Linoleic acid epoxide promotes the maintenance of mitochondrial function and active Na+ transport following hypoxia

Article abstract of DOI:10.1016/j.toxlet.2003.11.002

Low concentrations of arachidonic acid monoepoxides protect against ischemia/reperfusion injury. This study examined whether low concentrations of the linoleic acid monoepoxide, cis-12,13-epoxy-9-octadecenoic acid (12,13-EOA), protect renal cells against decreases in mitochondrial and transport functions induced by hypoxia/reoxygenation. Primary cultures of rabbit renal proximal tubular cells (RPTC) were pretreated with diluent or 1, 5, or 10μM 12,13-EOA for 1h and exposed to 2h hypoxia/0.5h reoxygenation in the absence of 12,13-EOA. Basal respiration, oligomycin-sensitive oxygen consumption (QO₂), and ATP content decreased 31, 35 and 65%, respectively, following hypoxia/reoxygenation. Hypoxia/reoxygenation also increased mitochondrial membrane potential (ΔΨ_m). Pretreatment with 12,13-EOA prevented decreases in basal and oligomycin-sensitive QO₂s and increases in ΔΨ_m. Despite the protection against decreases in mitochondrial function, 12,13-EOA pretreatment did not prevent the initial decrease in intracellular ATP content following hypoxia. However, pretreatment did accelerate the recovery of intracellular ATP levels during reoxygenation. Pretreatment with 12,13-EOA also prevented hypoxia-induced decreases in active Na⁺ transport. Ouabain-sensitive QO₂ (a marker of active Na⁺ transport) decreased 38% following hypoxia/reoxygenation but was maintained in RPTC pretreated with 1, 5 or 10μM 12,13-EOA prior to hypoxia. Pretreatment of RPTC with the hydrolyzed product of 12,13-EOA, 12,13-dihydroxyoctadecenoic acid, did not have any protective effects against mitochondrial dysfunction and decreases in active Na⁺ transport. Thus, this is the first report demonstrating that preconditioning of RPTC with low concentrations of 12,13-EOA, but not its hydrolyzed product, maintains mitochondrial respiration, accelerates restoration of ATP levels, and prevents decreases in active Na⁺ transport following hypoxia/reoxygenation.

Full text of DOI:10.1016/j.toxlet.2003.11.002

Toxicology Letters 147 (2004) 161–175
Linoleic acid epoxide promotes the maintenance of mitochondrial
function and active Na+ transport following hypoxia
Graz˙yna Nowaka,∗, David F. Grantb, Jeffery H. Moranc
a
Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 4301 West Markham St.,
Little Rock, AR 72205-7199, USA
b
Department of Pharmaceutical Sciences, University of Connecticut, 372 Fairfield Road, HGH-390A, U-92, Storrs, CT 06269-2092, USA
c
Center for Toxicology and Environmental Health, L.L.C. 615 West Markham St., Little Rock, AR 72201-1396, USA
Received 27 May 2003; received in revised form 4 November 2003; accepted 5 November 2003
Abstract
Low concentrations of arachidonic acid monoepoxides protect against ischemia/reperfusion injury. This study examined
whether low concentrations of the linoleic acid monoepoxide, cis-12,13-epoxy-9-octadecenoic acid (12,13-EOA), protect renal
cells against decreases in mitochondrial and transport functions induced by hypoxia/reoxygenation. Primary cultures of rabbit
renal proximal tubular cells (RPTC) were pretreated with diluent or 1, 5, or 10 ␮M 12,13-EOA for 1 h and exposed to 2 h hy-
poxia/0.5 h reoxygenation in the absence of 12,13-EOA. Basal respiration, oligomycin-sensitive oxygen consumption (QO2), and
ATP content decreased 31, 35 and 65%, respectively, following hypoxia/reoxygenation. Hypoxia/reoxygenation also increased
mitochondrial membrane potential (ꢀΨm). Pretreatment with 12,13-EOA prevented decreases in basal and oligomycin-sensitive
QO2s and increases in ꢀΨm. Despite the protection against decreases in mitochondrial function, 12,13-EOA pretreatment did not
prevent the initial decrease in intracellular ATP content following hypoxia. However, pretreatment did accelerate the recovery of
intracellular ATP levels during reoxygenation. Pretreatment with 12,13-EOA also prevented hypoxia-induced decreases in active
Na+ transport. Ouabain-sensitive QO2 (a marker of active Na+ transport) decreased 38% following hypoxia/reoxygenation but
was maintained in RPTC pretreated with 1, 5 or 10 ␮M 12,13-EOA prior to hypoxia. Pretreatment of RPTC with the hydrolyzed
product of 12,13-EOA, 12,13-dihydroxyoctadecenoic acid, did not have any protective effects against mitochondrial dysfunction
and decreases in active Na+ transport. Thus, this is the first report demonstrating that preconditioning of RPTC with low con-
centrations of 12,13-EOA, but not its hydrolyzed product, maintains mitochondrial respiration, accelerates restoration of ATP
levels, and prevents decreases in active Na+ transport following hypoxia/reoxygenation.
© 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Renal proximal tubular cells; Mitochondria; Hypoxia; Linoleic acid epoxide
Abbreviations: 12,13-EOA, cis-12,13-epoxy-9-octadecenoic acid; 12,13-DHOA, 12,13-dihydroxyoctadecenoic acid; NP-HPLC, normal
phase high pressure liquid chromatography; sEH, soluble epoxide hydrolase; RPTC, renal proximal tubular cells; ꢀm, mito-
chondrial membrane potential; CYP, cytochrome P450; EETs, cis-epoxyeicosatrienoic acids; JC-1, 5,5ꢀ,6,6ꢀ-Tetrachloro-1,1ꢀ,3,3ꢀ-
tetraethylbenzimidazolylcarbo-cyanine iodide; DMEM:F12, Dulbecco’s Modified Eagle’s Essential medium (DMEM): Ham’s F-12 nutrient
mix; Hepes, N-2-hydroxyethylpiperazine-Nꢀ-2-ethanesulfonic acid; PBS, phosphate buffered saline; QO2, oxygen consumption; FCCP, p-
trifluoromethoxyphenyl-hydrazone; INF-␥, interferon-␥.
∗
Corresponding author. Tel.: +1-501-686-6345; fax: +1-501-686-6057.
E-mail address: gnowak@uams.edu (G. Nowak).
0378-4274/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2003.11.002

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G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
1. Introduction
in intracellular ion homeostasis, mitochondrial and
plasma membrane potentials, and decreased cellular
functions (Jones, 1986). Phospholipase A2 activation
and changes in intracellular fatty acid concentrations,
including linoleic and arachidonic acids concentra-
tion, also occur early in ischemic injury (Jones, 1986).
Low concentrations of arachidonic acid metabolites
such as 12-lipoxygenase metabolites and arachidonic
acid monoepoxides (EETs) regulate cardiac L-type
Ca2+ channels and have potent cardioprotective prop-
erties against ischemia/reperfusion injury (Chen et al.,
1999; Murphy et al., 1995; Node et al., 1999; Wu
et al., 1997; Zeldin, 2001). Low concentrations of
EETs (1–5 ␮M) and the over-expression of CYP2J2,
a specific arachidonic and linoleic acid epoxygenase,
protect against hypoxia and reoxygenation injury in
cardiac myocytes and aortic endothelial cells (Wu
et al., 1997; Yang et al., 2001). EETs affect renal
function either by regulating tubular transport pro-
cesses or by mediating the effects of renal hormones
such as renin, angiotensin II, and arginine vasopressin
(Zeldin, 2001). In the proximal tubular segment of the
nephron, EETs inhibit Na+ transport and mediate the
angiotensin II-induced increases in cytosolic Ca2+
concentrations (Zeldin, 2001). The potent effects that
EETs have on electrolyte transport in the kidney and
lungs suggest that the protective properties of these
metabolites may be mediated through maintaining
intracellular ion homeostasis (Harris et al., 1990;
Jacobson et al., 1984; Madhun et al., 1991; Pascual
et al., 1998; Zeldin, 2001). We have shown that
linoleic acid (100 ␮M) prevents chloride influx dur-
ing the late phases of cellular death and protects RPT
from cellular lysis induced by hypoxia/reoxygenation.
However, linoleic acid does not promote the mainte-
nance of physiological functions during reoxygena-
tion following hypoxia (Moran et al., 2001a).
Linoleic acid is an essential fatty acid metabo-
lized by many cytochrome P450s (CYP) to produce
cis-epoxyoctadecenoic acids (EOAs). These are sub-
sequently hydrolyzed by soluble epoxide hydrolase
(sEH) to dihydroxyoctadecenoic acids (DHOAs)
(Bylund et al., 1998a,b; Moran et al., 2000; Oliw
et al., 1996). Multiple cytochrome P450s including
CYP1A2, CYP2C2, CYP2C8, CYP2C9, CYP2C19,
CYP2J2, CYP2J3, CYP2J5, CYP2J9, CYP2E1, and
CYP3A4 metabolize linoleic acid to produce EOAs
(Bylund et al., 1998a,b; Moran et al., 2000; Oliw et al.,
1996). The physiological levels of these epoxides in
humans are unknown, however, in pathological con-
ditions such as adult respiratory distress syndrome,
severe burns, and systemic shock, the blood con-
centrations of EOAs can reach levels above 100 ␮M
(Fukushima et al., 1988; Kosaka et al., 1994; Ozawa
et al., 1991; Sugiyama et al., 1987). The role of
EOAs and DHOAs in regulating physiological and/or
pathological functions in different organs is unknown.
Previously, we have shown that linoleic acid is me-
tabolized in renal proximal tubules in vitro to the
DHOA metabolites, presumably via EOA formation
(Moran et al., 2000). At pathological concentrations,
the EOAs are more potent than the DHOAs, uncouple
oxidative phosphorylation, and induce renal proximal
tubular cell (RPTC) death (Moran et al., 1997, 2000,
2001b). However, EOAs and DHOAs are less potent
than linoleic acid, and the production of EOAs and
DHOAs represents a detoxification pathway that de-
creases the deleterious effects of pathological concen-
trations of linoleic acid in RPTC (Moran et al., 2000,
2001b). The effects and role of low concentrations of
EOAs and DHOAs in the kidney are unknown.
Ischemia/reperfusion injury in the kidney is com-
mon during systemic shock, heart failure, vascular
obstruction, in clinical settings including surgery and
transplantation, and in many pathological conditions
associated with elevated levels of unesterified fatty
acids and their metabolites (Jones, 1986; Southard
et al., 1992; Weinberg et al., 1995). The temporal se-
quence of the early events during ischemia is well de-
fined and characterized by mitochondrial dysfunction,
reduction in pyridine nucleotides and ATP content, and
a decrease in intracellular pH (Jones, 1986; Weinberg,
1991). These changes are accompanied by alterations
Analogous to the protective properties of low mi-
cromolar concentrations of EETs, it is likely that
EOAs produced from linoleic acid during the early
stages of ischemia protect the kidney against the is-
chemia/reperfusion injury. Previous studies suggest
that the protective mechanisms of EETs and linoleic
acid are initiated during the reversible phases of is-
chemic injury and promote the recovery of normal
cellular functions (Moran et al., 2001a; Wu et al.,
1997; Zeldin, 2001). The goal of the present study
was to determine if low concentrations (≤ 10 ␮M) of

G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
163
12,13-EOA and 12,13-DHOA protect RPTC against
2.3. Synthesis of cis-12,13-epoxy-9-octadecenoic
the early changes in mitochondrial and transport
functions induced by hypoxia/reoxygenation and to
examine if these metabolites promote the recovery
of RPTC functions following hypoxia/reoxygenation.
Our study was also designed to determine the poten-
tial mitochondrial targets for the protective effects of
12,13-EOA and 12,13-DHOA.
acid (12,13-EOA) and 12,13-dihydroxyoctadecenoic
acid (12,13-DHOA)
Linoleic acid was epoxidized by dissolving in
methylene chloride and incubating with methyltri-
oxorhenium (0.5 mol%), pyridine (12 mol%), and
hydrogen peroxide (1.5 mol equivalents) at ambi-
ent temperature for 30 min (Moran et al., 2000;
Rudolph et al., 1997). The reaction was quenched
by the addition of manganese (IV) oxide and ex-
tracted twice with methylene chloride. 12,13-EOA
was purified using normal phase high pressure liquid
chromatography (NP-HPLC) as described (Moran
et al., 2000) dried under nitrogen, and stored at
−20 ◦C. Silica purified monoepoxides were hy-
drolyzed to the vicinal diols at room temperature
using HClO4 (1.25%). 12,13-DHOA was puri-
fied using NP-HPLC as described (Moran et al.,
2000), dried under nitrogen, and stored at −20 ◦C.
12,13-EOA and 12,13-DHOA are stable under these
previously established storage conditions, contained
less than 1% of the corresponding 9,10-isomers
and were greater than 99% pure (Moran et al.,
2000).
2. Materials and methods
2.1. Materials
Female New Zealand White rabbits (2.0–2.5 kg)
were purchased from Myrtle’s Rabbitry (Thompson
Station, TN). 5,5ꢀ,6,6ꢀ-Tetrachloro-1,1ꢀ,3,3ꢀ-tetraethyl-
benzimidazolylcarbocyanine iodide (JC-1) was
obtained from Molecular Probes (Eugene, OR).
l-Ascorbic acid-2-phosphate magnesium salt and
cell culture media were obtained from Wako Bio-
Products (Richmond, VA) and Life Technologies
(Grand Island, NY), respectively. Carbonyl cyanide
p-trifluoromethoxyphenyl-hydrazone (FCCP) and
ouabain were obtained from Aldrich (Milwaukee,
WI) and Fluka Chemical Corp. (Milwaukee, WI),
respectively. Cell culture hormones, oligomycin, and
other reagents were purchased from Sigma (St. Louis,
MO). Other chemicals were of HPLC or reagent
grade and were purchased from either Sigma–Aldrich
(St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
2.4. Exposure to 12,13-EOA, 12,13-DHOA and
hypoxia/reoxygenation
RPTC monolayers reached confluence within 6
days and were used for experiments on the sev-
enth day of culture. Quiescent, confluent monolayers
were treated with 12,13-EOA (1, 5, and 10 ␮M),
12,13-DHOA (5 ␮M) or diluent (ethanol, 0.25%)
for 1 h prior to hypoxia. Hypoxia was induced in
RPTC by replacing culture media with fresh warm
(37 ◦C) media (without 12,13-EOA and 12,13-DHOA)
pre-gassed with 95% N2/5%CO2. The dishes were
placed in an air-tight chamber and gassed with 95%
N2/5% CO2. This method resulted in decreasing the
O2 content in the media to <5% without changing
CO2 content and pH (data not shown). Following 2 h
of hypoxia, RPTC were returned to 95% air/5% CO2
atmosphere and incubated with shaking for 30 min
prior to taking samples and measurement of differ-
ent functions. The recovery of media O2 content
occurred within 5 min after reoxygenation (data not
shown).
2.2. RPTC culture
Renal proximal tubules were isolated and purified
according to the iron oxide perfusion and differen-
tial sieving method as described earlier (Nowak and
Schnellmann, 1996). RPTC were cultured in modified
DMEM/Ham’s F12 medium (50:50) without phenol
red, pyruvate, glucose, and supplemented with 15 mM
Hepes, 15 mM NaHCO3, 6 mM lactate, 50 nM hy-
drocortisone, 5 ␮g/ml transferrin, 5 ng/ml selenium,
10 nM insulin, and 0.05 mM l-ascorbic acid phos-
phate (pH 7.4, 295 mmol/l) as described previously
(Nowak and Schnellmann, 1996). RPTC were cul-
tured at 37 ◦C, in a 95% air/5% CO2 atmosphere
with constant swirling (60 rpm) to improve media
oxygenation.

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G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
2.5. Measurement of mitochondrial function
for 30 min (37 ◦C) in the presence of 10 ␮M JC-1. Af-
ter loading, the media were aspirated, and the mono-
layers were kept on ice, washed twice with ice-cold
phosphate buffered saline (PBS), scraped off culture
dishes, washed once more, and resuspended in PBS.
Fluorescence was analyzed by flow cytometry (Beck-
ton Dickinson’s FACSCalibur) using excitation by a
488 nm argon-ion laser. The JC-1 monomer (green)
and J-aggregate (red) were detected separately in FL1
(em. 525 nm) and FL2 (em. 590 nm) channels, respec-
tively. The changes ꢀΨm in RPTC were assessed us-
ing ratio of JC-1 aggreggate/JC-1 monomer. RPTC
treated with FCCP (3 ␮M; 30 min) and oligomycin
(10 ␮g/ml; 30 min) were included with each set of
samples to induce the loss of ꢀΨm (0.12 0.04 versus
2.0 0.2 in FCCP-treated and control RPTC, respec-
tively) and hyperpolarization (3.5 0.6 versus 2.0 0.2
in oligomycin-treated and control RPTC, respectively)
of the mitochondrial membrane.
Mitochondrial function in RPTC was assessed by
measurements of: (1) cellular oxygen consumption,
(2) intracellular ATP content, (3) F0F1-ATPase activ-
ity, and (4) the mitochondrial membrane potential.
2.6. Oxygen consumption (QO2)
QO2 was measured in RPTC using our stan-
dard procedure described previously (Nowak and
Schnellmann, 1995; Nowak and Schnellmann, 1996).
Briefly, RPTC monolayers were gently scraped off
culture dishes with the use of rubber policeman,
suspended in the warm (37 ◦C) culture media, and
transferred to a QO2 chamber. QO2 was measured
polarographically with a Clark-type electrode, as
described previously (Nowak and Schnellmann,
1995; Nowak and Schnellmann, 1996). Oligomycin-
sensitive QO2 was measured as the difference between
basal and oligomycin-insensitive QO2, which was de-
termined following addition of oligomycin (10 ␮g/ml).
Uncoupled QO2 was measured following the addi-
tion of carbonyl cyanide p-trifluoromethoxyphenyl-
hydrazone (1.5 ␮M). Approximately 106 cells were
used in every measurement of cell respiration.
2.9. Active Na+ transport
Active Na+ transport was measured using the
ouabain-sensitive QO2 as a marker as described pre-
viously (Nowak and Schnellmann, 1995; Nowak and
Schnellmann, 1996). Ouabain-sensitive QO2 was
measured as the difference between basal QO2 and
QO2 in the presence of ouabain (1 mM).
2.7. Measurement of intracellular ATP content
ATP content in RPTC was measured by the lu-
ciferase method in freshly prepared cellular lysates
using the ATP bioluminescence assay kit supplied by
Roche (Indianapolis, IN) and instructions provided by
the manufacturer.
2.10. Isolation of RPT mitochondria
Mitochondria were isolated from RPTC as de-
scribed by Lash and Sall (1993). RPTC were homog-
enized in ice-cold isolation buffer (225 mM sucrose,
10 mM K2HPO4 (pH 7.4), 5 mM MgCl2, 20 mM
KCl, 20 mM triethanolamine hydrochloride, 0.1 mM
phenylmethanesulfonyl fluoride, and 2 mM EGTA),
and the homogenate centrifuged at 1000 × g for
10 min at 4 ◦C. Supernatant resulting from this cen-
trifugation was saved, and the resulting pellet was
washed in the isolation buffer and centrifuged at
1000 × g for 10 min at 4 ◦C. Both supernatants were
combined and centrifuged at 15,000 × g for 5 min
at 4 ◦C. The resulting pellet was washed twice by
re-suspending in the isolation buffer and centrifuging
at 15,000 × g for 5 min at 4 ◦C. Mitochondrial pellets
were resuspended in 10 mM Tris–HCl buffer (pH 7.4)
containing 25 mM sucrose, 75 mM mannitol, 100 mM
2.8. Mitochondrial membrane potential (∆Ψm)
ꢀΨm was assessed using JC-1, a cationic fluores-
cent dye that exhibits potential-dependent accumu-
lation in mitochondria. At lower ꢀΨm, JC-1 exists
as a green fluorescing monomer (525 nm). At high
ꢀΨm characteristic of energized mitochondria, JC-1
forms red fluorescing aggregates in mitochondria re-
sulting in a fluorescence emission shift from green
(525 nm) to red (590 nm). Mitochondrial depolariza-
tion is indicated by a decrease in the red/green flu-
orescence intensity ratio. RPTC were exposed to 2 h
hypoxia followed by 0.5 h reoxygenation and loaded

G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
165
KCl, 0.05 mM K2EDTA, and 5 mM H3PO4 and were
used for measurement of F0F1-ATPase activity.
was observed at 24 h of reoxygenation (Fig. 1B
and D). Furthermore, LDH release from monolay-
ers to the culture media was equivalent in control
RPTC and RPTC following hypoxia/reoxygenation
(Fig. 1E). Pretreatment with 12,13-EOA (5 ␮M) or
12,13-DHOA (5 ␮M) had no effect on LDH re-
lease either in controls (Fig. 1E) or following hy-
poxia/reoxygenation (1.9 0.4, 3.0 0.05, and 4.1
1.3% following hypoxia/reoxygenation, 12,13-EOA
+ hypoxia/reoxygenation, and 12,13-DHOA + hypo-
xia/reoxygenation, respectively).
2.11. Measurement of F0F1-ATPase activity
ATPase activity of the F0F1-ATPase was carried out
in freshly isolated RPT mitochondria by measuring
the release of Pi from ATP as described by Law et al.
(1995). The assay was performed at 30 ◦C in 10 mM
Tris–HCl, pH 8.2, containing 200 mM KCl, 3 mM
MgCl2, and RPTC mitochondria (approximately 1 mg
protein). In brief, the reaction was initiated by the
addition of ATP (5 mM), performed under linear con-
ditions, and terminated after 5 min by adding 3 M
trichloroacetic acid followed by centrifugation. Inor-
ganic phosphate concentration in the supernatant was
determined using the Sumner reagent. Each sample
was run in the absence and presence of oligomycin
(10 ␮g/ml) and the F0F1-ATPase activity was ex-
pressed as the oligomycin-sensitive activity.
3.2. Mitochondrial function
RPTC were treated with 12,13-EOA (1, 5, and
10 ␮M) or 12,13-DHOA (5 ␮M) for 1 h followed
by washing out these compounds prior to hypoxia
and reoxygenation. In the initial set of experi-
ments we determined that 1 h treatment of control
RPTC with 12,13-EOA had no effect on basal QO2
(20.8 1.6 nmol versus 20.7 1.1 nmol O2/min/mg
protein in RPTC treated with 0 and 5 ␮M 12,13-EOA,
respectively). In contrast, 1 h treatment of control
RPTC with 5 ␮M 12,13-DHOA decreased basal QO2
2.12. Assessment of cell injury
RPTC injury was assessed by the release of lac-
tate dehydrogenase (LDH) from the monolayer into
the culture media according to the method published
previously (Moran and Schnellmann, 1996).
by 24% (20.8
1.6 nmol versus 15.9
0.5 nmol
O2/min/mg protein in RPTC treated with 0 and 5 ␮M
12,13-DHOA, respectively).
Exposure of confluent quiescent RPTC to 2 h hy-
poxia followed by 0.5 h reoxygenation decreased basal
oxygen consumption (basal QO2) by 31% in compar-
ison with controls (Fig. 2). Treatment of RPTC with
12,13-EOA prior to hypoxia resulted in the mainte-
nance of basal QO2 at 2 and 24 h following hypoxia
and reoxygenation (Fig. 2, data not shown). RPTC
were also treated with 12,13-DHOA (1, 5, 10 ␮M)
prior to hypoxia/reoxygenation to determine whether
protective effects of 12,13-EOA are mediated through
the hydrolyzed metabolite. Pretreatment of RPTC with
12,13-DHOA had no protective effect against the de-
crease in basal QO2 following hypoxia and reoxygena-
tion (Fig. 2, data not shown).
2.13. Statistical analysis
Data are presented as a mean
standard error
(S.E.) and were analyzed for significance using
ANOVA. Multiple means were compared using the
Student–Newman–Keuls test. Statements of signifi-
cance were based on P < 0.05. Rabbit renal proximal
tubules isolated on a given day represent a separate
experiment (n = 1).
3. Results
3.1. Cell injury
In the initial set of experiments we determined
that 1 h exposure of confluent quiescent RPTC to
12,13-EOA had no effect on oligomycin-sensitive
QO2 (a marker of oxidative phosphorylation)
(13.0 1.0 nmol versus 13.9 1.2 nmol O2/min/mg
protein in RPTC treated with 0 and 5 ␮M 12,13-EOA,
respectively). In contrast, 12,13-DHOA (5 ␮M)
Morphological examination of RPTC monolay-
ers showed no cell detachment and loss or rounding
up following 2 h hypoxia and 0.5 h reoxygena-
tion, which suggested that no cell death occurred
in these conditions (Fig. 1A and C). No cell loss

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G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
Fig. 1. The effects of 2 h hypoxia + 0.5 h reoxygenation (C) and 2 h hypoxia + 24 h reoxygenation (D) on RPTC monolayers. (A) Control
RPTC at 2.5 h. (B) Control RPTC at 24 h. Magnification 100×.

G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
167
Fig. 2. The effects of preconditioning of RPTC with 1, 5, and 10 ␮M 12,13-EOA and 5 ␮M 12,13-DHOA on basal oxygen consumption
(QO2) following 2 h hypoxia and 0.5 h reoxygenation (H/R). RPTC were treated with different concentrations of 12,13-EOA and 5 ␮M
12,13-DHOA for 1 h, culture media changed, and RPTC subjected to hypoxia in the absence of 12,13-EOA or 12,13-DHOA. The
measurements were performed after 30 min reoxygenation. Data are presented as means S.E., n = 4 separate experiments. Values with
different superscripts are significantly different (P < 0.05) from each other.
decreased oligomycin-sensitive QO2 by 60% in con-
trol RPTC (13.0 1.0 nmol versus 5.2 2.8 nmol
O2/min/mg protein in RPTC treated with 0 and 5 ␮M
12,13-DHOA, respectively).
Hypoxia/reoxygenation decreased uncoupled QO2
by 15% (Fig. 3B). Treatments with 12,13-EOA or
12,13-DHOA prior to hypoxia had no protective effect
on uncoupled QO2 in hypoxic RPTC (Fig. 3B).
The results above demonstrate that low micro-
molar concentrations of 12,13-EOA protect against
decreases in mitochondrial function following hy-
poxia/reoxygenation and suggest that protective ef-
fects of 12,13-EOA are mediated through maintaining
the integrity of oxidative phosphorylation and not
electron flow through the respiratory chain. These
results also show that the 12,13-EOA exerts protec-
tive effects directly and not through the 12,13-diol
metabolite.
Oligomycin-sensitive QO2 was reduced by 35%
in RPTC exposed to hypoxia and reoxygenation
(Fig. 3A). Pretreatment with 12,13-EOA prevented
the decreases in oligomycin-sensitive QO2 induced
by hypoxia and reoxygenation in RPTC (Fig. 3A). In
contrast, pretreatment of RPTC with 12,13-DHOA of-
fered no protection against the decrease in oligomycin-
sensitive QO2 caused by hypoxia/reoxygenation
(Fig. 3A). Neither hypoxia/reoxygenation nor 12,13-
EOA or 12,13-DHOA affected oligomycin-insensitive
QO2 (data not shown).
In the initial set of experiments we determined that
uncoupled QO2 (a marker of mitochondrial electron
transport rate) in confluent quiescent RPTC was unaf-
fected by 12,13-EOA (57.9 1.8 nmol versus 56.9
2.7 nmol O2/min/mg protein in RPTC treated with
0 and 5 ␮M 12,13-EOA, respectively) but decreased
27% in the presence of 12,13-DHOA (57.9 1.8 nmol
versus 42.3 8.0 nmol O2/min/mg protein in RPTC
treated with 0 and 5 ␮M 12,13-DHOA, respectively).
3.3. ATP content and the activity of F0F1-ATPase
Intracellular ATP content in control RPTC was
11.4
1.1 nmol/mg protein. Treatment of control
RPTC with 12,13-EOA had no effect on intracellular
levels of ATP (Fig. 4). The exposure of RPTC to hy-
poxia decreased ATP content by 68% (Fig. 4). ATP
levels in hypoxic RPTC reoxygenated for 0.5 h were
equivalent to ATP levels in hypoxic RPTC (Fig. 4).

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G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
Fig. 4. The effect of preconditioning of RPTC with 5 ␮M
12,13-EOA on the intracellular ATP content in RPTC at 0.5 h and
24 h of reoxygenation following 2 h hypoxia. RPTC were treated
as indicated in Fig. 2. ATP samples were taken at 0.5 h and 24 h
of reoxygenation following 2 h hypoxia. Data are presented as
means S.E., n = 3–5 separate experiments.
The activity of F0F1-ATPase was unaffected by
12,13-EOA treatment in control RPTC (Fig. 5). Hy-
poxia/reoxygenation or pretreatment of RPTC with
5 ␮M 12,13-EOA prior to hypoxia had no effect on
F0F1-ATPase activity (Fig. 5). However, pretreatment
with 12,13-DHOA decreased F0F1-ATPase activity in
hypoxic RPTC by 24% (Fig. 5).
These results show that the protective effects
of 12,13-EOA in RPTC are not mediated through
maintaining ATP levels. However, pretreatment with
12,13-EOA accelerates recovery of ATP levels fol-
lowing hypoxia/reoxygenation injury, which sug-
gests that pretreatment with 12,13-EOA maintains
functional integrity of RPTC mitochondria during
hypoxia/reoxygenation. In contrast, 12,13-DHOA de-
creases F0F1-ATPase activity and does not promote
the recovery of respiration and ATP levels following
hypoxia in RPTC.
Fig. 3. The effects of preconditioning of RPTC with 1, 5,
and 10 ␮M 12,13-EOA and 5 ␮M 12,13-DHOA on oligomycin-
sensitive (A) and uncoupled (B) QO2s following 2 h hypoxia
and 0.5 h reoxygenation (H/R). RPTC were treated and measure-
ments carried out as indicated in Fig. 2. Data are presented as
means S.E., n = 4 separate experiments. Values with different
superscripts are significantly different (P < 0.05) from each other.
Pretreatment with 5 or 10 ␮M 12,13-EOA prior to
hypoxia did not prevent decreases in ATP levels
in hypoxic RPTC following 0.5 h of reoxygenation
(Fig. 4, data not shown). However, pretreatment with
12,13-EOA accelerated recovery of ATP levels at
24 h of reoxygenation following hypoxia (Fig. 4). In
contrast, pretreatment with 12,13-DHOA decreased
3.4. Mitochondrial membrane potential (∆Ψm)
ATP levels in control RPTC (11.5
1.1 nmol/mg
Mitochondrial respiration results in the genera-
tion of a proton (and pH) gradient across the inner
mitochondrial membrane and produces the mem-
brane potential (ꢀΨm), which represents most of
the energy of the proton gradient. Lipophilic cations
such as JC-1 accumulate in the mitochondrial ma-
trix driven by the electrochemical gradient (negative
versus 7.3 2.1 nmol/mg protein in RPTC treated
with 0 and 5 ␮M 12,13-DHOA, respectively) and did
not promote the recovery of ATP levels in hypoxic
RPTC (6.1 0.3 nmol/mg versus 5.2 1.2 nmol/mg
protein in hypoxic RPTC pretreated with 0 and 5 ␮M
12,13-DHOA, respectively).

G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
169
Fig. 5. The effect of preconditioning of RPTC with 5 ␮M 12,13-EOA on the F0F1-ATPase activity in mitochondria isolated from RPTC
following 2 h hypoxia and 0.5 h reoxygenation (H/R). RPTC were treated as indicated in Fig. 2. Data are presented as means S.E., n = 3
separate experiments. Values with different superscripts are significantly different (P < 0.05) from each other.
inside mitochondrion). The higher the ꢀΨm, the
more polarized is the mitochondrial membrane, and
more JC-1 is taken up into the mitochondrial matrix.
Once taken up into the mitochondrial matrix, JC-1
forms aggregates that fluoresce red. JC-1 in the cy-
tosol and at low ꢀΨm in mitochondria exists in a
monomeric form that produces green fluorescence.
Thus, an increase in intensity of red fluorescence of
JC-1 indicates higher ꢀΨm, whereas the loss of red
and increased green fluorescence indicates decreased
ꢀΨm and mitochondrial membrane depolarization.
The changes ꢀΨm in RPTC were assessed using ratio
of JC-1 aggreggate/JC-1 monomer.
Treatment of RPTC with 5 ␮M 12,13-EOA alone
had no effect on ꢀΨm in RPTC (Figs. 6A and
7). In contrast, exposure of RPTC to 5 ␮M 12,13-
DHOA increased JC-1 aggreggate/JC-1 monomer
ratio by 30% (Figs. 6B and 7). JC-1 aggreggate/JC-1
monomer ratio in RPTC mitochondria increased also
30% following 2 h hypoxia and 0.5 h reoxygenation
(Figs. 6C and 7). The increase in ꢀΨm following hy-
poxia/reoxygenation was accompanied by decreases
in basal and oligomycin-sensitive respiration of RPTC
(Figs. 2, 3 and 7). Pretreatment with 12,13-EOA prior
to hypoxia prevented increases in JC-1 aggreggate/JC-
1 monomer ratio following hypoxia/reoxygenation
(Figs. 6D and 7). However, pretreatment with 12,13-
DHOA had no effect on hypoxia-induced increases
in ꢀΨm (Figs. 6E and 7). These data suggest that
12,13-EOA, but not its metabolite 12,13-DHOA,
prevents increases in the mitochondrial membrane
potential in RPTC during reoxygenation following
hypoxia.
3.5. Active Na+ transport
Treatment of RPTC for 1 h with 12,13-EOA had
no effect on ouabain-sensitive QO2 (11.0 0.4 nmol
versus 11.3 0.6 nmol O2/min/mg protein in RPTC
treated with 0 and 5 ␮M 12,13-EOA, respectively).
Treatment of RPTC for 1 h with 5 ␮M 12,13-
DHOA decreased ouabain-sensitive QO2 by 86%
(11.0 0.4 nmol versus 1.5 1.3 nmol O2/min/mg
protein in RPTC treated with 0 and 5 ␮M 12,13-
DHOA, respectively).
Exposure of RPTC to hypoxia followed by re-
oxygenation decreased ouabain-sensitive QO2 by
38% (Fig. 8). Pretreatment of RPTC with 12,13-
EOA prior to hypoxia resulted in the maintenance of
ouabain-sensitive QO2 during reoxygenation (Fig. 8).
In contrast to the protective effects of 12,13-EOA
in hypoxic RPTC, pretreatment with 12,13-DHOA
had no effect on decreases in ouabain-sensitive QO2
following hypoxia and reoxygenation (Fig. 8). These
results demonstrate that 12,13-EOA pretreatment, but
not its metabolite 12,13-DHOA, protects against the

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G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
Fig. 6. The effect of preconditioning of RPTC with 5 ␮M 12,13-EOA and 5 ␮M 12,13-DHOA on JC-1 accumulation(red fluorescence) in
RPTC following 2 h hypoxia and 0.5 h reoxygenation. RPTC were treated as indicated in Fig. 2 and loaded with 10 ␮M JC-1 for 30 min
at 37 ◦C following 2 h hypoxia. Fluorescence was analyzed by flow cytometry using excitation by a 488 nm argon-ion laser. The JC-1
monomer (green fluorescence) and J-aggregate (red fluorescence) were detected in FL1 (em. 525 nm) and FL2 (em. 590 nm) channels,
respectively. (A) The effect of vehicle and 5 ␮M 12,13-EOA exposure on JC-1 accumulation in control RPTC. (B) The effect of vehicle
and 5 ␮M 12,13-DHOA exposure on JC-1 accumulation in control RPTC. (C) The effect of 2 h hypoxia and 0.5 h reoxygenation on JC-1
accumulation in RPTC mitochondria. (D) The effect of pretreatment of RPTC with 5 ␮M 12,13-EOA on JC-1 accumulation in RPTC
following 2 h hypoxia and 0.5 h reoxygenation. (E) The effect of pretreatment of RPTC with 5 ␮M 12,13-DHOA on JC-1 accumulation
in RPTC following 2 h hypoxia and 0.5 h reoxygenation. Experiments were performed five times with comparable results.
decrease in active Na+ transport in RPTC caused by
hypoxia and reoxygenation.
2001b; Schonfeld and Bohnensack, 1997; Schonfeld
et al., 2000). In renal proximal tubules, linoleic acid
induces cytotoxicity and metabolism of linoleic acid
to the corresponding monoepoxides and diols repre-
sents a detoxification pathway (Moran et al., 2000,
2001b). Interestingly, lower concentrations of linoleic
acid are cytoprotective in hepatocytes exposed to
IFN-␥ and in renal proximal tubules exposed to mito-
chondrial inhibitors and hypoxia (Liang and Akaike,
1998; Moran et al., 2001b). Linoleic acid and arachi-
donic acid have both been shown to prevent cellular
lysis in renal proximal tubules exposed to hypoxia but
these fatty acids do no protect against cellular damage
upon reoxygenation. It is thought that the mechanism
of cytoprotection of linoleic acid in renal proximal
4. Discussion
The physiological and pathological significance
of linoleic acid metabolism to the monoepoxides by
cytochrome P450 dependent pathways and to linoleic
acid diols by soluble epoxide hydrolase remains
unknown. In many models, pathological concentra-
tions of linoleic acid and other non-esterified fatty
acids uncouple oxidative phosphorylation, induce
mitochondrial permeability transition, and activate
processes leading to cell death (Moran et al., 2000,

G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
171
Pathological concentrations of 12,13-EOA and
12,13-DHOA are toxic in renal proximal tubules
(Moran et al., 2000, 2001b). However, it is possible
that lower concentrations of these active compounds
are cytoprotective against ischemia/reoxygenation in-
jury or enhance recovery processes following injury
in renal proximal tubules. Our study was specifically
designed to determine whether 12,13-EOA and/or
12,13-DHOA protect against the early cellular events
leading to hypoxic injury in RPTC and to examine the
subcellular targets of 12,13-EOA and/or 12,13-DHOA
in RPTC.
No RPTC death occurred in response to hypoxia
and reoxygenation. However, hypoxia produced a
significant degree of sublethal injury to RPTC that
manifested as mitochondrial dysfunction, a decrease
in intracellular ATP levels, and a decrease in active
Na+ transport. Our results show that pretreatment of
RPTC with low concentrations of 12,13-EOA, but
not 12,13-DHOA, protects against mitochondrial dys-
function following hypoxia and reoxygenation. Fur-
thermore, these data show that the protective effects
of 12,13-EOA do not require the presence of 12,13-
EOA during hypoxia and reoxygenation. Despite the
maintenance of basal and oligomycin-sensitive QO2s
in hypoxic RPTC pretreated with 12,13-EOA, intra-
cellular ATP content in hypoxic RPTC was depleted
to the same extend as in hypoxic RPTC that had not
been treated with 12,13-EOA. However, 12,13-EOA
promoted the repletion of intracellular ATP follow-
ing hypoxia/reoxygenation. These results suggest that
linoleic acid epoxides do not exert their protective ef-
fects by decreasing ATP consumption and preserving
ATP concentration during hypoxia. These metabo-
lites appear to maintain the functional integrity of the
mitochondria and promote ATP synthesis during the
recovery process. Our findings are consistent with pre-
vious reports, which showed that following prolonged
ischemia, ATP is repleted more rapidly in hearts
preconditioned by brief periods of ischemia than in
non-preconditioned hearts (Green et al., 1998). In
contrast, arachidonic acid did not promote the recov-
ery of ATP levels in renal proximal tubules following
hypoxia and reoxygenation (Zager et al., 1996). Lack
of recovery of ATP at 24 h following hypoxia may be
caused by consumption of ATP by F0F1-ATPase that
operates in a reverse manner and pumps H+ out of
the mitochondrial matrix to maintain ꢀΨm.
Fig. 7. The effect of preconditioning of RPTC with 5 ␮M
12,13-EOA and 5 ␮M 12,13-DHOA on JC-1 aggregate/monomer
ratio in RPTC following 2 h hypoxia and 0.5 h reoxygenation
(H/R). RPTC monolayers were treated and analyzed as indicated
in Fig. 6. Data are presented as means S.E., n = 9 separate
experiments. Values with different superscripts are significantly
different (P < 0.05) from each other.
tubules is not through promoting the recovery of mi-
tochondrial function and maintaining ATP levels but
preventing chloride influx during the late phases of
oncosis (Moran et al., 2001a; Zager et al., 1996).
Fig. 8. The effects of preconditioning of RPTC with 1, 5, and
10 ␮M 12,13-EOA and 5 ␮M 12,13-DHOA on ouabain-sensitive
QO2 following 2 h hypoxia and 0.5 h reoxygenation (H/R). RPTC
were treated and measurements carried out as indicated in Fig. 2.
Data are presented as means S.E., n = 4−−6 separate experi-
ments. Values with different superscripts are significantly different
(P < 0.05) from each other.

172
G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
Because 12,13-EOA pretreatment had no protective
ꢀΨm in hypoxic RPTC by maintaining intracellular
ion homeostasis. The decrease in Na+/K+-ATPase
activity during hypoxia results in increases in in-
tracellular Na+ concentration and decreases in K+
concentration. The increase in intracellular Na+ may
lead to increases in intracellular Ca2+ and H+ due
to changes in the activity of Na+/H+ exchanger on
the plasma and/or mitochondrial membrane. Thus, by
maintaining Na+/K+-ATPase activity during hypoxia
and reoxygenation, 12,13-EOA may prevent changes
in Na+, K+, and H+ homeostasis and subsequently
ꢀΨm.
effect on the reductions in the mitochondrial electron
transport rate induced by hypoxia/reoxygenation, we
concluded that the protection of mitochondrial func-
tion in RPTC preconditioned by 12,13-EOA was not
mediated through the maintenance of the electron
transfer rate nor through the increased supplementa-
tion of reducing equivalents. It was more plausible that
the cytoprotective effects of 12,13-EOA are mediated
through the preservation of the oxidative phosphory-
lation complex or the maintenance of the mitochon-
drial membrane potential (ꢀΨm). The measurements
of the F0F1-ATPase activity, the major component of
the oxidative phosphorylation complex, demonstrated
that hypoxia/reoxygenation and 12,13-EOA had no ef-
fect on the specific activity of F0F1-ATPase in RPTC
mitochondria. Thus, it appears that pretreating RPTC
with 12,13-EOA did not sustain the mitochondrial
function through maintaining F0F1-ATPase activity.
Similarly, Green et al. (1998) showed that the mech-
anism of ischemic preconditioning in the heart is not
dependent on mitochondrial F0F1-ATPase. However,
it is possible that 12,13-EOA maintained the func-
tion of the ADP/ATP translocase, abolished adenine
nucleotide degradation, or prevented the disruption
of mitochondrial ion homeostasis. In contrast, the
lack of protection against mitochondrial dysfunction
in hypoxic RPTC pretreated with 12,13-DHOA was
associated with decreased activity of F0F1-ATPase,
which suggests that 12,13-EOA and 12,13-DHOA
have differential effects on mitochondrial function.
The decreases in basal and oligomycin-sensitive
respirations in hypoxic RPTC were accompanied
by increases in ꢀΨm. We observed an increase in
both the fluorescence of JC-1 aggregate and the JC-1
aggregate/monomer ratio in RPTC following hy-
poxia/reoxygenation, which was consistent with more
JC-1 accumulating in mitochondria and suggested a
higher ꢀΨm. Pretreatment of RPTC with 12,13-EOA
prevented the increase in ꢀΨm. Increases in the pro-
ton gradient across the mitochondrial membrane in
RPTC subjected to hypoxia/reoxygenation were not
generated by increases in the electron transport rate as
the uncoupled QO2 was decreased in hypoxic RPTC.
Further, the protection against the changes in ꢀΨm
offered by 12,13-EOA was not through increasing
electron transport rate through the respiratory chain.
It is likely that 12,13-EOA attenuates the changes in
In contrast, 12,13-DHOA did not maintain
Na+/K+-ATPase activity in hypoxic RPTC and did
not prevent increases in ꢀΨm. Moreover, the treat-
ment with 12,13-DHOA alone decreased Na+/K+-
ATPase activity in control RPTC and increased ꢀΨm
to the level similar to ꢀΨm in RPTC subjected to
2 h hypoxia and 0.5 h reoxygenation. This suggests
that the inhibition of Na+/K+-ATPase and increases
in ꢀΨm occurred prior to hypoxia. The inhibition of
Na+/K+-ATPase and changes in ion homeostasis may
be a mechanism that mediates the increase in ꢀΨm.
Alternatively, the inhibitory effect of 12,13-DHOA
on F0F1-ATPase may be responsible for the increases
in ꢀΨm and lack of protective effects against hy-
poxia/reperfusion. In most eukaryotic cells, ꢀΨm is
generated by the respiratory chain or through ATP-
dependent reversal of mitochondrial ATP synthase.
The energy released by the electron transport pumps
protons across the mitochondrial inner membrane,
generating an electrochemical and pH gradient. Our
results suggest that the 12,13-DHOA-induced increase
in proton gradient was not generated by an accelerated
rate of electron transport but was rather caused by
decreased activity of H+ pump of F0F1-ATPase. Hy-
perpolarization of the inner mitochondrial membrane
could be explained by the inability of F0F1-ATPase to
efficiently pump protons back into the mitochondrial
matrix through the proton channel of ATP synthase.
This would result in the accumulation of protons
in the mitochondrial intermembrane space and an
increase in the proton gradient. An alternative mito-
chondrial mechanism that could cause an increase in
ꢀΨm is the closure of the voltage-dependent anion
channel (VDAC) localized on the outer mitochondrial
membrane. The closure of VDAC results in a loss of
permeability of the outer mitochondrial membrane to

G. Nowak et al. / Toxicology Letters 147 (2004) 161–175
173
various ions, inhibits mitochondrial respiration and
oxidative phosphorylation, and causes mitochondrial
hyperpolarization. An inhibitory effect of 12,13-
DHOA on VDAC could explain both the decreased
oxidative phosphorylation and the elevated ꢀΨm in
control RPTC.
regulate ion homeostasis in renal cells. Our results
show that pretreatment of RPTC with 12,13-EOA pre-
vents the detrimental effects of hypoxia/reoxygenation
on active Na+ transport. These effects appear to be
specific for 12,13-EOA because 12,13-DHOA has
no protective effects against hypoxia/reoxygenation-
induced decreases in active Na+ transport. Whether
these in vitro results are directly applicable to in vivo
models of ischemia-reperfusion remains unknown.
In conclusion, our data show that pretreatment with
low concentrations of 12,13 EOA, prevents mitochon-
drial dysfunction and decreases in active Na+ trans-
port in RPTC induced by hypoxia and reoxygenation.
The protective effects are specific for 12,13-EOA and
are not observed using 12,13-DHOA, which suggests
that these two metabolites of linoleic acid have differ-
ent targets in RPTC. The 12,13-EOA does not prevent
ATP depletion during hypoxia, however, it maintains
the integrity of the oxidative phosphorylation com-
plex and accelerates the repletion of intracellular ATP
during reoxygenation. Our data also suggest that the
cytoprotection of 12,13-EOA is mediated through the
maintenance of mitochondrial membrane potential and
intracellular ion homeostasis.
Our data are in contrast with reports suggesting that
hypoxia/reperfusion injury in freshly isolated renal
proximal tubules (used in suspension) is associated
with a loss of ꢀΨm (Weinberg et al., 2000). However,
our study showing that 2 h hypoxia/0.5 h reoxygena-
tion increases ꢀΨm was carried out using a different
experimental model (cultured renal proximal tubular
cells) and incubation media. Furthermore, recent stud-
ies have shown that mitochondrial dysfunction is not
always associated with mitochondrial permeability
transition and that mitochondrial membrane hyperpo-
larization is an early event that precedes cytochrome c
release from mitochondria and the initiation of apop-
totic events (Poppe et al., 2001). These studies also
showed that dissipation of mitochondrial K+ and H+
gradients prevents hyperpolarization of the mitochon-
drial membrane, inhibits cytochrome c release and at-
tenuates apoptosis (Poppe et al., 2001). Our data show
that preventing increases in ꢀΨm and decreases in ac-
tive Na+ transport by 12,13-EOA in hypoxic RPTC is
associated with maintained mitochondrial respiration
and improved recovery of intracellular ATP levels.
Our results also demonstrate that the effects of 12,13-
EOA are specific because 12,13-DHOA does not pre-
vent hypoxia-induced increases in ꢀΨm, decreases in
active Na+ transport, and mitochondrial dysfunction.
Regulation of intracellular ion homeostasis is a
potential mechanism for preventing mitochondrial
dysfunction. It has been suggested that the metabo-
lites of the 12-lipoxygenase pathway of arachidonic
acid metabolism mediate ischemic preconditioning
through the regulation of K+ and/or Ca2+ channel
activities (Chen et al., 1999; Murphy et al., 1995).
Also, arachidonic acid metabolites of the cytochrome
P450 epoxygenase pathway alter electrolyte transport
in kidney cells. In cortical tubular cells, these metabo-
lites increase cytosolic Ca2+ concentrations, inhibit
Na+ reabsorption and K+ secretion, and activate
Na+/H+ exchange (Harris et al., 1990; Jacobson et al.,
1984; Madhun et al., 1991). The present study shows
that oxidized linoleic acid metabolites derived form
cytochrome P450 epoxygenase dependent pathways
Acknowledgements
This study was supported by the American
Heart Association (Heartland Affiliate) Grant-in-Aid
0151416Z (to GN), by NIH grant GM56708 (DFG),
and by Department of Defense Grants N00014-99-1-
0905 and N00014-99-1-0606 (DFG).
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