JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2010, 61, 3, 325-332
www.jpp.krakow.pl
J.M. MORENO1, I. RODRIGUEZ GOMEZ1, R. WANGENSTEEN2, R. PEREZ-ABUD3, J. DUARTE4, A. OSUNA3, F. VARGAS1
MECHANISMS OF HYDROGEN PEROXIDE-INDUCED VASOCONSTRICTION
IN THE ISOLATED PERFUSED RAT KIDNEY
1
Departamento de Fisiologia, Facultad de Medicina, Granada, Spain; 2Departamento de Ciencias de la Salud, Universidad
de Jaen, Spain; 3Servicio de Nefrologia, Unidad Experimental, Hospital Virgen de las Nieves, Granada, Spain;
4Departamento de Farmacologia, Facultad de Farmacia, Granada, Spain
The vasoconstrictor effect of hydrogen peroxide (H2O2) on isolated perfused rat kidney was investigated. H2O2 induced
vasoconstriction in the isolated rat kidney in a concentration-dependent manner. The vasoconstrictor effects of H2O2
were completely inhibited by 1200 U/ml catalase. Endothelium-removal potentiated the renal response to H2O2. The
H2O2 dose-response curve was not significantly modified by administration of the NO inhibitor L-NAME (10-4 mol/l),
whereas it was increased by the non-specific inhibitor of K+-channels, tetraethylammonium (3·10-3 mol/l). Separately,
removal of extracellular Ca2+, administration of a mixture of calcium desensitizing agents (nitroprusside, papaverine,
and diazoxide), and administration of a protein kinase C (PKC) inhibitor (chelerythrine, 10-5 mol/l) each significantly
attenuated the vasoconstrictor response to H2O2, which was virtually suppressed when they were performed together.
The pressor response to H2O2 was not affected by: dimethyl sulfoxide (7·10-3 mol/l) plus mannitol (3·10-3 mol/l);
intracellular Ca2+ chelation using BAPTA (10-5 mol/l); calcium store depletion after repeated doses of phenylephrine (105 g/g kidney); or the presence of indomethacin (10-5 mol/l), ODYA (2·10-6 mol/l) or genistein (10-5 mol/l). We conclude
that the vasoconstrictor response to H2O2 in the rat renal vasculature comprises the following components: 1)
extracellular calcium influx, 2) activation of PKC, and 3) stimulation of pathways leading to sensitization of contractile
elements to calcium. Moreover, a reduced pressor responsiveness to H2O2 in female kidneys was observed.
K e y w o r d s : kidney, hydrogen peroxide, Ca2+, protein kinase C, sexual dimorphism, renal perfusion pressure, catalase,
vasoconstriction
INTRODUCTION
Hydrogen peroxide is produced in many different human
cell types, including fibroblast, vascular endothelial, smooth
muscle, and inflammatory cells (1). It is known to act as a
cellular signaling molecule within blood vessels, and it plays key
roles in regulating vascular smooth muscle cell (VSMC) growth,
differentiation, migration, and vascular inflammation (1-5).
Studies have demonstrated the importance of H2O2 in
regulating vascular tone, but its role not well understood since it
can modify vascular tone in complex ways. Thus, studies have
demonstrated both a contractile and relaxant response to H2O2
depending on the species, vascular bed, and contractile state (2,
4, 6, 7). However, the effects of H2O2 on the complete renal
vasculature are not known. H2O2 has been shown to cause
constriction in a variety of vascular beds under quiescent
conditions, and it can induce vasoconstriction in a number of
arteries in vitro, including rat aorta (8, 9), vena cava (7, 10) and
pulmonary artery (11), canine basilar artery (12), and human
placental arteries (13). Several mechanisms contribute to H2O2induced vasoconstriction in these vessels, including: increase in
Ca2+ influx or Ca2+ release from intracellular stores in smooth
muscle cells (14, 15); formation of cyclooxygenase-derived
prostanoids (8, 16); activation of enzymes such as phospholipase
A2 (17), phospholipase C (18), tyrosine kinases (10) and protein
kinase C (19); activation of potassium (K+) channels (7); and
generation of hydroxyl radicals (9).
Increased production of free radicals is closely associated
with an enhanced arteriolar tone (20, 21), which is in turn
associated with an elevation in blood pressure. Thus, Swei et al.
(20) reported a correlation between arteriolar tone and plasma
hydrogen peroxide levels in hypertensive rats. In mice, vascular
overexpression of catalase reduced the pressor response to
vasoconstrictor agents (22) and decreased systolic blood
pressure and vascular constriction per se, indicating the
importance of endogenous H2O2 as a vasoconstrictor and
regulator of blood pressure (23).
The role of hydrogen peroxide in regulating vascular tone
would be especially relevant in the kidney, since this organ
receives 20% of cardiac output, and renal hemodynamics play an
essential role in the control of renal sodium excretion and blood
pressure (24). Thus, Chen et al. (25) demonstrated that the shortterm administration of H2O2 into the renal medulla significantly
reduces renal medullary blood flow and sodium excretion, and
Makino et al. (26) found that increased renal medullary H2O2
leads to hypertension.
Taking account of the above findings on the importance of
the tone of the renal vascular bed in the regulation of total
326
peripheral resistance, renal sodium excretion, and blood
pressure, the present experiments were designed to test the
hypothesis that H2O2 may play a significant role in the control of
renal vascular tone. To this end, the ability of H2O2 to contract
the complete renal vasculature in the isolated perfused rat kidney
was evaluated, studying the main mechanisms responsible for
this effect. Gender-associated differences in the renal
responsiveness to H2O2 were also investigated.
MATERIALS AND METHODS
Animals and isolated perfused kidney preparation
This investigation conformed to the Guide for the Care and
Use of Laboratory Animals published by the US National
Institute of Health (NIH Publication No 85-23, revised 1996).
Ninety-six male (300±10 g) and six female Wistar age matched
rats (250±6 g) were maintained on standard chow and tap water
ad libitum. The isolated perfused kidney preparation was
performed following a previously reported procedure (27, 28).
The animals were anesthetized with pentobarbital sodium (40
mg/kg, i.p.), the abdomen was opened through a midline
incision, and the renal arteries were exposed and cannulated with
a beveled 18-gauge needle and secured with ligatures. The
kidneys were perfused at a constant flow rate (5 ml/g of kidney
weight per min) by means of a roller pump (IPS-4, Ismatec S.A.
Zurich) with Tyrode solution (37°C) of the following
composition (mM): NaCl, 137; KCl, 2.7; CaCl2, 1.8; MgCl2, 1.1;
NaHCO3, 12.0; NaH2PO4, 0.42; and D(+) glucose, 5.6, all
aerated with 5% CO2 in O2. The kidney was then dissected clear
from its surrounding tissues and placed in a chamber containing
the Tyrode solution at 37°C. Renal vascular responses were
recorded (TRA-021 transducer connected to a two-channel
Letigraph 2000 recorder, Letica S.A., Barcelona) as changes in
the renal perfusion pressure downstream from the pump.
Experimental protocol
The experiments evaluated the renal response to H2O2 (2.2 to
22 x 10-5 mol) under the following conditions (n=6, each group):
1) no treatment; 2) after administration of catalase (1200 U/ml)
to evaluate the specificity of the vasoconstrictor effect; 3) after
administration of DMSO/mannitol (7/3 10-3 mol/l) as hydroxyl
radical scavenger; 4) after endothelium removal; 5) after LNAME (10-4 mol/l) pretreatment to inhibit NO synthesis; 6) after
pretreatment with tetraethylammonium (3·10-3 mol/l) as nonspecific K+ channel inhibitor; 7) without calcium plus EGTA
(5·10-4 mol/l) to evaluate the role of extracellular calcium; 8)
after verapamil (10-5 mol/l) to analyze calcium influx; 9) without
calcium plus BAPTA (10-5 mol/l); 10) after verapamil plus
BAPTA to evaluate the role of intracellular calcium; 11) without
calcium plus four successive bolus doses (10-5 g/g kidney) of
phenylephrine to deplete intracellular calcium stores; 12) after
indomethacin (10-5 mol/l) to inhibit prostaglandins; 13) after
ODYA (2·10-6 mol/l) to inhibit cytochrome P450
monooxygenase; 14) after chelerythrine (10-5 mol/l) to inhibit
protein kinase C; 15) after genistein (10-5 mol/l) to inhibit protein
tyrosine kinase; 16) after combined pretreatment without
calcium plus nitroprusside (10-4 mol/l), papaverine (10-4 mol/l),
and diazoxide (10-4 mol/l), using these three drugs to decrease
the sensitivity to calcium and reduce the contractility; and 17)
after this last treatment (condition 16) plus chelerythrine.
The endothelium was removed by passing air through the
isolated kidney for 4.5 min. Endothelium removal was assessed
by measuring the vasodilator response to a bolus dose of 10-6 g/g
kidney acetylcholine (ACh) in the vascular bed after its
constriction with phenylephrine (10-6 mol/l). Preparations with a
vasodilator response to ACh of >10% were rejected. The above
inhibitors were added to the perfusate after the stabilization
period, followed by a 30-min interval before dose-response
curves were recorded. The inhibitors were present throughout
the experiment. Dose-response curves were constructed by
injecting boluses of 50 µl/g kidney of the agonist. Each injection
produced a small and transient increase in renal perfusion
pressure (RPP) that preceded the agonist-induced response. The
minimum time interval between successive doses of an agonist
was 10 min, which could be extended when necessary until the
previous response had disappeared. Dose-response curves in the
presence of the inhibitors were compared with the respective
dose-response curves under normal conditions. The peak
response was used to construct these curves. Dose-response
curves were performed in untreated kidneys throughout the
experimental period. From these curves, we randomly took n=6
for statistical purposes as control values. The responsiveness to
H2O2 was also determined in isolated kidneys from female rats
in order to explore any gender-related differences in the
vasoconstriction induced by this agent.
Drugs
The following drugs were used: l-phenylephrine
hydrochloride, acetylcholine, catalase, dimethyl sulfoxide
(DMSO), H2O2, indomethacin, mannitol, tetraethylammonium
chloride (TEA), diazoxide, L-phenylephrine hydrochloride, Nωnitro-L-arginine methyl ester (L-NAME), ethyleneglycol-bis (βaminoethyl ether)N,N’-tetraacetic acid (EGTA), acetyl methyl
ester of bis (o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid
(BAPTA-AM), genistein, chelerythrine, sodium nitroprusside,
papaverine hydrochloride, and verapamil hydrochloride,
purchased from Sigma-Aldrich Quimica S.A. (Alcobendas,
Madrid, Spain); pentobarbital sodium, purchased from Serva
(Heidelberg, Germany); and ODYA, from Cayman Chemical
(Ann Arbor, MI, USA). The drugs were prepared daily in deionized water from stock solutions kept at - 20°C. Stock solutions
were prepared in water, with the exception of indomethacin,
which was prepared in absolute ethanol and BAPTA-AM in
NaHCO3 (0.3N). H2O2 was purchased as 30% w/w concentrated
solution and subsequently diluted in distilled water.
Statistical analysis
Dose-response curves in the presence or absence of the
inhibitors were compared using a three-factor random block
design (rat, dose, and treatment). When the results of the analysis
of variance were significant, the Tukey’s t and Neumann-Keuls
tests were applied (29).
RESULTS
The baseline RPP value was 42±2 mmHg (n=96). Baseline
RPP values were not significantly modified by any treatment
with the exception of endothelium removal, which was followed
by a transient elevation of vascular tone that returned to baseline
values within 15 min. The control dose-response curve to H2O2
is the same in the different Figures.
H2O2 induction of vasocontriction in the isolated perfused
kidney and influence of sex
Fig. 1A shows that H2O2 elicited a concentration-dependent
vasoconstriction of isolated rat kidney. The concentration of
H2O2 needed to induce near-maximal constriction was 22·10-5
327
Fig. 1. (A) Representative tracing of the vasoconstrictor responses to bolus doses of H2O2 in the isolated rat kidney in comparison to
the maximal response to a bolus dose of phenylephrine, expressed as absolute changes in renal perfusion pressure (RPP). The control
dose-response curve to H2O2 is the same in all Figures. Control dose-response curves to H2O2 were randomly performed throughout
the experimental period. (B) Vascular response to increasing bolus doses of H2O2 in kidneys from male and female rats. * P<0.01
compared with the kidneys of male rats. Data are expressed as means±SEM (n=6 in each group). Doses of H2O2 are expressed per
gram of kidney.
mol. The maximal peak response to H2O2 was 74±5% of the
peak response to phenylephrine.
A sexual dimorphic pattern of response to H2O2 was
observed. Renal vasculature from female rats showed markedly
reduced responsiveness to H2O2 (Fig. 1B). The concentrationresponse curve was characterized by a shift towards the right,
with decreased responses to medium concentrations and a
similar maximal response.
Effect of catalase and of hydroxyl radical scavengers on the
renal response to H2O2
The treatment of isolated kidneys with 1200 u/ml catalase,
which decomposes H2O2, produced an almost complete
disappearance of the pressor response to H2O2 (Fig. 2A).
Experiments were carried out in the presence of DMSO plus
mannitol (hydroxyl radical scavengers) to examine the possible
role of hydroxyl radical formation in the vasoconstriction effects
of H2O2. These effects on the renal vascular bed were not
affected by administration of DMSO plus mannitol (Fig. 2A),
suggesting that the contractile response to H2O2 is not associated
with the production of hydroxyl radicals.
Effects of endothelium removal, nitric oxide blockade, and K+channel blockade on the renal response to H2O2
Analysis of the dose-response curves showed a significantly
greater vasoconstriction at medium doses in kidneys with
endothelium removed than in those with intact endothelium.
Administration of the NO inhibitor L-NAME did not
significantly modify the dose-response curve to H2O2, whereas
administration of the non-specific K+-channel inhibitor
tetraethylammonium produced a significant shift to the left in the
dose-response curve to H2O2 (Fig. 2B).
Effects of indomethacin and CYP-450 inhibition on H2O2induced vasoconstriction in the isolated kidney
Administration of the prostaglandin inhibitor indomethacin
did not modify the dose-response curves to H2O2 in the isolated
kidneys (Fig. 2C). The effects of the P450 cytochrome inhibitor
ODYA were tested to investigate the possible involvement of the
metabolites of arachidonic acid generated by cytochrome P450dependent enzymes. As shown by Fig. 5, the presence of ODYA
had no effect on the vasoconstrictor response to any of the tested
concentrations of H2O2 (Fig. 2C).
Involvement of Ca2+ in H2O2-induced vasoconstriction of rat
kidney
Results obtained demonstrated that extracellular Ca2+ is
required for the vasoconstriction produced by increasing
concentrations of H2O2. The response of isolated kidney to H2O2
was assessed after equilibrating the renal vasculature in Ca2+free Tyrode solution containing 2.10-4 mol/l EGTA for at least 30
min before the experiments (Fig. 3A) and after calcium channel
blockade with verapamil (Fig. 3B). Removal of Ca2+ from the
extracellular medium and verapamil administration both had an
appreciable inhibitory effect on the vasoconstriction caused by
all tested concentrations of H2O2.
For the intracellular Ca2+-buffered experiment, the
membrane-permeable Ca2+ chelator BAPTA was added to the
medium in the presence of verapamil. The role of intracellular
Ca2 stores was also analyzed by administering four doses of
phenylephrine (10-5 g/g kidney), which led to the disappearance
of the response to this agonist. The pressor response to H2O2 in
the isolated kidney was not significantly modified by
intracellular chelation or by store depletion (Fig. 3A and B), and
intracellular chelation with BAPTA did not significantly modify
the inhibitory effect produced by verapamil, suggesting that
intracellular Ca2+ does not play a role in the H2O2-induced
vasoconstriction observed.
Role of PKC and protein tyrosine phosphorylation in H2O2induced vasoconstriction in the isolated kidney
The protein tyrosine kinase inhibitor genistein were tested to
investigate the involvement of PKC and/or protein tyrosine
phosphorylation in the H2O2-induced vasoconstriction observed.
Contractile responses of the arteries to H2O2 were suppressed by
328
Fig. 2. (A) Effects of the administration of catalase (1200 U/ml)
and hydroxyl radical scavengers DMSO/mannitol (7/3 10-3
mol/l), (B) Effects of endothelium removal, L-NAME (10-4
mol/l), or TEA (3·10-3 mol/l) and (C) Effects of indomethacin
(10-5 mol/l) and ODYA (2·10-6 mmol/l) on the renal response to
increasing bolus doses of H2O2. Data are expressed as
means±SEM (n=6 in each group). Doses of H2O2 are expressed
per gram of kidney. * P<0.05; ** P<0.001 vs. normal conditions.
Data are expressed as means±SEM (n=6 in each group).
Fig. 3. (A). Effects of calcium-free Thyrode plus EGTA (5·10-4
mmol/l), calcium-free Thyrode plus BAPTA (10-5 mol/l), and
calcium-free Thyrode plus 4 successive bolus doses (10-5 g/g kidney)
of phenylephrine. (B) Effects of verapamil (10-5 mol/l) and of
verapamil plus BAPTA (10-5 mol/l) and C Effects of chelerythrine
(10-5 mol/l), genistein (10-5 mmol/l), calcium-free Thyrode plus
calcium desensitizing agents (nitroprusside, 10-4 mol/l; papaverine,
10-4 mol/l; and diazoxide, 10-4 mol/l) and calcium-free Thyrode plus
chelerythrine plus calcium-desensitizing agents on the renal response
to increasing bolus doses of H2O2. * P<0.01; ** P<0.001 vs. normal
conditions. Data are expressed as means±SEM (n=6 in each group).
Doses of H2O2 are expressed per gram of kidney.
329
Table 1. Maximal response in renal perfusion pressure (RPP) produced by the highest dose of H2O2 (22.10-5 mol) under the different
experimental conditions. Data are given in absolute values.
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treatment of the renal vascular bed with chelerythrine but were
not modified by genistein treatment (Fig. 3C). These data
strongly suggest that PKC activation but not protein tyrosine
phosphorylation is an important step in H2O2 -induced
vasoconstriction in the isolated kidney.
Influence of calcium-desensitizing agents on H2O2-induced
vasoconstriction of isolated rat kidney.
To analyze the influence of H2O2 on calcium sensitivity, the
response to this agent was studied in the presence of a cocktail
of calcium-desensitizing agents composed of papaverine (PKA
activator), nitroprusside (PKG activator) and diazoxide (K+
channel activator) in the absence of extracellular calcium. The
mixture of desensitizing agents produced an important
attenuation in the dose response curve to H2O2 (Fig. 3C).
Moreover, the addition of chelerythrine to these agents in the
calcium-free solution virtually abolished the pressor response to
H2O2 in the isolated kidney (Fig. 3C).
Table 1 summarizes the differences in renal perfusion
pressure attained by the highest dose of H2O2 under normal
conditions and after the administration of the different inhibitors.
DISCUSSION
H2O2 is known to modify the vascular tone of various
preparations, but no data have been published on the effect of
H2O2 on the complete renal vascular bed. This study aimed to
define the effects of H2O2 as a regulator of vascular tone in the
renal vasculature and to gain insight into the mechanisms of
H2O2-induced vessel contractility. The isolated perfused rat
kidney was selected for the experiments because it comprises the
complete renal vasculature including small resistance arteries
and arterioles, which are physiologically more relevant to the
control of vascular resistance. Results obtained demonstrate that
H2O2 produces a concentration-dependent vasoconstriction in
the isolated rat kidney. The maximal vasoconstrictor response
was around 74% of that obtained with phenylephrine but lasted
longer (180% increase in duration). Thakali et al. (30) also
observed that the aortic and venous contraction produced by
H2O2 was lower than the maximal contraction induced by an
adrenergic agonist.
The concentration of hydrogen peroxide used in this and
other studies (7-9), which resulted in contraction, could
conceivably occur in vivo, especially under certain
pathophysiological conditions. H2O2 levels can reach 0.8 mmol/l
in rat venular endothelium during neutrophil activation (31), and
vascular H2O2 levels in the µmolar range can be found in the
plasma of hypertensive patients (32, 33). Antihypertensive
therapy is accompanied not only by a decrease in blood pressure
but also by a fall in plasma H2O2 levels (33). Moreover, an
increased vasoconstrictor response to H2O2 has been reported in
genetic (8) and secondary (7) forms of hypertension. Considered
alongside the above findings, the present observation that H2O2
is a potent vasoconstrictor in the renal vasculature suggests that
H2O2 generated in the vascular wall can modulate renal vascular
tone and contribute to the pathogenesis of various vascular
diseases. This may be of special importance in hypertension, due
to the key role of renal hemodynamics in this disease (24).
Experimental evidence suggests that oxidative stress is
greater in the male sex (34). The increased plasma H2O2 levels in
hypertensive patients are lower in women than in men (33), and
female SHR showed reduced urinary excretion of H2O2 in
comparison to male SHR (35). Female sex is also associated
with decreased renal vascular reactivity to vasoconstrictors (27).
We found a lower pressor responsiveness to H2O2 in the kidneys
from female rats than in those from male rats. This reduced
330
responsiveness to H2O2 and its lower production might
contribute to the reduced blood pressure of female animals.
With respect to the mechanism by which H2O2 induced renal
vasoconstriction, research into the role of Ca2+ has yielded
controversial findings in different preparations. Thus, H2O2induced contraction was independent of extracellular Ca2+ influx
in pulmonary arteries (11, 36) but dependent on extracellular
Ca2+ influx and release of intracellular Ca2+ in canine basilar
arteries and rat thoracic aorta (15). Thakali et al. (7) observed
that aortic H2O2-induced contraction requires extracellular Ca2+
influx but venous H2O2-induced contraction does not. These
studies indicate possible species-specific, experimental
condition–specific, and vessel-specific differences in the role of
extracellular influx of Ca2+ and release of calcium from
intracellular stores in H2O2-induced contraction. The present
study demonstrated that removal of extracellular Ca2+
significantly depressed the constrictor effects of H2O2 on renal
vessels. These contractile responses to H2O2 were not
significantly modified when the intracellular Ca2+ in smooth
muscle cells was buffered by 10-5 mol/l BAPTA, a membranepermeable Ca2+ chelator, or when the internal stores of calcium
were depleted by the administration of successive boluses of
phenylephrine. Therefore, our data indicate that extracellular
Ca2+ but not Ca2+ release from intracellular stores is required for
H2O2-induced vessel constrictions in the isolated kidney.
Oxidants such as H2O2 are known to participate in PKC
mobilization in vascular smooth muscle cell membranes (37).
PKC is involved in the signaling pathways by which
vasoconstrictor stimuli induce activation of vascular smooth
muscle. PKC may increase the myofilament force sensitivity to
(Ca2+) and myosin light-chain kinase phosphorylation, thereby
maintaining vascular contraction (38). In the present study, the
PKC inhibitor chelerythrine significantly attenuated the H2O2induced vasoconstriction of rat renal vasculature, indicating the
involvement of PKC in this effect of H2O2. These data are
consistent with reports that PKC activation plays an essential
role in the development of contractile responses in aortic rings
(9) and endothelial cells (39).
Treatment of vascular smooth muscle cells with oxidants,
including H2O2, has been reported to enhance protein tyrosine
phosphorylation (37). The present results demonstrate that the
protein tyrosine kinase genistein did not significantly modify the
H2O2-produced constriction of rat renal vasculature, suggesting
that protein tyrosine phosphorylation is unlikely to be involved
in this vasoconstriction. These findings contrast with a report by
Yang et al. (9) that genistein significantly suppressed the H2O2produced contraction of rat aortic rings.
Intrarenal microcirculation is under hormonal, paracrine
and neural control, and renal vascular tone results of the
interaction of vasoconstrictors and vasodilators (40, 41). In this
balance is specially important the contribution of three major
members of the vasodilator family: nitric oxide, prostaglandins
(40) and epoxides (41), CYP-450 dependent arachidonic acid
(AA) metabolites, identified with the endothelium-derived
hyperpolarizing factor (EDHF), all released from endothelial
cells, to the buffering of the intrarenal vasoconstrictor
influences (28). We found a shift to the left of the dose-response
curve to H2O2 in endothelium-denuded kidneys with respect to
intact kidneys, indicating that the H2O2-elicited
vasoconstriction is negatively modulated by endothelium, as
previously reported in rat aorta (8, 9). However, the dose
response curve to H2O2 in the renal vasculature was not
significantly affected by nitric oxide (NO) blockade. Hence, the
negative modulatory role of NO found in the responsiveness to
other vasoconstrictors in the isolated kidney (27, 28) is not
observed in the vasoconstrictive response to H2O2, possibly
because H2O2 can impair NO-mediated signaling in blood
vessels (42), stimulate NAD(P)H oxidase in vascular cells (43),
reduce levels of tetrahydrobiopterin (44), and increase
expression of arginase I (45). Moreover, H2O2 decreases NO
production by inactivating eNOS cofactors without affecting
eNOS activity (46). L-NAME may be ineffective due to these
potential anti-NO effects of H2O2, therefore the increased
responsiveness to H2O2 produced by endothelium removal
would be due to blockade of the endothelium-derived
hyperpolarizing factor. In fact, H2O2-induced renal
vasoconstriction was significantly enhanced when K+ channels
were inactivated with tetraethylammonium in the present study,
in agreement with reports that K+ channel activity plays an
important role in the vasoconstrictor response to H2O2. Thus,
Thakali et al. (7) observed that aorta under quiescent conditions
contracted minimally to exogenous H2O2 and that aorta
depolarization, which induces K+ channel blockade, potentiated
aortic contraction to H2O2; Ardanaz et al. (2) obtained similar
results in the abdominal aorta and superior mesenteric artery. K+
channel blockade did not enhance renal H2O2 vasoconstriction
to the same degree as reported in aortic tissue (7). These intertissue differences in H2O2 responsiveness have been attributed
to differences in K+ channel expression (7). Thus, rat aorta
expressed low levels of KATP channel mRNA, whereas the vena
cava, which is almost unresponsive to K+ channel blockade, had
no detectable KATP channel mRNA (47).
H2O2 has been shown to induce indomethacin-sensitive
contractions in pial arterioles from newborn pigs (48), in strips
of guinea-pig trachea (49), and in rat aortic tissue (8, 9). In our
experiment, H2O2-induced vasoconstriction of the renal vessels
was not significantly affected in the presence of indomethacin,
indicating that the contractile effects of H2O2 in the renal
vascular bed are not mediated or modulated by an increased
metabolism of arachidonic acid via the cyclooxygenase pathway.
Previous results in rat aorta suggested a role for the release of
cytochrome P450-dependent metabolites in response to H2O2 (9),
and the administration of proadifen (an inhibitor of cytochrome
P450 monooxygenase inhibitor) markedly potentiated H2O2induced aortic contractions in rat. However, the administration of
ODYA (cytochrome P450 monooxygenase inhibitor) in the
present study did not significantly modify the renal response to
H2O2, suggesting that CYP-450 derivatives do not play a role.
Several authors have suggested that the generation of
hydroxyl radicals contributes to the vascular smooth muscle
contraction induced by H2O2 (50), and the hydroxyl radical
scavengers deferoxamine and DMSO significantly attenuated
the contractile response to H2O2 in aortic rings (9). However, the
vasoconstrictor effect of H2O2 in the renal vascular bed was not
modified by the association of the radical scavengers DMSO and
mannitol, suggesting that the formation of hydroxyl radicals
plays no part in this effect. Similar results have been reported in
pial arterioles of newborn pigs using deferoxamine (48), and in
the aortic tissue using DMSO (40) or DMSO plus mannitol (6).
In summary, this study demonstrates that hydrogen peroxide
can induce vasoconstriction in the vasculature of isolated
perfused rat kidney. Our results suggest that the vasoconstrictor
response to H2O2 in the rat renal vasculature comprises the
following components: 1) extracellular calcium influx, 2) PKC
activation, and 3) stimulation of pathways leading to the
sensitization of contractile elements to calcium. Our data also
indicate that no role is played by calcium release from
intracellular stores, prostaglandins, CYP-450 products, or
tyrosine phosphorylated products in the vasoconstrictor response
to H2O2 in the rat renal vascular bed.
Acknowledgments: This study was supported by a grant (CTS1659) Proyecto de Excelencia from Consejería de Innovación
Ciencia y Empresa of the Junta de Andalucía and by the Ministerio
331
de Sanidad y Consumo, Instituto de Salud Carlos III (Red de
Investigación Renal, REDinREN RD06/0016/0017 and Red
HERACLES RD06/0009). Fondo Europeo de Desarrollo
Regional (FEDER) “Una manera de hacer Europa”. We thank R.
Arcas and M. Quintana for expert technical assistance.
Conflict of interests: None declared.
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R e c e i v e d : August 20, 2009
A c c e p t e d : May, 2010
Author´s address: Dr. F. Vargas, Departamento de Fisiologia,
Facultad de Medicina, E-18012, Granada, Spain; Phone:
+34958243520; Fax: +34958246179; E-mail: [email protected]