the plates were treated with streptavidin-eroxidase (45 min) and o-phenylendiamin-containing substrateolution, the absorbance being read at 490 nm with Stat-Fax 2100icroplate reader.

. Results

.1. The involvement of mitochondrial protein kinases initochondrial pore formation and its regulation by ˛7 nAChR

To elucidate signaling pathways of �7 nAChR in mitochondriae considered the possible involvement of mitochondrial pro-

ein kinases. The cytoplasmic domains of nAChRs expressed in thelasma membrane of neurons are coupled with multiple proteininases phosphorylating them on serine/threonine and tyrosineesidues (Pacheto et al., 2003; Wang et al., 2004; Parada et al.,010) to regulate cell proliferation or survival (Dajas-Bailador andonnacott, 2004). Experimental data published quite recently

howed the presence of various protein kinases in mitochondriaKowalczyk and Zabłocka, 2008; Antico Arciuch et al., 2009) andheir connection to mitochondrial permeability transition poreMPTP) through which cyt c is released in response to apop-ogenic stimuli (Miura et al., 2010). We used a set of specificnhibitors to reveal which kinases were involved in MPTP forma-ion upon the effect of either Ca2+ or H2O2 and which of them wereffected upon �7 nAChR ligation. As shown in Fig. 1A, inhibition ofalcium/calmodulin-dependent kinase (CaKMII, inhibitor KN62) orrotein kinase C (PKC, inhibitor Bis) diminished the amount of cyt

released upon 90 �M Ca2+ addition to mitochondria. The effectsf p38 and Src kinase inhibitors (SB202190 and PP2, respectively)ere at the level of DMSO, in which they were diluted. PNU 282987

ttenuated cyt c release stimulated by Ca2+ in the presence of p38r Src inhibitors and did not further change the effects of CaKMIInd PKC inhibitors. This data indicated that Ca2+ stimulated MPTPormation through CaMKII and PKC, while �7 nAChR could inhibithe pathway(s) involving these enzymes.

Cyt c release stimulated by H2O2 was not affected with CaKMIInhibitor KN62, but was efficiently attenuated with PKC and Srcnhibitors (Bis and PP2, respectively). The effect of p38 inhibitorB202190 was again at the level of DMSO (Fig. 1B). PNU 282987ttenuated the effect of H2O2, did so in the presence of p38 and CaK-II inhibitors and potentiated the effects of Src and PKC inhibitors.

herefore, MPTP induction upon H2O2 did not involve CaMKII, butas dependent on Src kinase(s) and PKC. The effect of PNU 282987

as similar to those of PKC and Src inhibitors, so �7 nAChR could

nhibit PKC- and Src-dependent pathway(s).Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortman-

in did not influence cyt c release stimulated by either Ca2+ or H2O2;

was added just before, 3 or 10 min after wortmannin application. Ctrl – mitochon-dria without Ca2+ or wortmannin. Each column corresponds to mean ± S.E. (n = 3).*p < 0.05; **p < 0.005; ***p < 0.0005.

moreover, it induced cyt c release itself. PNU 282987 prevented cytc release only when added to mitochondria before wortmannin orsoon after it (3 min, Fig. 2A). It was concluded that MPTP forma-tion was negatively regulated by mitochondrial PI3K, and inhibitingthis enzyme was sufficient to induce cyt c release in the absence ofany other apoptogenic stimuli. PNU 282987 prevented the effect ofwortmannin, therefore, �7 nAChR helped keep PI3K active.

In the whole cell, PI3K interacts with protein kinase B (Akt)to enable its phosphorylation by mTORC2 and phosphoinositide-dependent kinase 1 (PDPK1). Expression of active Akt targeted tomitochondria was found to be sufficient to significantly reducethe release of cyt c from mitochondria (Mookherjee et al., 2007).We studied the level of mitochondrial Akt phosphorylation uponthe effects of Ca2+ or wortmannin in the presence or absence ofPNU 282987. In the sandwich assay, Akt was captured from themitochondria detergent lysate with Akt-specific antibody and itsphosphorylation was revealed with the antibody against phospho-rylated Ser 473, the first site of Akt phosphorylation by mTORC2.Such an approach is advantageous compared to immunoprecipitat-ion and Western blot because its results can be easily calculated.As shown in Fig. 2B, Akt was phosphorylated in intact mitochon-dria and the level of phosphorylation was significantly decreasedby either Ca2+ or wortmannin. PNU 282987 restored the Aktphosphorylation state decreased by both Ca2+ and wortmannin

and even non-significantly increased its phosphorylation in non-treated mitochondria. This data indicated that MPTP formationwas accompanied with Akt dephosphorylation (due to PI3K inhibi-tion), whereas the signal from �7 nAChR generated by PNU 282987

G. Gergalova et al. / The International Journal of Biochemistry & Cell Biology 49 (2014) 26–31 29

Fig. 3. The effects of PNU 282987, MLA or PNU 282987 in the presence of allostericmodulator PNU 120596 (1 �M) (A) and of �7(1–208)- or �7(179–190)-specific anti-b 2+

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Fig. 4. The effect of ACh (1 mM), MLA (10 nM) or �7(179–190)-specific antibody

odies (Ab) (B) on cyt c released from mitochondria under the effect of Ca (90 �M).S – non-specific rabbit IgG. Each point on the curve corresponds to mean ± S.E.

n = 3).

inding resulted in restoration of PI3K activity and, subsequently,kt phosphorylation.

.2. The mechanism of anti-apoptotic ˛7 nAChR functioning initochondria

The �7 nAChRs are permeable to both mono- and bivalentations including Ca2+, which can trigger multiple intramitochon-rial processes. However, our preliminary data demonstrated thatnti-apoptotic effect of �7 nAChR in mitochondria did not dependn extramitochondrial Ca2+ (Gergalova et al., 2012) and nAChR-pecific ligands did not affect mitochondria membrane potentialGergalova and Skok, 2011). This data suggested that �7 nAChRsrigger intramitochondrial events by ion-independent mechanism.o explore this in more details, we applied �7-specific ligandsffecting the nAChR in different ways in the assay of cyt c releaserom isolated mitochondria stimulated with 90 �M Ca2+. This assays highly specific for mitochondria and allows neglecting the poten-ial contamination of mitochondria preparation with other liverissue components.

In our initial studies, �7-specific agonists (choline orNU282987) inhibited cyt c release from mitochondria stim-lated with either 90 �M CaCl2 or 0.5 mM H2O2 and their effectsere withdrawn with �7-specific antagonist MLA (Gergalova et al.,

012). Further experiments demonstrated that MLA could alsottenuate cyt c release from mitochondria. The dose-dependenturves presented in Fig. 3A show that PNU282987 taken at 30 nMthat is close to its binding constant to �7 nAChR (26 nM; Sharplesnd Wonnacott, 2001)) inhibited about 70% of cyt c release that

as close to its plateau effect (80% of inhibition). MLA effect at

0 nM (specific to both homomeric and heteromeric �7 nAChRs)as significantly smaller than that of PNU282987 but still quite

vident (about 50%). The allosteric modulator PNU 120596, which

(6 �g/ml) on superoxide anion release from isolated mitochondria in Ca2+-containing RPMI 1640 medium (measured according to the medium pH change).Each column corresponds to mean ± S.E. (n = 3). *p < 0.05; **p < 0.005.

was shown to significantly prolong the �7 ion channel openstate (Williams et al., 2011, 2012) did not improve the effect ofPNU 282987 at any dose. Moreover, the combined effect of theagonist and allosteric modulator was smaller than that of theagonist alone. However, PNU 120596 itself slightly inhibited cyt crelease (about 13%). This data indicated that the open state of �7nAChR ion channel was not critical to attenuate cyt c release frommitochondria.

Finally, we showed that �7 nAChR-specific antibodies alsoinhibited cyt c release from mitochondria stimulated by Ca2+

(Fig. 3B). The antibody effect was dose-dependent and was strongerwith the antibody generated against the whole extracellulardomain �7(1–208) compared to that against a definite epitope�7(179–190). The �7(179–190) fragment belongs to the nAChRloop C critical for the binding of both agonists and competi-tive antagonists (Brejc et al., 2001). Previously we found that�7(179–190)-specific antibody reduced ACh-induced membranecurrents in the neurons of superior cervical ganglia of the rat (Skoket al., 1999) and competed with MLA for the binding to the neu-rons of submucosal plexus of the guinea-pig (Glushakov et al.,2004). The antibody raised against �7(1–208) domain could bindmultiple extracellular epitopes including 179–190 (Lykhmus et al.,2010). The data presented here demonstrate that binding the �7nAChR with the antibody was sufficient to prevent cyt c releasefrom mitochondria.

3.3. The effect of nAChR ligation on superoxide release frommitochondria

MPTP formation and cyt c release from mitochondria are facil-itated by reactive oxygen species (ROS) generation and releaseupon Ca2+ overload (Circu and Aw, 2010). Superoxide anion (O2

−)reacts with water to generate OH− ions resulting in media alkaliza-tion. We incubated isolated mitochondria in RPMI 1640 mediumcontaining 0.6 mM Ca2+ (Ca(NO3)2) in the presence or absence ofdifferent nAChR ligands and monitored pH of the medium by thelight absorbance of phenol red. As shown in Fig. 4, mitochondriaincubated in the medium during 10 min raised its pH compared tocontrol medium by �pH = 0.3. Both ACh (1 mM) and MLA (10 nM)

decreased this difference almost twice. �7(179–190)-specific anti-body did not affect the pH itself but prevented the effect of ACh.These data were in good accordance with the effect of �7 nAChRligands on cyt c release. No direct antibody effect on O2

− release

30 G. Gergalova et al. / The International Journal of Bi

Fig. 5. The suggested scheme of �7 nAChR involvement in MPTP-related signalingin mitochondria. Ca2+ penetrates the mitochondria intermembrane space throughVDAC and affects CaKMII to stimulate (through PKC) MPTP formation and cyt crelease. H2O2 penetrates mitochondria directly through the outer membrane (OM)and affects Src-kinase(s) to stimulate MPTP formation and cyt c release (also throughPKC). For simplicity, VDAC is presented as a part of MPTP. PI K prevents MPTP for-mr

(dt

4

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ninnc

of the pentameric ligand-gated ion channel superfamily. J Biol Chem

3

ation and cyt c release. �7 nAChR helps keep PI3K active and, possibly, negativelyegulates Src kinase(s) and CaKMII.

compared to obvious effect on cyt c release) could indicate thatifferent kinds of �7 nAChR signaling were required to preventhese two processes (to be discussed below).

. Discussion

The main finding presented in this paper is that mitochondrial7 nAChRs regulate cyt c release by engaging intramitochondrialrotein kinases involved in MPTP formation. The latter occurredhrough CaKMII upon Ca2+ overload and through Src kinase(s)pon H2O2 application, involved PKC and PI3K. �7 nAChR obviouslyctivated mitochondrial PI3K/Akt pathway and might also inhibitaKMII or Src kinase(s) pathways. This is in accord with the databtained in the whole cells where �7 nAChR signaling up-regulatedcl-2 and down-regulated caspase-3 expression through PI3K andkt resulting in cell survival (Dajas-Bailador and Wonnacott, 2004).7 nAChR agonists also attenuated cyt c release stimulated bythanol in rat hippocampal neuronal cultures demonstrating thenvolvement of mitochondria (Li et al., 2002). Our data indicate that7 nAChRs expressed in mitochondria activate signaling cascadesimilar to those engaged by the nAChRs of the plasma membrane.he suggested scheme of �7 nAChR involvement in regulating cyt

release from mitochondria is presented in Fig. 5.Obviously, the targets of mitochondrial protein kinases are dif-

erent from those of cytosolic ones. Recent investigations havehown that mitochondrial protein kinases directly phosphory-ate pro- and anti-apoptotic proteins, as well as VDAC and ANTKowalczyk and Zabłocka, 2008). Beside other targets, Akt phos-horylates Bcl-xS, a pro-apoptotic protein located in the outerembrane of mitochondria, to prevent its association with VDAC

nd involvement in MPTP (Wei et al., 2009). This can explain why7 nAChR prevents MPTP formation and cyt c release from iso-

ated mitochondria by maintaining the Akt active (phosphorylated)tate.

Another important finding is that the mechanism by which �7AChR engages mitochondrial kinases and prevents ROS generation

s ion-independent and could be triggered by the agonist, antago-ist or antibody binding. Such mode of the nAChR functioning isot completely new, because it was previously shown that intra-ellular Ca2+ raise could be induced with MLA or �-bungarotoxin

ochemistry & Cell Biology 49 (2014) 26–31

in non-excitable cells like the leukocytes or monocytes (Razani-Boroujerdi et al., 2007; Hecker et al., 2009). In both cases, therole of nicotinic receptors was to regulate intracellular signalingof either T cell antigen-specific receptor or purinergic P2X7 recep-tor in obviously ion-independent manner. The data presented hereallow suggesting that this is also the case with mitochondrial �7nAChRs. Previously we reported that �7 nAChRs were closely con-nected to voltage-dependent anion channels (VDAC), the maincomponents of mitochondria outer membrane (Gergalova et al.,2012). VDAC itself functions as an ion channel and influencesMPTP formation either directly, by oligomerization and couplingwith ANT, cyclophillin D and Bcl-2 family members (Keinan et al.,2010), or indirectly, in a yet non-identified way. Superoxide anionis also released into cytosol through VDAC (Han et al., 2003;Park et al., 2011) that facilitates cyt c release from mitochondria(Ott et al., 2007). Our data allow suggesting that mitochondrial�7 nAChRs influence VDAC functioning, possibly, through con-formational changes within the nAChR intramitochondrial partcaused by the external (cytoplasmic) ligand binding. Low molec-ular weight ligands (ACh, PNU282987 and MLA) could affect bothVDAC permeability (superoxide release) and MPTP formation (cyt crelease), while the antibody affected MPTP formation but not VDACpermeability itself. This kind of signaling does not contend thatmitochondrial �7 nAChRs are invaluable ion channels. The ion per-meability can be critical for their other, yet non-identified activities.

In whole, the data presented here demonstrate that �7 nAChRsexpressed in the outer membrane of mitochondria control theearly events of mitochondria-driven apoptosis by regulating theactivity of mitochondrial kinases, primarily of PI3K/Akt path-way. This means that mitochondrial nAChRs form an additionalline of defense to support the cellular viability depending onthe availability of intracellular cholinergic ligands. The lattercould be either choline present in the cytosol at 30–50 �M(Alkondon and Albuquerque, 2006) or ACh formed by mitochon-drial choline acetyltransferase. Also, we cannot exclude the roleof endogenous peptide regulators like SLURP-1 (secreted mam-malian Ly6/urokinase plasminogen-type activator receptor-relatedprotein-1) shown to trigger the �7 nAChR-mediated pathways innon-excitable cells (Moriwaki et al., 2007; Tjiu et al., 2011). Thepresence of intracellular ligands is expected to stimulate mito-chondrial �7 nAChRs, engage intramitochondrial kinases and keepmitochondria resistant to apoptogenic agents. The demonstratedincreased susceptibility of mitochondria to calcium-induced per-meability transition upon choline deficiency (Teodoro et al., 2008)supports this scenario.

References

Albuquerque EX, Pereira EF, Alkondon M, Rogers SW. Mammalian nicotinic acetyl-choline receptors: from structure to function. Physiol Rev 2009;89:73–120.

Alkondon M, Albuquerque EX. Subtype-specific inhibition of nicotinic acetyl-choline receptors by choline: a regulatory pathway. J Pharmacol Exp Ther2006;318:268–75.

Antico Arciuch VG, Alippe Y, Carreras MC, Poderoso JJ. Mitochondrial kinases in cellsignaling: facts and perspectives. Adv Drug Deliv Rev 2009;61(14):1234–49.

Arias HR, Richards VE, Ng D, Ghafoori ME, Le V, Mousa SA. Role of non-neuronalnicotinic acetylcholine receptors in angiogenesis. Int J Biochem Cell Biol2009;41(7):1441–51.

Bocquet N, Prado de Carvalho L, Cartaud J, Neyton J, Le Poupon C, Taly A, et al. Aprokaryotic proton-gated ion channel from the nicotinic acetylcholine receptorfamily. Nature 2007;445:116–9.

Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van der Oost J, Smit AB, et al.Crystal structure of Ach-binding protein reveals the ligand-binding domain ofnicotinic receptors. Nature 2001;411:269–76.

Changeux JP. The nicotinic acetylcholine receptor: the founding father

2012;287(48):40207–15.Chernyavsky AI, Arredondo J, Marubio LM, Grando SA. Differential regulation of

keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptorsubtypes. J Cell Sci 2004;117:5665–79.

l of Bi

C

D

D

G

G

G

H

H

H

K

K

K

K

K

K

K

L

L

M

M

G. Gergalova et al. / The International Journa

ircu ML, Aw TY. Reactive oxygen species, cellular redox systems and apoptosis.Free Radic Biol Med 2010;48(6):749–62.

ajas-Bailador F, Wonnacott S. Nicotinic acetylcholine receptor and the regulationof neuronal signaling. Trends Pharmacol Sci 2004;25(6):317–24.

e Jonge WJ, Ulloa L. The alpha7 nicotinic acetylcholine receptor as a pharmacolog-ical target for inflammation. Br J Pharmacol 2007;151:915–29.

ergalova G, Skok M. Study of nicotine effects on mitochondria membrane poten-tial: participation of nicotinic acetylcholine receptors. Ukrainskiy BiochimichniyZhurnal 2011;83(5):13–21 [Ukrainian].

ergalova GL, Lykhmus OYu, Kalashnyk OM, Koval LM, Chernyshov VO, KryukovaEA, et al. Mitochondria express �7 nicotinic acetylcholine receptors to regulateCa2+ accumulation and cytochrome c release: study on isolated mitochondria.PLoS ONE 2012;e7(2):e31361.

lushakov AV, Voytenko LP, Skok MV, Skok VI. Distribution of neuronal nicotinicacetylcholine receptors containing different alpha-subunits in the submucosalplexus of the guinea-pig. Auton Neurosci 2004;110(1):19–26.

an D, Antunes F, Canali R, Rettori D, Cadenas E. Voltage-dependent anion channelscontrol the release of the superoxide anion from mitochondria to cytosol. J BiolChem 2003;278(8):5557–63.

arlow E, Lane D. Antibodies. A laboratory manual. Cold Spring Harbor, NY: ColdSpring Harbor Laboratory; 1988.

ecker A, Mikulski Z, Lips KS, Pfeil U, Zakrzewicz A, Wilker S, et al. Upregulationof acetylcholine synthesis and paracrine cholinergic signaling in intravascu-lar transplant leukocytes during rejection of rat renal allografts. J Leukoc Biol2009;86:13–22.

alamida D, Poulas K, Avramopoulou V, Fostieri E, Lagoumintzis G, Lazaridis K, et al.Muscle and neuronal nicotinic acetylcholine receptors. Structure, function andpathogenicity. FEBS J 2007;274(15):3799–845.

alashnyk OM, Gergalova GL, Komisarenko SV, Skok MV. Intracellular localization ofnicotinic acetylcholine receptors in human cell lines. Life Sci 2012;91:1033–7.

awashima K, Fujii T. Basic and clinical aspects of non-neuronal acetylcholine:overview of non-neuronal cholinergic systems and their biological significance.J Pharmacol Sci 2008;106(2):167–73.

einan N, Tyomkin D, Shoshan-Barmatz V. Oligomerization of the mitochondrialprotein voltage-dependent anion channel is coupled to the induction of apopto-sis. Mol Cell Biol 2010;30:5698–709.

oval L, Lykhmus O, Zhmak M, Khruschov A, Tsetlin V, Magrini E, et al. Differen-tial involvement of �4�2�7 and �9�10nicotinic acetylcholine receptors in Blymphocyte activation in vitro. Int J Biochem Cell Biol 2011;43:516–24.

owalczyk JE, Zabłocka B. Protein kinases in mitochondria. Postepy Biochem2008;54:209–16.

ummer W, Lips KS, Pfeil U. The epithelial cholinergic system of the airways. His-tochem Cell Biol 2008;130(2):219–34.

i Y, Meyer EM, Walker DW, Millard WJ, He YJ, et al. Alpha7 nicotinic receptor activa-tion inhibits ethanol-induced mitochondrial dysfunction, cytochrome c releaseand neurotoxicity in primary rat hippocampal neuronal cultures. J Neurochem2002;81:853–8.

ykhmus O, Koval L, Pavlovych S, Zouridakis M, Zisimopoulou P, Tzartos S, et al.Functional effects of antibodies against non-neuronal nicotinic acetylcholinereceptors. Immunol Lett 2010;128:68–73.

iura T, Tanno M, Sato T. Mitochondrial kinase signalling pathways in myocar-dial protection from ischemia/reperfusion-induced necrosis. Cardiovasc Res

2010;88(1):7–15.

ookherjee P, Quintanilla R, Roh M-S, Zmijewska AA, Jope RS, JohnsonGVW. Mitochondrial-targeted active akt protects sh-sy5y neuroblastomacells from staurosporine-induced apoptotic cell death. J Cell Biochem2007;102(1):196–210.

ochemistry & Cell Biology 49 (2014) 26–31 31

Moriwaki Y, Yoshikawa K, Fukuda H, Fujii YX, Misawa H, Kawashima K. Immunesystem expression of SLURP-1 and SLURP-2, two endogenous nicotinic acetyl-choline receptor ligands. Life Sci 2007;80:2365–8.

Ortells M, Lunt G. Evolutionary history of the ligand-gated ion channel superfamilyof receptors. Trends Neurosci 1995;18:121–7.

Ott M, Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria, oxidative stress andcell death. Apoptosis 2007;12(5):913–22.

Pacheto MA, Pastoor TE, Wecker L. Phosphorylation of the a4 subunit of human a4b2nicotinic receptor:role of cAMP-dependent protein kinase A (PKA) and proteinkinase C (PKC). Brain Res Mol Brain Res 2003;114(1):65–72.

Parada E, Egea J, Romero A, del Barrio L, García AG, López MG. Poststress treatmentwith PNU282987 can rescue SH-SY5Y cells undergoing apoptosis via �7 nicotinicreceptors linked to a Jak2/Akt/HO-1 signaling pathway. Free Radic Biol Med2010;49(11):1815–21.

Park J, Lee J, Choi C. Mitochondrial network determines intracellular ROS dynam-ics and sensitivity to oxidative stress through switching inter-mitochondrialmessengers. PLoS ONE 2011;6(8):e23211.

Razani-Boroujerdi S, Boyd RT, Davila-Garcia MI, Nandi JS, Mishra NC, Singh SP, et al.T cells express alpha7-nicotinic acetylcholine receptor subunits that requirea functional TCR and leukocyte-specific protein tyrosine kinase for nicotine-induced Ca2+ response. J Immunol 2007;179(5):2889–98.

Resende RR, Adhikari A. Cholinergic receptor pathways involved in apoptosis, cellproliferation and neuronal differentiation. Cell Commun Signal 2009;7:20–5.

Sharples CGV, Wonnacott S. Neuronal nicotinic receptors. Tocris Rev 2001;19:1–12.

Skok MV, Voitenko LP, Voitenko SV, Lykhmus EY, Kalashnik EN, Litvin T, et al. Studyof � subunit composition of nicotinic acetylcholine receptor in the neuronsof autonomic ganglia of the rat with subunit-specific anti-�(181–192) peptideantibodies. Neuroscience 1999;93(4):1437–46.

Sottocasa GL, Kuylenstierna B, Ernster L, Bergstrand AJ. An electron-transport systemassociated with the outer membrane of liver mitochondria. A biochemical andmorphological study. Cell Biol 1967;32:415–38.

Sugiyama K, Tezuka T. Acetylcholine promotes the emergence and elonga-tion of lateral roots of Raphanus sativus. Plant Signal Behav 2011;6(10):1545–53.

Teodoro JS, Rolo AP, Duarte FV, Simoes AM, Palmeira CM. Differential alterations inmitochondrial function induced by a choline-deficient diet: understanding fattyliver disease progression. Mitochondrion 2008;8:367–76.

Tjiu JW, Lin PJ, Wu WH, Cheng YP, Chiu HC, Thong HY, et al. SLURP1 mutation-impaired T-cell activation in a family with mal de Meleda. Br J Dermatol2011;164(1):47–53.

Wang K, Hackett JT, Cox ME, van Hoek M, Linstrom JM, Parsons SJ. Regulation of theneuronal nicotinic acetylcholine receptor by Src family tyrosine kinases. J BiolChem 2004;279(10):8779–86.

Wei Z, Qi J, Dai Y, Bowen WD, Mousseau DD. Haloperidol disrupts Akt signalling toreveal a phosphorylation-dependent regulation of pro-apoptotic Bcl-XS func-tion. Cell Signal 2009;21(1):161–8.

Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: the non-neuronal cholin-ergic system in humans. Br J Pharmacol 2008;154(8):1558–71.

Williams DK, Peng C, Kimbrell MR, Papke RL. Intrinsically low open probability of �7nicotinic acetylcholine receptors can be overcome by positive allosteric mod-ulation and serum factors leading to the generation of excitotoxic currents at

physiological temperatures. Mol Pharmacol 2012;82(4):746–59.

Williams DK, Wang J, Papke RL. Investigation of the molecular mechanism ofthe �7 nicotinic acetylcholine receptor positive allosteric modulator pnu-120596 provides evidence for two distinct desensitized states. Mol Pharmacol2011;80:1013–32.