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FEBS Letters 585 (2011) 429–434
journal homepage: www.FEBSLetters.org
Anandamide inhibits oxidative phosphorylation in isolated liver mitochondria
Patrizia Zaccagnino a, Angela Corcelli a, Maristella Baronio a, Michele Lorusso a,b,⇑
a
b
Department of Medical Biochemistry, Biology and Physics, University of Bari, Policlinico, Piazza G. Cesare, 70124 Bari, Italy
Institute of Biomembrane and Bioenergetics, CNR, Bari, Italy
a r t i c l e
i n f o
Article history:
Received 2 December 2010
Revised 21 December 2010
Accepted 21 December 2010
Available online 25 December 2010
Edited by Peter Brzezinski
Keywords:
Anandamide
Mitochondrial respiratory chain
Membrane potential
FoF1 ATP synthase
a b s t r a c t
A study on the effect of anandamide (AEA) in energy coupling of rat liver mitochondria is presented.
Micromolar concentrations of AEA, while almost ineffective on substrate supported oxygen consumption rate and on uncoupler stimulated respiration, strongly inhibited the respiratory state
III. AEA did not change the rate and the extent of substrate generated membrane potential, but
markedly delayed rebuilding by respiration of the potential collapsed by ADP addition. Overall,
these data suggest that anandamide inhibits the oxidative phosphorylation process. Direct measurement of the FoF1 ATP synthase activity showed that the oligomycin sensitive ATP synthesis was
inhibited by AEA, (IC50, 2.5 lM), while the ATP hydrolase activity was unaffected. Consistently,
AEA did not change the membrane potential generated by ATP hydrolysis.
Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
Endocannabinoids are lipid mediators present in the central
nervous system (CNS) and in peripheral tissues at concentration
ranging from about 0.1 to 20 nmol/g [1]. They mimic the pharmacological effects exerted by D-9 tetrahydrocannabinol, the psycoactive component of the plant Cannabis sativa and its derivatives
marijuana and hashish [2].
N-Arachidonylethanolamine, named anandamide (AEA) from
‘‘ananda’’ the Sanskrit word for ‘‘bliss’’ is an important endocannabinoid belonging to the N-acyl-ethanolamine family (NAEs). It
is a derivative of arachidonic acid in which the carboxylic group
is bound to the amino group of ethanolamine by an amide bond.
AEA free concentration can increase markedly in tissues subjected to various insults [3–6], when a stimulus-dependent activation of N-acylphosphatidylethanolamine-specific phospholipase D
will release AEA from the lipid precursor N-arachidonoyl-phosphatidyl-ethanolamine.
AEA action is mediated by type-1 and type-2 cannabinoid
receptors. These receptors belong to the family of the seven
trans-membrane spanning receptors and are coupled to Gi/o proteins [7–9]. AEA is also considered a true ‘‘endovanilloid’’ because
Abbreviations: AA, arachidonic acid; AEA, N-Arachidonylethanolamine or
anandamide; NA-Gly, N-arachidonylglycine; CCCP, carbonyl cyanide-3 chlorophenylhydrazone; FAAH, fatty acid amide hydrolase; Ap5, p1,p2-di (adenosine-50 )
pentaphosphate pentalithium salt
⇑ Corresponding author. Fax: +39 080 5448538.
E-mail address: [email protected] (M. Lorusso).
it binds with low affinity to the transient receptor potential cation
channel, subfamily V, member 1 [10].
AEA and other endocannabinoids play a central role in many
pathophysiological processes including the immune regulation,
the neurotransmission in the CNS [11,12] and the maintenance of
energy balance. In addition AEA is able to promote cell death by
apoptosis and to inhibit cell proliferation in different types of cells
[13–17].
AEA action is believed to be terminated upon its entrance into
the intracellular space where it is degraded by the enzyme fatty
acid amide hydrolase (FAAH) to arachidonic acid (AA) and ethanolamine, thus losing its biological activity. FAAH is a membranebound protein localised in cell membranes of various tissues such
as liver, brain, kidney, testis and small intestine where exhibits a
different degree of hydrolytic activity [18–21].
On the other hand, AEA appears to exhibit definite action within
the cell, based on the observation that many of the effects induced
by AEA occur independently of cannabinoid and vanilloid receptors
[22–24]. The finding that endocannabinoids and AEA in particular
are able to control both energy homeostasis and cell death by
the intrinsic pathway of apoptosis [25,26] has addressed the attention to a possible involvement of mitochondria in these effects.
Several reports have indeed shown that in some cell types, including neuronal and sperm cells, endocannabinoids influence directly
the functionality of mitochondria altering their integrity [27,28].
Furthermore it was reported that in rat heart mitochondria AEA
causes decrease in oxygen consumption and mitochondrial membrane potential, also altering the membrane permeability [29–31].
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2010.12.032
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P. Zaccagnino et al. / FEBS Letters 585 (2011) 429–434
This work was aimed at better understanding the interactions
of AEA with isolated mitochondria and its effect on mitochondrial
bioenergetics. We show here that AEA, used at low micromolar
concentrations, caused a strong and direct inhibition of the oligomycin sensitive FoF1 ATP synthase activity in isolated rat liver
mitochondria, without affecting the ATP hydrolase activity. Physiopathological implications and dysfunctions caused by AEA interaction with the liver ATP synthase are discussed.
2. Materials and methods
2.1. Chemicals
Phenylmethanesulfonyl fluoride (PMSF), oligomycin, carbonyl
cyanide-3 chlorophenylhydrazone (CCCP), rotenone, AA, NADP,
p1,p2-di (adenosine-50 ) pentaphosphate pentalithium (Ap5) salt
and bovine serum albumin (BSA) were purchased from Sigma (St.
Louis, MO). Arachidonyl ethanolamide (AEA) and N-arachidonylglycine (NA-Gly) were obtained from Cayman Chemical Company
(Ann Arbor, MI, USA). Hexokinase, phosphoenolpyruvate (PEP),
L-lactate dehydrogenase (LDH), pyruvate kinase (PK) and glucose6-phosphate dehydrogenase (G6P-DH) were from Roche Diagnostics Corporation (Indianapolis, IN, USA).
2.2. Isolation of mitochondria
Rat liver mitochondria were isolated by differential centrifugation using an isolation buffer containing 0.22 M mannitol, 75 mM
sucrose, 10 mM HEPES (pH 7.4), 1 mM EDTA and 0.25 mM PMSF
[32].
Protein concentration was determined by the Biuret method
using BSA as standard.
were suspended in 1 ml of Buffer A, supplemented with 5 lM safranin-O at 25 °C. The potential was generated by the addition of respiratory substrates 10 mM succinate (in the presence of 2 lg/ml
rotenone) or glutamate plus malate (10 mM each) or by the addition
of 1.5 mM ATP. Other details are specified in the legend to figures.
2.6. Measurement of FoF1 ATP synthase activity
Liver mitochondria were suspended at 0.25 mg protein/ml in
200 mM sucrose, 3 mM MgCl2, 1 mM EDTA, 10 mM KH2PO4, pH
7.4, 20 mM glucose, 5 units hexokinase, 300 lM Ap5, 2 lg/ml of
rotenone, at 25 °C. Five minutes after the addition, under aerobic
conditions, of 10 mM succinate and 0.3 mM ADP, the mitochondrial suspension was treated with 28% perchloric acid (w/v) and
centrifuged at 16 000g. After centrifugation, the supernatant
was neutralized with 60% KOH (w/v) and stored frozen at 20 °C.
After thawing and centrifugation at 16 000g, the supernatant
was added to a mixture containing 1 mM MgCl2, 150 mM Tris–Cl,
pH 7.4 and 7 units of glucose-6-phosphate dehydrogenase. 1 mM
NADP was added and the concentration of glucose-6-phosphate
was determined following NADP reduction at 340 nm using a Beckmann DU 7400 spectrophotometer.
2.7. Measurement of FoF1 ATPase activity
ATP hydrolase activity was measured by an ATP-regenerating
system [35]. Liver mitochondria were frozen and thawed three times
and suspended (at 0.1 mg protein/ml) in a buffer consisting of
375 mM sucrose, 75 mM KCl, 30 mM Tris–Cl, pH 7.4, 3 mM MgCl2,
2 mM PEP, 55 U/ml lactate dehydrogenase, 40 U/ml pyruvate kinase,
0.3 mM NADH. The reaction was started by the addition of 1 mM ATP
and the oxidation of NADH was followed at 340 nm.
3. Results
2.3. Measurement of oxygen consumption rate
3.1. Lipid analysis of rat liver mitochondria
The respiratory activity of liver mitochondria was measured
polarographically with a Clark-type electrode, in an all-glass reaction chamber magnetically stirred, at 25 °C. Rat liver mitochondria
were suspended at a final concentration of 0.25 mg/ml in Buffer A
(75 mM sucrose, 50 mM KCl, 30 mM Tris–Cl, (pH 7.4), 2 mM
KH2PO4, 2 mM MgCl2 and 10 lM EGTA). Resting state respiration
was started on the addition of 10 mM succinate in the presence
of 2 lg/ml rotenone or glutamate plus malate (10 mM each). State
III and uncoupled respiration were obtained by adding ADP
(0.5 mM, unless otherwise specified) and CCCP (0.5 lM), respectively. Other additions are specified in the legend to figures.
2.4. Lipid extraction and analysis
Rat liver mitochondria were suspended in Buffer A, supplemented with 2 lg/ml rotenone, 10 mM succinate and 0.5 mM
ADP and incubated for 0–8 min with AEA and NA-Gly at concentration of 40 nmol/mg prot. Total lipids were extracted using Bligh
and Dyer method [33]. The extracts were carefully dried under
N2 before being weighed and then dissolved in chloroform. Total
lipid extracts were analysed by TLC on silica gel 60A plates (Merck,
20 10 cm, layer thickness 0.2 mm). Lipids were eluted with chloroform/methanol/acetic acid 27:3:0.3 (v/v) and detected by exposure to iodine vapours.
2.5. Measurement of membrane potential
The mitochondrial membrane potential (DW) was measured as
previously described [34] following the safranin-O fluorescence
quenching at 525 nm (excitation), 575 nm (emission) with a Jasco
FP 6200 spectrofluorimeter. Rat liver mitochondria (0.25 mg/ml)
Rat liver contains a definite activity of FAAH that hydrolyses different NAEs including anandamide to AA and ethanolamine [19]. It
is a membrane bound enzyme which is found predominantly associated to the mitochondrial and microsomal fractions [18–20].
Thus preliminary experiments were carried out aimed at finding
out whether and to what extent anandamide could be hydrolyzed
when exposed to mitochondria isolated from rat liver, under our
test conditions.
AEA and AA were analyzed by TLC analysis in the lipid extract
from mitochondria incubated with AEA at 40 nmol/mg protein
(Fig. 1). The lipid to protein ratio of our preparation was found to
be 0.34 ± 0.05. Under the selected conditions for TLC, free and
esterified cholesterol migrated close to the solvent front, AEA and
AA were in the middle, while the polar lipids remained close to
the deposition line. The intensity of the AEA band did not decrease
after 8 min incubation with respiring mitochondria, indicating that
no hydrolysis occurred. Under the same experimental conditions,
NA-Gly, which is also substrate for FAAH, underwent partial hydrolysis, as revealed by a net decrease of its intensity band and by the
appearance of the AA corresponding band [36]. This was used as
internal positive control.
3.2. Mitochondrial respiration
The effects of micromolar concentrations of AEA on oxygen consumption activity of rat liver mitochondria were examined. AEA
caused a small inhibitory effect on succinate (plus rotenone) supported respiration (resting state) (Fig. 2A) and a stronger, concentration dependent, inhibition of ADP stimulated respiration (state
III) (Fig. 2A and B). BSA addition to AEA (10 lM, 40 nmol/mg
P. Zaccagnino et al. / FEBS Letters 585 (2011) 429–434
431
Fig. 1. Lipid analysis of rat liver mitochondria. Lipids were extracted from liver mitochondria incubated for 0 and 8 min with AEA and Na-Gly at 40 nmol/mg protein (final
concentration 60 lM). Thin-layer chromatography was performed with a solvent containing chloroform/methanol/acetic acid (27:3:0.3 v/v/v). 100 lg of total lipid extract
was loaded in each lane. Standards of cholesterol (CHOL), arachidonic acid (AA), anandamide (AEA) and N-arachidonylglycine (NA-Gly) are shown.
protein) rescued almost completely the inhibition of state III mitochondrial respiration (not shown). In the presence of CCCP, added
after oligomycin, the oxygen consumption rate was only slightly
affected (Fig. 2A and B). As a consequence, AEA caused a marked
drop of the respiratory control ratio (RCR) calculated as ADPstimulated vs succinate supported respiration, while it had almost
no effect on the respiratory control ratio measured as uncouplerstimulated vs oligomycin-inhibited respiration (Fig. 2C).
Similar effects were observed when glutamate + malate was
used as respiratory substrate (not shown).
3.3. Mitochondrial membrane potential
Fig. 3A shows traces of safranin fluorescence quenching ensuing
upon addition of respiratory substrates to mitochondria. It is
shown that the rate and extent of membrane potential generated
by succinate (plus rotenone) addition to mitochondria were unaffected by treatment with 5 lM (20 nmol/mg prot) AEA. ADP addition at the steady-state caused an atractyloside and oligomycin
sensitive transient drop of the membrane potential. The time required for the potential to be rebuilt by respiration was, on other
hand, largely increased by AEA in the concentration dependent
fashion (see inset in Fig. 3A). The extent of membrane potential
generated by glutamate + malate addition (in the absence of nigericin) was much lower than that measured with succinate (see the
fluorescence intensity scale in Fig. 3A and B). Again AEA delayed
the rebuilding of the potential which was almost completely collapsed by ADP addition at the steady-state (Fig. 3B).
The effects observed on the membrane potential were reproduced during oxygen consumption experiments, when the state
III to state IV transition was followed (Fig. 3C). Again, the state III
phosphorylation time (i.e., the time required for added ADP to be
phosphorylated) was markedly increased in mitochondria treated
with AEA (Fig. 3C).
Overall, the data obtained from experiments illustrated in Figs.
2 and 3 indicate that AEA slows down the ADP consumption rate,
that is, the FoF1 ATP synthase activity.
3.4. ATP synthase and ATPase activities
The effect of AEA on the FoF1 ATP synthase activity in liver mitochondria is reported in Fig. 4. The activity measured as oligomycin
sensitive ATP synthesis was progressively inhibited by AEA in the
concentration range of 1–20 lM, with IC50 value of 2.5 lM
(10 nmol/mg protein) (Fig. 4A). AEA had, on the other hand, no
effect on the oligomycin sensitive ATP hydrolase activity (Fig. 4B).
The ability of AEA to inhibit the ATP synthase activity without
affecting the hydrolase activity was further confirmed by the
experiment reported in Fig. 5. It is shown that AEA (10 lM,
40 nmol/mg protein) did not cause any change in the rate and extent of oligomycin sensitive membrane potential generated by ATP
hydrolysis, which was stable over time (Fig. 5). An obvious corollary from this experiment is that AEA did not affect the atractyloside sensitive mitochondrial transport of ATP, i.e., the ADP/ATP
carrier activity.
4. Discussion
Some effects produced by NAEs interaction with mitochondria
isolated from different tissues have been described, with different
results. Epps et al. [29] reported that N-oleoylethanolamine
reduced (IC50, 30 lM) the inner membrane permeability increase
induced by Ca2+ plus phosphate in heart mitochondria, while at
higher concentrations inhibited uncoupled respiration and the
development of membrane potential. Subsequently, Wasilewski
et al. [30], by exposing rat heart mitochondria to AEA concentrations in the range 40–180 lM, showed the cannabinoid to exhibit
a protonophoric effect, inhibition of uncoupled respiration, also
causing opening of the mitochondrial permeability transition pore
(MPTP), not accompanied by matrix swelling. Others [37] have
reported that AEA although causing matrix swelling in mice liver
mitochondria, decreased substantially the Ca2+ induced cytochrome c release, an early event of the apoptotic programme. It
is possible that the different effects observed could arise in part
from differences in the concentration range used. Furthermore,
functions of mitochondria from various tissues could be differently
influenced by AEA, due to a different protein to lipid ratio or to tissue specific activity of the amide hydrolyzing enzyme FAAH [29].
The present results show that in liver mitochondria AEA did not
affect substantially the resting state respiration (Fig. 2), neither did
it decrease the substrate generated membrane potential (no protonophoric effect) (Fig. 3). At few micromolar concentrations AEA
inhibits, on the other hand, selectively the FoF1 ATP synthase complex as shown by the inhibition of state III respiration (Figs. 2 and
3) and, directly, by the inhibition of the oligomycin sensitive ATP
synthesis, with IC50 of 2.5 lM (Fig. 4A). The ATP hydrolase activity
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P. Zaccagnino et al. / FEBS Letters 585 (2011) 429–434
Fig. 2. Effects of AEA on oxygen consumption in rat liver mitochondria. Respiration
was initiated by the addition of succinate (plus rotenone) to mitochondria
preincubated with the indicated concentrations of AEA. (A) Traces of oxygen
consumption in AEA treated mitochondria respiring with succinate. Where
indicated 0.5 mM ADP, 2 lg/ml oligomycin and 0.5 lM CCCP were added. Numbers
on the traces refer to the rate of oxygen consumption as nmol O2 min1 mg protein1. (B) Effect of AEA on ADP or CCCP stimulated respiration rate. (C) Respiratory
Control Ratio (RCR) measured as ADP-stimulated vs succinate respiration or CCCP
stimulated vs oligomycin-inhibited respiration. Values reported are means ± S.D.
from five to ten separate experiments.
was clearly unaffected (Fig. 4). Importantly, under our test conditions, AEA did not undergo hydrolysis by FAAH (Fig. 1).
The FoF1 ATP synthase is a protein complex located in the mitochondrial inner membrane which synthesizes ATP but can also
operate in the reverse direction hydrolysing ATP. ATP synthesis
by FoF1 is driven by the proton flow through the trans-membrane
Fo sector from the cytosol to the mitochondrial matrix, while ATP
hydrolysis is associated with proton flow in the opposite direction,
from the mitochondrial matrix to the cytosol. The selective inhibitory effect exerted by AEA on ATP synthesis, without any effect on
the ATPase activity, reminds observations showing a differential
effect of a pathological mutation in the gene coding for the ATPase
6 of Fo, which causes inhibition of ATP synthesis but has no effect
on ATP hydrolysis [38].
Fig. 3. Effects of AEA on respiration dependent transmembrane potential in liver
mitochondria. DW was generated by succinate (A) or glutamate + malate (B)
addition to mitochondria supplemented with the indicated concentrations of AEA.
Where indicated 0.1 mM ADP, 0.2 mM atractyloside (Atr), 2 lg/ml oligomycin and
0.25 lM CCCP, were added. The time required for the potential to be rebuilt as a
function of AEA concentration is reported in the inset. (C) State III to State IV
transition in succinate respiring liver mitochondria suspended in a Buffer A also
supplemented with 0.3 mM Ap5. Numbers on the traces refer to the rate of oxygen
consumption as nmol O2 min1 mg protein1.
ADP addition to state IV respiring mitochondria results in rapid
drop of the respiratory membrane potential, negative in the inner
matrix space, which is regenerated by respiration dependent phosphorylation of ADP to ATP. An interesting observation is that AEA
whilst inhibited the recovery of the membrane potential, accompanying ADP consumption by oxidative phosphorylation, had no
effect on the initial rapid drop of the membrane potential observed
upon ADP addition. It is conceivable that the initial drop of the
membrane potential results apparently from rapid induction of
P. Zaccagnino et al. / FEBS Letters 585 (2011) 429–434
Fig. 4. Effects of AEA on oligomycin sensitive ATP synthase and ATP hydrolase
activities in liver mitochondria. (A) ATP synthesis was measured as nmol of glucose
6-phosphate produced during 5 min incubation in mitochondria respiring with
succinate. (B) ATP hydrolase activity. Where indicated 2 lg/ml oligomycin (oligo)
was present. Values reported are means ± S.D. from three to five experiments. For
other details see under Sections 2.6 and 2.7.
433
natural IF1 protein which inhibits ATP hydrolysis, AEA inhibits
selectively the F1 catalytic step(s) of ADP phosphorylation to ATP.
This candidates AEA as a useful tool to analyze the structural/functional events in the rotary mechanism(s) of ATP synthesis and
hydrolysis.
In conclusion the present study could show that the cytotoxic
action of AEA involves a direct action on the mitochondrial FoF1
ATP synthase complex. The consequent decline in ATP production
by the oxidative phosphorylation system would be expected to
cause mitochondrial dysfunction, thus affecting numerous cell
functions such as membrane ion transport, macromolecule synthesis as well as cell signalling. The concentration range we used in
the present work has been shown by Siegmund et al. to block, in
the presence of receptor antagonists, hepatic stellate cells proliferation and, consequently, the liver fibrogenesis [24]. Subsequently it
has been reported that AEA may also cause hepatocyte cell death
under conditions of oxidative stress and GSH depletion [39]. These
conditions are typical of several pathological states, including alcoholic liver disease and biliary obstruction.
Similar to the mitochondrial enzyme, as far as the subunit composition and the sensitivity to inhibitors are concerned, a FoF1 type
of ATP synthase has been described to be ectopically expressed on
the plasma membranes of several tumour cell lines and human
umbilical vein endothelial cells [40]. This synthase complex represents a binding protein for angiostatin, a strong inhibitor of angiogenesis and cell proliferation [41]. Based on the results we
presented, AEA could also play a similar role by binding the plasma
membrane ATP synthase. This hypothesis is substantiated by
recent findings indicating that endocannabinoids may function as
suppressors of angiogenesis and tumour spreading in different
types of cancer [42].
Acknowledgements
This work was financially supported by a Grant from the National Research Project (PRIN) on ‘‘Mitochondrial Bioenergetics:
Redox Mechanisms, ROS Production, Redox Control of Cell Differentiation’’. We thank Professor Sergio Papa for suggestions and
for critical reading of the manuscript.
References
Fig. 5. Effects of AEA on ATP generated membrane potential in liver mitochondria.
Membrane potential was generated by the addition of 1.5 mM ATP to rat liver
mitochondria. Where indicated 0.2 mM atractyloside (Atr) and 2 lg/ml oligomycin
were present. CCCP was used at concentration of 0.25 lM.
H+ influx trough the Fo channel upon ADP binding (in the presence
of inorganic phosphate) to the Fi binding sites at the matrix side of
the complex. This rapid H+ influx is, in fact, suppressed by atractyloside inhibition of ADP entry or by block of proton conduction in
Fo by oligomycin. AEA has no effect on this initial step of ADP induced rapid proton influx, but apparently inhibits the subsequent
step(s) of ADP phosphorylation to ATP at the catalytic sites in F1.
Thus at difference of the classical ATP synthase inhibitor oligomycin, which inhibits both ATP synthesis and hydrolysis, and the
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