Effects of nitrosopropofol on mitochondrial energy-converting system

Article abstract of DOI:10.1016/S0006-2952(02)01253-4

Nitrosopropofol (NOPR) is a relatively stable compound obtained from the reaction between the general anesthetic 2,6 diisopropylphenol (propofol) and nitrosoglutathione (GSNO) and bearing a more acidic phenol group than propofol. It interfered with mitoch

Full text of DOI:10.1016/S0006-2952(02)01253-4

Biochemical Pharmacology 64 (2002) 1133±1138
Effects of nitrosopropofol on mitochondrial energy-converting system
Roberto Stevanatoa, Federico Momoa, Michela Marianb, Maria Pia Rigobellob,
Alberto Bindolic, Marcantonio Bragadind, Ezio Vincentie, Guido Scutarib,*
aDepartment of Physical Chemistry, University of Venice, Venice, Italy
bDepartimento di Chimica Biologica, Universita di Padova, Viale G. Colombo 3, 35121 Padova, Italy
cCNR Center for the Study of Biomembranes, Padova, Italy
dDepartment of Environmental Sciences, University of Venice, Venice, Italy
eAnaesthesiology and Intensive Care Unit of Camposampiero Hospital, Padova, Italy
Received 7 March 2002; accepted 14 June 2002
Abstract
Nitrosopropofol (NOPR) is a relatively stable compound obtained from the reaction between the general anesthetic 2,6 diisopro-
pylphenol (propofol) and nitrosoglutathione (GSNO) and bearing a more acidic phenol group than propofol. It interfered with
mitochondrial energetic metabolism in a concentration-dependent manner. Concentrations as high as 100 or 200 mM disrupted both
oxidative phosphorylation and electron transport. Low concentrations of NOPR (50 mM) markedly slowed down the electron transport rate
which was insensitive both to ADP and uncoupler stimulation and spontaneously gradually stopped. Consequently, both the
transmembrane potential production and the ATP synthesis system were affected. In the presence of 10 or 20 mM NOPR, mitochondria
respired but showed a worsening of the respiratory control and produced a transmembrane potential useful to respond to a phosphorylation
pulse, but were not able to restore it. These results were consistent with ATP synthesis and swelling experiments. NOPR was effective at
concentrations lower than those required by the combination of propofol and GSNO, suggesting that mitochondria might be able to
catalyze the reaction between GSNO and propofol and that the resulting metabolite was more active on mitochondrial membrane structure
than the parent compounds. Although the details of the process are yet unknown, the mechanism presented may be of potential relevance
to rationalize the pathophysiological effects of propofol.
# 2002 Elsevier Science Inc. All rights reserved.
Keywords: Anesthetics; Nitrosopropofol; Rat liver mitochondria; Respiration; Transmembrane potential; ATP synthesis
1. Introduction
oxidative phosphorylation [5±7]. In addition, an NO
synthase activity has been recently characterized also in
mitochondria [8] where NO appears to modulate the
energetic metabolism [8±13]. Following the hypothesis
that, in mitochondria, propofol might interfere with the
regulatory function of NO, we studied the combined action
of the anesthetic and the physiological NO donor GSNO,
on the mitochondrial energy metabolism. We have demon-
strated that an evident synergistic effect occurs, leading, in
the presence of propofol, to a GSNO concentration-depen-
dent restraint of the energy production [14].
Propofol is an alkyl-substituted phenol widely used as an
intravenous general anesthetic [1]. Recently, it has been
suggested that the marked vasodilation observed during
propofol treatment might be mediated by the production of
NO by the endothelial cells [2,3]. Also the negative
chronotropic effects induced by propofol in cultured rat
ventricular myocytes, which is in part mediated by M-2
acetylcholine receptors activation, involves an enhance-
ment of NO production [4].
We have previously reported that propofol may interfere
with cellular energy availability by affecting mitochondrial
It has been reported by Mouithys-Mickalad et al. [15]
that, in suitable conditions, propofol is able to react with
peroxynitrite to form a phenoxyl radical and, more
recently, by Cudic and Ducrocq [16], with NO or its
derivatives to give addition products. Considering this
possibility, we have synthesized and characterized the
2,6-diisopropyl-4-nitrosophenol. On the assumption that
* Corresponding author. Tel.: ‡39-49-8276148; fax: ‡39-49-8073310.
E-mail address: bioclin@civ.bio.unipd.it (G. Scutari).
Abbreviations: CCCP, carbonylcyanide-m-chlorophenylhydrazone;
RLM, rat liver mitochondria; NOPR, nitrosopropofol; GSNO, nitrosoglu-
tathione.
0006-2952/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 0 6 - 2 9 5 2 ( 0 2 ) 0 1 2 5 3 - 4

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R. Stevanato et al. / Biochemical Pharmacology 64 (2002) 1133±1138
such a compound might be produced also in the mitochon-
drial matrix and be active there, we have studied the effects
of NOPR on the mitochondrial energy metabolism.
tor the rate of proton concentration decrease due to the shift
of ATP, ADP, and phosphate anion dissociation equilibria
[20]. The rate of ATP synthesis was taken as the difference
between the rates obtained in the absence and presence of
1 mg oligomycin/mg of mitochondrial proteins. The
experiments were performed in the basal medium (from
which 3-N-morpholinopropanesulfonic acid was omitted)
supplemented with 5 mM K-succinate, 1.25 mM rotenone,
and 240 mM ADP.
2. Materials and methods
Mitochondria were isolated from liver of albino Wistar
rats in 0.25 M sucrose, 10 mM Na-HEPES (pH 7.4), and
0.25 mM EGTA by differential centrifugation [17], omit-
ting EGTA in the ®nal washing. Protein content of the
mitochondrial suspension was assayed by the biuret
method [18]. Experiments on respiration, membrane
potential, and ATP synthesis were carried out at 208 using
1 mg/mL of mitochondrial protein in a basal medium
containing 0.25 M sucrose, 15 mM 3-N-morpholinopropa-
nesulfonic acid, 1.5 mM rotenone, 1.6 mM MgSO4, 2 mM
KH2PO4, 6 mM NaCl, 1 mg/mL BSA, and 5 mM Na-
succinate, pH 7.0.
Swelling experiments were followed spectrophotometri-
cally by the decrease in absorbance at 540 nm in the presence
of 0.25 mg/mL mitochondrial protein in 213 mM mannitol,
71 mM sucrose, 5 mM HEPES±Tris (pH 7.4), 5 mM succi-
nate, 3 mg/mL rotenone, and 3 mM oligomycin. Oxygen
consumption was followed using a Clark oxygen electrode
(Yellow Springs Instruments Co) in the basal medium
supplemented with 5 mM K-succinate and 1.5 mM rotenone.
Transmembrane electrical potential (DC) was measured
by monitoring the distribution of tetraphenylphosphonium
cation across the inner mitochondrial membrane with a
tetraphenylphosphonium-selective electrode in the basal
medium supplemented with 5 mM K-succinate and 1.5 mM
rotenone [19].
NOPR was synthesized following the procedure utilized
for the preparation of nitrosophenol [21]. Two milliliters of
concentrated hydrochloric acid were slowly added to 3 g of
2,6-diisopropylphenol dissolved in 25 mL ethanol main-
tained at À58. A solution of 1.1 g sodium nitrite in 5 mL
water was gradually added to the mixture under vigorous
stirring and maintaining the temperature below 08. After
15 min, the yellow product was stirred for 30 min and then
poured into iced water. The yellow precipitate was crystal-
lized from toluene and characterized by GC/MS analysis
and UV±Vis. The latter spectrum shows a characteristic
absorption band at 310 nm [22]. Interestingly, NOPR pre-
pared with the earlier reported procedure or after reaction
of propofol with GSNO in the presence of copper(II) gives
rise to superimposable GC/MS and UV±Vis spectra [22].
In Fig. 1, the proton NMR spectrum of NOPR is reported
and the chemical shifts are consistent with the structure of
the compounds. The spectrum presents distinct resonances
for the aromatic protons 3 and 5 as well as for the methynic
and methylic protons of the isopropyl groups. This is
attributed to the hindered rotation of the nitroso group,
due to the order (between 1 and 2) of the C±N bond [23]. A
purity grade higher than 95% was con®rmed by thin layer
chromatography. NOPR was used as an ethanol solution
and appropriate blanks were run to exclude possible etha-
nol effects.
ATP synthesis was evaluated by using a semi-micro
combined pH electrode (Ingold Messtechnik AG) to moni-
Fig. 1. Proton NMR spectrum of NOPR. 1H NMR (CDCl3, 300 MHz), d: 8.7 (1H, OH, broad singlet), 7.52 and 6.91 (2H, 3H, and 5H, singlets), 3.06 and 3.1
(2H, methynic, septets, J ˆ 8:5 Hz), 1.14 and 1.12 (12H, methyls, doublets, J ˆ 8:5 Hz).

R. Stevanato et al. / Biochemical Pharmacology 64 (2002) 1133±1138
1135
Traces reported in the ®gure correspond to the most
representative experiment among those performed on 15
different mitochondrial preparations. It was chosen con-
sidering the closest to the mean of the experiments showing
the maximal values of DC, state 3 respiration, and ATP
synthesis.
The values were compared to each other by the paired
Student's t-test. and P-values resulted <0.001 except for
those obtained in the presence of 100 or 200 mM NOPR
and compared between themselves (P < 0:01).
min/mg prot, respectively), a true state 4 cannot be restored
and CCCP induces a limited stimulation of respiration
which spontaneously stops. The respiration inhibition is
almost complete when concentrations as high as 100 or
200 mM of NOPR are used (31.1 and 19.4 ngat oxygen/
min/mg prot, respectively) and the system appears insen-
sitive to any kind of stimulation.
Control mitochondria produce and maintain an electrical
transmembrane potential of about 180 mV (Fig. 3). The
addition of a limited amount of ADP induces a phosphor-
ylation pulse observed as a transient protonic gradient
utilization that, however, is rapidly rebuilt at the same
level present before ADP addition (Fig. 3a). In the presence
of 10 or 20 mM NOPR, mitochondria still establish a
transmembrane potential, although lower of the control
of about 10 or 20 mV, respectively. In addition, they are not
able to restore it after the phosphorylation pulse (Fig. 3b
and c). NOPR, at concentrations of 50 mM or higher
completely prevents mitochondria from producing any
signi®cant transmembrane potential (Fig. 3d±f).
The rate and extent of ATP synthesis are consistent with
those of the transmembrane potential (Fig. 4). ATP synth-
esis of control mitochondria is of 42 nmoles/min/mg prot.
In the presence of 10 mM NOPR the synthesis rate is about
halved (19 nmoles/min/mg prot), with respect to the con-
trol, while it is reduced to about a seventh (6.2 nmoles/min/
mg prot) with 20 mM NOPR. The synthesis of ATP is
undetectable with 50 mM NOPR, while the results obtained
with 100 and 200 mM NOPR were not reported since ATP
formation is completely inhibited.
Fig. 5 shows the mitochondrial swelling induced by the
addition of 10 mM Ca2‡ in the presence of NOPR in the
range 10±200 mM. It appears that the mitochondrial swel-
ling induced by calcium ions uptake is completely abol-
ished by propofol concentrations as high as 100 mM and
3. Results
Fig. 2 shows the time course of mitochondrial oxygen
consumption during resting respiration and ADP- and
carbonylcyanide-m-chlorophenylhydrazone (CCCP)-sti-
mulated respiration in the presence of increasing concen-
trations of NOPR in the range 2±200 mM.
Control mitochondria exhibit a state 3 rate of 102 ngat
oxygen/min/mg prot, a state 4 rate of 13.8 ngat oxygen/
min/mg prot, and an uncoupled respiration of 104.3
ngat Oxygen/min/mg prot (Fig. 2a). Consequently, a respi-
ratory control ratio of 7.3 and an ADP/O ratio of 1.5 may be
calculated. In the presence of 10 mM NOPR (Fig. 2b), ADP
addition stimulates respiration, although to a lower rate
(74.1 ngat oxygen/min/mg prot) with respect to the con-
trol. In the same conditions, the resting respiration is faster
(51 ngat oxygen/min/mg prot), the restoration of state 4 is
delayed, and the addition of the strong uncoupler CCCP
evokes a transient increase of the respiration that, after-
wards, gradually stops.
In the presence of 20 or 50 mM NOPR (Fig. 2c and d) the
respiration, although still slightly sensitive to the ADP
addition, is strongly inhibited (57.1 and 52.6 ngat oxygen/
Fig. 2. Effect of NOPR on resting and ADP- or CCCP-stimulated respiration of isolated rat liver mitochondria. Additions (indicated by the arrows): rat liver
mitochondria (RLM), 0.3 mM ADP, 1.6 mM CCCP. NOPR was present in the incubation medium before mitochondria addition. NOPR concentrations in
(mM): (a) none; (b) 10; (c) 20; (d) 50; (e) 100; and (f) 200.

1136
R. Stevanato et al. / Biochemical Pharmacology 64 (2002) 1133±1138
that the effect is concentration dependent, being the swel-
ling in the presence of 10, 20 or 50 mM NOPR proportion-
ally inhibited.
4. Discussion
NOPR is the main product of the nitrosation reaction of
the general anesthetic propofol. Its presence in the incuba-
tion medium of respiring mitochondria interferes with both
the oxidative phosphorylation and the electron transport in
a concentration-dependent manner. Concentrations as high
as 100 mM or more completely disrupt the mitochondrial
aerobic metabolism (Fig. 2). Also 50 mM NOPR has a
dramatic effect on respiration since the electron transport
rate is strongly slowed down and afterwards spontaneously
stops. Besides, it is insensitive to both ADP and CCCP
stimulation, indicating that a major structural damage has
occurred. In addition, 10 mM NOPR induces a worsening
of the mitochondrial energetic system, although maintain-
ing a good ef®ciency of electron transport and a signi®cant
residual functioning of oxidative phosphorylation. Higher
concentrations such as 20 mM NOPR evokes a behavior
qualitatively similar to that of 50 mM but quantitatively
milder suggesting that the effect is concentration depen-
dent.
These experiments indicate that the major effect of
NOPR is referable to an alteration of the mitochondrial
membrane structures and that, compared to propofol, the
molecular modi®cation due to introduction of the NO
group might play a critical role. In fact, titration of propofol
or NOPR shows that the presence of the NO group shifts
Fig. 3. Effect of NOPR on mitochondrial DC formation. NOPR was
present in the medium before mitochondria. At the arrows, 0.3 mM ADP
was added. The curves have been redrawn using a linear millivolt scale.
NOPR concentrations in (mM): (a) none; (b) 10; (c) 20; (d) 50; (e) 100; and
(f) 200.
Fig. 4. Effect of NOPR on ATP synthesis by isolated rat liver mitochondria. NOPR, at the concentration indicated, was present in the incubation medium
before mitochondria addition. Mitochondria (1 mg protein/mL) and 0.3 mM ADP were also present. The reported rates represent the difference between the
rates measured in the absence and in the presence of oligomycin (1 mg/mg protein).

R. Stevanato et al. / Biochemical Pharmacology 64 (2002) 1133±1138
1137
Fig. 5. Mitochondrial swelling induced by calcium addition in the presence of increasing NOPR concentrations. Mitochondria were preincubated for 1 min
with NOPR and 10 mM Ca2‡ was added at the arrow indicated. NOPR concentration in (mM): (a) none; (b) 5; (c) 10; (d) 20; (e) 50; (f) 100; and (g) 200.
the pKa value of the phenol group from about 11 for
propofol to about 7.5 [22], indicating that, at physiological
pH, NOPR is more dissociated than propofol, and, conse-
quently, is present mostly in the charged form.
matrix volume due to the in¯ux of a solute leads to a
proportional decrease of the matrix refractive index, which
approaches that of the medium, and results in a decrease of
the light scattered. Hence, the reported results may be
regarded as a consequence of the NOPR effect on the DC
discussed before and con®rm those observations. Indeed,
in the experimental conditions adopted, the swelling is
quantitatively dependent on the calcium entering the
matrix and the associated water ¯ux, which, in turn,
depends on the membrane potential strength which is
decreased by NOPR, according to its concentration.
In a previous paper [14], we have reported that the
associated action of relatively high concentrations of pro-
pofol and GSNO resulted in an inhibition of the mitochon-
drial energetic ef®ciency and suggested that the effects
might be ascribed to little amounts of a new molecule
possibly derived from the interaction of propofol and
GSNO in the mitochondrial environment.
NOPR is the main product of the in vitro nitrosation of
propofol but may be obtained in the presence of GSNO and
of copper ions [16]. It is a stable compound and its phenol
group is more dissociable than that of propofol. NOPR is
able to impair the mitochondrial energetic system at con-
centrations far lower than those required, to obtain the
same effects, by the combination of GSNO and propofol.
Mitochondria are potentially able to catalyze the reac-
tion between GSNO and propofol and the derived product,
NOPR, might be a metabolite more active than the parent
compound. Moreover, further work is required to unam-
biguously detect the presence of NOPR in the matrix of
mitochondria supplied with propofol and GSNO.
Conceivably, the transmembrane potential formation
re¯ects the respiration behavior and low NOPR concentra-
tions (10 or 20 mM) allow mitochondria to build up a
potential lower than that of control, but suf®cient to
support a phosphorylation pulse. However, after the phos-
phorylation pulse, mitochondria are unable to restore DC.
Higher NOPR concentrations (100 or 200 mM) prevent the
formation of any signi®cant potential. The rate of ATP
synthesis, which utilizes the energy of the transmembrane
potential, is consistent with the membrane potential value.
Moreover, the addition of NOPR to the incubation medium
after the production of a stable potential induces a decrease
of the voltage value whose extent appears to be propor-
tional to the NOPR concentration (data not shown).
This indicates that NOPR action might be mainly refer-
able to a derangement of the membrane tightness. How-
ever, if the effect would be merely an increase of the proton
permeability, respiration should be uncoupled and, conse-
quently, the oxygen consumption rate increased. On the
contrary, as apparent in Fig. 2, the respiration is inhibited,
i.e. the electron transport seems hindered in a concentra-
tion-dependent manner, as con®rmed by the uncoupler
ineffectiveness.
Calcium ions, taken up by the electrical gradient of
metabolically active mitochondria, induce mitochondrial
swelling. As apparent in Fig. 5, 100 or 200 mM NOPR
completely prevents mitochondrial swelling while lower
NOPR concentrations proportionally limit the mitochon-
drial shape change as may be detected by following the
light scattering shift of the suspension. An increase of the
In the cell, where NO production is relevant and GSH is
abundant, the proposed mechanism might be of some
importance in rationalizing the way of action of propofol

1138
R. Stevanato et al. / Biochemical Pharmacology 64 (2002) 1133±1138
tion of isolated rat liver mitochondria. Biochem Pharmacol 1991;
42:87±90.
and in elucidating some clinical effects reported in recent
years. Among the latter, bradycardia and hypotension due
to the marked vasodilation elicited by propofol and
mediated by NO production and release from the endothe-
lial cells should be mentioned [2,3]. In addition, the
stimulation of the ciliary beat frequency elicited by pro-
pofol in cultured tracheal epithelial cells via an NO±cGMP
pathway was also observed [24].
Concerning the concentrations tested, propofol is
usually administered by the ``Target Controlled Infusion''
which allows to obtain controlled plasma concentrations
of propofol (expressed as mg/mL) on the basis of a
pharmacokinetic program controlled by a computerized
pump. Currently, deep sedation requires a plasmatic con-
centration level higher than 1.8 mg/mL (i.e. about 10 mM),
while maintenance of deep hypnosis needs plasmatic
concentrations between 3.6 and 5.3 mg/mL (20±30 mM)
and induction of anesthesia about 35±40 mM [25,26].
When the target chosen is not a plasmatic concentration
but a ``site effect concentration'', which is another option
of the Target Controlled Infusion system, plasmatic levels
may reach peaks very high (70±80 mM), even though for a
very short time. Therefore, the concentrations of NOPR
used in our work (10±200 mM) are in the concentration
range clinically employed.
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