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Vol. 303, Issue 2, 823-830, November 2002
-Hydroxy-L-arginine and
Other Compounds Bearing a C=NOH Function in the Rat Aorta
Pharmacology and Physico-Chemistry, Centre National de la Recherche Scientifique (Unité Mixte Recherche 7034) and University Louis Pasteur, Strasbourg, France (P.V., C.S., P.B., K.C., N.C., B.M., J.-C.S.); Laboratory of Pharmacological and Toxicological Chemistry and Biochemistry, Centre National de la Recherche Scientifique (Unité Mixte Recherche 8601) and University René Descartes, Paris, France (J.L.B., D.M.); and Department of Biochemistry, Charles University, Prague, Czech Republic (P.V., P.B., K.C., G.E.)
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Abstract |
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The mechanisms of vasorelaxation elicited by
N
-hydroxy-L-arginine
(L-NOHA) and other compounds bearing a C=NOH function and the structural determinants governing this effect were investigated in
rat aorta. L-NOHA, formamidoxime, five aromatic
monosubstituted amidoximes, and one aromatic monosubstituted ketoxime
elicited relaxation in endothelium-denuded rings.
N-Hydroxyguanidine and substituted
N-hydroxyguanidines were markedly less active.
Relaxations induced by L-NOHA and by the most active
studied compound, 4-chlorobenzamidoxime (ClBZA), were unmodified by the
presence of endothelium. In endothelium-denuded rings, they were
blunted by the NO scavenger
2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (300 µM) and
by the inhibitor of guanylyl-cyclase activation
1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (1 µM). In addition, L-NOHA- and ClBZA both caused cGMP
accumulation. L-Arginine, but not
D-arginine (1 mM), antagonized the effect of
L-NOHA but not ClBZA. Both L-NOHA- and
ClBZA-induced relaxations were inhibited by the NAD(P)H-dependent
enzymes inhibitor diphenyliodonium (30 µM) and the NAD(P)H-dependent
reductases inhibitor 7-ethoxyresorufin (10 µM), but they were
unmodified by the cytochrome P450 (P450) inhibitor proadifen (10 µM) and by the NO synthase inhibitor
N
-nitro-L-arginine methyl
ester (L-NAME, 300 µM). These results show that
L-NOHA and other compounds with a C=NOH function can cause
endothelium-independent relaxation in the rat aorta. They suggest that
activation of guanylyl cyclase and NO formation is implicated in
relaxation and that a 7-ethoxyresorufin-sensitive NAD(P)H-dependent
pathway is involved. On one hand, L-NOHA and amidoximes may
be useful tools for characterizing this pathway in blood vessels and,
on the other, may offer a novel approach for treating vascular diseases
with impaired endothelial NO activity.
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Introduction |
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N
-Hydroxy-L-arginine
(L-NOHA) is a stable intermediate in the two step
reaction catalyzed by NO synthase, the enzyme family involved in the
formation of NO and L-citrulline from
L-arginine and O2 in
mammals (Forstermann et al., 1994
; Marletta, 1994
; Stuehr, 1997
). The
first step of the reaction, N-oxygenation of the guanidine function of L-arginine, is specifically and
exclusively catalyzed by NO synthase isoforms (Stuehr et al., 1991
;
Klatt et al., 1993
). The second step of the reaction consists in
oxidative cleavage of the C=NOH bond of the
N-hydroxyguanidine function, with formation of the free
radical NO and L-citrulline. Recently, it has
been reported that non-
-amino acid
N-aryl-N-hydroxyguanidines are also oxidized by
NO synthase with formation of NO (Renodon-Cornière et al., 2002
).
It has been known for several years, however, that oxidative cleavage
of the C=N bond of C=NOH functions of various compounds, including
L-NOHA, can be catalyzed also by hemoproteins like horse radish peroxidase, rat liver microsomal cytochrome P450
(P450), hemoglobin, and catalase. This results in generation of stable
nitrogen oxides, with the possible intermediate formation of NO
(Boucher et al., 1992a
,b
; Jousserandot et al., 1998
; Caro et al.,
2001
). The latter reactions provide alternative pathways to the second
step of the reaction catalyzed by NO synthases. The existence of such
pathway(s) might be important in blood vessels to restore NO formation
in pathological situations in which endothelial NO synthase expression
or activity is impaired. This is especially the case of
atherosclerosis, diabetes, hypertension, and various situations of
increased coronary risk such as cigarette smoking (for reviews see
Harrison, 1997
; Li and Forstermann, 2000
).
Being a substrate for NO synthase (Stuehr et al., 1991
; Klatt et al.,
1993
; Moali et al., 2000
), L-NOHA can produce
endothelium-dependent vascular relaxation as a result of its
metabolization to NO and L-citrulline by the endothelial NO
synthase. This mechanism, which is antagonized by NO synthase
inhibitors, has been reported to account for the major part of
relaxation caused by L-NOHA in bovine pulmonary and porcine
coronary arteries (Wallace et al., 1991
; Abdul-Hussain et al., 1996
).
In other vessels such as the rabbit aorta, however, L-NOHA
produced no endothelium-dependent relaxation by itself; it did not
influence endothelium-dependent relaxation caused by acetylcholine, but
it induced modest endothelium-independent relaxation (Zembowicz et al.,
1992
). Robust endothelium-independent relaxations, which were reversed
by NO synthase inhibitors, were also produced by L-NOHA in
arginine-depleted isolated pulmonary arteries (Wallace et al., 1991
).
These relaxations were probably due to metabolization by the inducible
isoform of NO synthase because they only occurred after prolonged
incubation and comparable relaxations were also produced by addition of
L-arginine. Thus, the mechanisms of
L-NOHA-induced relaxation appear variable from one vessel
to the other.
The existence of an NO synthase-independent pathway capable of
oxidizing L-NOHA or another compound with a C=NOH bond,
formamidoxime, to nitrogen oxides has been suggested in cultured smooth
muscle cells from the rat aorta (Schott et al., 1994
) and from the rat trachea (Jia et al., 1998
), respectively. In both cases, formation of
nitrite was blunted by a P450 inhibitor, supporting the involvement of
P450. Exposure of the isolated trachea to formamidoxime caused cGMP
accumulation and relaxation (Jia et al., 1998
), consistent with the
view that the pathway leading to NO formation was functional in the
tissue as well as in the cultured tracheal cells. In the present
investigation, we hypothesized that this could also be the case in the
freshly isolated aorta.
Therefore, effects of L-NOHA and nonamino acid compounds
bearing a C=NOH function (Fig. 1) were
studied here on vascular relaxation and cGMP accumulation in rat aortic
rings with or without endothelium. As L-NOHA competed with
L-arginine for transport into aortic smooth muscle by a
cationic amino acid carrier (Schott et al., 1994
), some experiments
were performed in the presence of L-(or D-)
arginine. The involvement of NO was explored using an NO scavenger
(2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; PTIO) and an inhibitor of guanylyl cyclase activation by NO
(1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ; 1 µM). The hypothesis that NAD(P)H-dependent enzymes, especially P450s or P450 reductase, were implicated in oxidation of C=NOH bonds
was investigated using the flavoprotein-dependent enzymes inhibitor
diphenyliodonium, nonselective inhibitors of P450 isoforms (proadifen
and miconazole), and 7-ethoxyresorufin, which is not only a suicide
substrate of the P450 1A1 isoform (Tassaneeyakul et al., 1993
) but also an inhibitor of NADPH-P450 reductase
(Dutton et al., 1989
) and other reductases (Jiang and Ichikawa, 1999
). In addition,
N
-nitro-L-arginine
methyl ester (L-NAME) was used to inhibit NO synthase, which is also a flavoprotein-dependent enzyme.
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Materials and Methods |
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Contraction-Relaxation Experiments in Rat Aortic Rings.
Thoracic aorta rings were obtained as described previously
(Andriambeloson et al., 1997
) from male 12- to 14-week-old Wistar rats
bred in our laboratory from Iffa-Credo (France) genitors. In some
rings, the endothelium was removed by gentle rubbing with blunt
forceps. Rings were then mounted under 2 g of tension in organ
baths containing Krebs' solution of the following composition: 119 mM
NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.18 mM
KH2PO4, 11.0 mM glucose,
25.0 mM NaHCO3 at 37°C bubbled with 95%
O2/5% CO2 (pH 7.4). After
an equilibration period of 60 min during which the Krebs' solution was
changed every 15 min, basal tension was readjusted to 2 g. Then
contractile capacity was tested with norepinephrine or phenylephrine (1 µM) as indicated. The absence or the presence of a functional
endothelium was determined by the respective inability or ability of
acetylcholine (1 µM) to relax norepinephrine or phenylephrine
precontracted rings. Then a second washing period of 60 min followed
during which tension returned to baseline levels. Thereafter, rings
were contracted with a single dose of norepinephrine or phenylephrine
(0.1 µM, producing approximatively 80% of the maximum contractile
response, in both cases) in the presence or absence of the indicated
compounds. As there was no difference in relaxing effects of drugs with
C=NOH functions when contraction was elicited by norepinephrine or
phenylephrine (see Results), either agonist was used. In some cases,
however, phenylephrine was preferred because some drugs used to
investigate the mechanisms of relaxation altered the stability of
norepinephrine-induced contraction. In some rings,
L-arginine (1 mM) or D-arginine (1 mM) was
added just before norepinephrine. When contractions reached stable
levels, L-NOHA or compounds with C=NOH function were added cumulatively at the indicated final concentrations. In some
experiments, the following drugs (or corresponding solvent) were added
30 min before the contraction experiments and were also present
throughout the experiments: L-NAME (300 µM), ODQ (1 µM), PTIO (100 µM), diphenyliodonium (30 µM), proadifen (10 µM), miconazole (30 µM), or 7-ethoxyresorufin (10 µM).
Determination of Tissue cGMP Content.
Aortic rings without
endothelium (5 mm) were incubated for 30 min in Krebs' solution
supplemented with isobutylmethylxanthine (IBMX; 100 µM) at 37°C and
oxygenated with a gas mixture of 95% O2/5%
CO2. Thereafter, the Krebs' solution was
replaced by fresh solution, and rings were incubated in the absence or
presence of either L-NOHA (100 µM) or
4-chlorobenzamidoxime (ClBZA) (100 µM) for the next 30 min. The rings
were then put into 500 µl of ice-cold hydrochloric acid (0.1M) and
homogenized with a Potter glass/glass homogenizer for 30 s
followed by sonication (type 75 TS; Ultrason, Annemasse, France) for
15 s and centrifugation at 10,000g for 5 min. Pellet
and supernatant were frozen and maintained at
20°C until assayed.
The cGMP content was determined in thawed supernatant using a
radioimmunoassay described previously (Cailla et al., 1976
), modified
by separation of free cGMP with activated charcoal (Koch and
Lutz-Bucher, 1991
). DNA was determined in the pellet according to
Setaro and Morley (1976)
, and the cGMP content of the rings was
expressed as fmol · µg
1 DNA.
Expression of Results and Statistical Analysis.
Results were
expressed as mean ± S.E.M. of n experiments. When
possible (i.e., when full relaxation could be reached), the concentration value causing 50% relaxation of precontracted vessel (IC50) was determined by log-logit regression.
cGMP content is expressed as fmol · µg
1 DNA. Statistical comparisons of
concentration-effect curves were performed using multianalysis of
variance. A Student's t test for paired or unpaired data
(as appropriate) was used for other statistical comparisons, with
p values less than 0.05 considered to be statistically significant.
Drugs and Reagents.
L-NOHA was obtained from
Alexis Corporation (Läufelfingen, Switzerland) and
N
-hydroxy-D-arginine
from GlaxoSmithKline (Uxbridge, Middlesex, UK).
N-Hydroxyguanidines,
N-(4-chlorophenyl)-N'-hydroxyguanidine, N,N'-dicyclohexyl-N"-hydroxyguanidine,
N-hydroxydebrisoquine, benzamidoxime, ClBZA,
4-nitrobenzamidoxime (NO2BZA),
4-n-(hexyloxy)benzamidoxime (HXBZA),
4-(methoxy)benzamidoxime (MXBZA), and 4-chloroacetophenone-oxime (ClBK)
were synthesized following previously described procedures (Jousserandot et al., 1998
; Renodon-Cornière et al., 2002
).
20°C as a stock solution (10 mM) in Milli-Q water (Millipore Corporation, Bedford, MA).
Formamidoxime was dissolved in Milli-Q water. Other C=NOH compounds
were first dissolved in DMSO and then in Krebs' buffer in order
introduce less than 1% DMSO in organ baths. All other solutions were
prepared just before use, in Krebs' buffer. Controls were run using
the appropriate solvent.
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Results |
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Endothelium-Independent Relaxing Effects of
L-NOHA and Compounds with a C=NOH Function.
Contraction elicited by norepinephrine or phenylephrine
remained stable in control rings during the whole experiment, as shown in Fig. 2 in the case of norepinephrine
in endothelium-denuded rings. After the addition of L-NOHA,
relaxation developed slowly (within approximatively 15 min). The
concentration of L-NOHA was therefore cumulatively
increased each 15 min. Relaxations caused by nonamino acid compounds
with a C=NOH function developed more rapidly (within 3 to 5 min after
each drug addition), as illustrated in the case of the most active,
ClBZA. The concentration of these drugs was increased when relaxation
reached a plateau (each 3 to 5 min).
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-hydroxy-D-arginine
failed to relax norepinephrine precontracted rings (not shown). It was
significantly inhibited in the presence of
L-arginine but not
D-arginine (Fig.
3).
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Involvement of the NO-cGMP Pathway.
The data in Fig.
5 show that L-NOHA (100 µM)
and ClBZA (100 µM) were both able to cause cGMP accumulation in
endothelium-denuded aorta in the presence of the phosphodiesterase
inhibitor IBMX. In these experiments, the two compounds were used at
the same concentration (100 µM). The elevations in cGMP level
(1.9-fold in the case of L-NOHA and 4.2-fold in the case of
ClBZA) were consistent with the larger relaxing effect of ClBZA at this
concentration (Fig. 2).
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Involvement of P450s or Other NAD(P)H-Dependent Enzymes.
As illustrated in Fig. 7,
diphenyliodonium (30 µM) and 7-ethoxyresorufin (10 µM) markedly
inhibited relaxations elicited by L-NOHA and ClBZA. By
contrast, proadifen (10 µM) and L-NAME (300 µM) failed
to alter these relaxations. Miconazole (30 µM) did not either inhibit
relaxation caused by L-NOHA. Proadifen, miconazole, and
7-ethoxyresorufin did not affect precontraction level; however, it was
reduced by about 40% by diphenyliodonium (not shown).
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Discussion |
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The above data show that compounds bearing a C=NOH function, including L-NOHA, are able to produce endothelium-independent vasorelaxation in the rat aorta. They provide evidence that activation of guanylyl cyclase by NO released from these compounds is involved in this relaxation and that oxidative cleavage of the C=NOH function represents an NO synthase- and P450-independent pathway for NO production in the aorta.
Involvement of the NO-cGMP pathway in aortic relaxation caused by
L-NOHA and other compounds with a C=NOH function is
supported by inhibition by the NO scavenger PTIO and by the guanylyl
cyclase activation inhibitor ODQ, as well as by cGMP accumulation.
Whether NO was released in the cytosol (as a free radical or as a
related species) or directly transferred to guanylyl cyclase as
recently suggested in the case of nitroglycerin (Kleschyov et al.,
2002
) deserves further investigation. cGMP accumulation caused by
L-NOHA was relatively modest compared with the one produced
by ClBZA (this study) or other NO donors. Thus, despite inhibition by
ODQ, involvement of cGMP-independent relaxing mechanisms, such as
S-nitrosylation of K+ channels, cannot
be excluded, especially in the case of L-NOHA. The stereospecific inhibition by L-arginine,
however, is consistent with L-NOHA entering cells
via a cationic amino acid transporter (Schott et al., 1994
), whereas
ClBZA probably enters cells via another pathway. The transporter might
limit entry of L-NOHA in cells, accounting for a
relatively modest effect on cGMP accumulation and slowly developing and
moderate relaxation.
The finding that, in the rat aorta, neither the presence of endothelium
nor L-NAME (in endothelium-denuded rings) significantly influenced relaxations elicited by L-NOHA and ClBZA suggest
that metabolization by endothelial or nonendothelial NO synthase was of
minor importance, if any, in the formation of a relaxing compound from
C=NOH bonds in this particular tissue. This is different from what has
been reported in other vessels in which endothelial NO synthase plays a
major role in relaxation produced by L-NOHA (Wallace et
al., 1991
; Abdul-Hussain et al., 1996
). Tissue or species differences
might explain such discrepancy.
Failure of the P450 nonselective inhibitor proadifen to inhibit
relaxations caused by L-NOHA and ClBZA do not support the involvement of a proadifen-sensitive P450 in oxidation of C=NOH bond to
NO or a related relaxing compound in the freshly isolated rat aorta.
Another P450 inhibitor, miconazole, was also unable to blunt
L-NOHA-induced relaxation, whereas it inhibited formation of nitrite from L-NOHA in cultured aortic smooth muscle
cells (Schott et al., 1994
). This suggests that the mechanism of
oxidation of the C=NOH bonds was different in freshly dissected aorta
from that in cultured cells. Culture conditions might favor expression of P450 isoform(s) or other miconazole-sensitive enzymes which are not
expressed (or not expressed at the same level) in the tissue.
Structure activity relationships found in the present investigation
show that, in the rat aorta, the order of relaxant activity of
amidoxime, ketoxime and N-hydroxyguanidine derivatives is
opposite to the order of reactivity of the same compounds for
P450-dependent oxidative cleavage of the C=NOH bond, where
N-hydroxyguanidines are the most active and the ketoxime the
less active one (Jousserandot et al., 1998
). In addition, substitutions
that increase the ability of benzamidoximes to generate NO in liver
microsomes (by introducing an electron donating group in para position
of the phenyl group) have no clear effect on the relaxant activity of
these compounds (compare NO2BZA, ClBZA, MXBZA and
HXBZA, Fig. 4 and Table 1). The structural determinants required for
the relaxant effect are also entirely different from those required for
being an NO synthase substrate (Dijols et al., 2001
;
Renodon-Cornière et al., 2002
): the substituted
N-hydroxyguanidine, a poorly relaxant compound, is a
selective substrate for the inducible NO synthase; by contrast, the
identically substituted amidoxime ClBZA and ketoxime ClBK are potent
relaxant compounds, whereas they are not substrate for any of the three
NO synthase isoforms. Thus, structure activity relationships together
with the above-mentioned failure of P450 and NO synthase inhibitors to
blunt relaxation lend no support to the hypothesis of the involvement
of a P450 or NO synthase in the relaxant effect of compounds with a
C=NOH function in the rat aorta.
The present findings that both the nonspecific flavoprotein
enzyme inhibitor diphenyliodonium and the NADPH-reductases inhibitor 7-ethoxyresorufin abolished relaxations produced by L-NOHA
and ClBZA suggest that the reductase domain of a NAD(P)H-dependent enzyme might be implicated. Even though 7-ethoxyresorufin is regarded as a substrate and selective inhibitor of P450
1A1 (Tassaneeyakul et al., 1993
), it is also an
inhibitor of various reductases (Dutton et al., 1989
; Jiang and
Ichikawa, 1999
). Interestingly, it has been previously reported that
7-ethoxyresorufin and diphenyleneiodonium (an analog of
diphenyliodonium), but not classical P450 inhibitors such as proadifen,
caused inhibition of glyceryl trinitrate-induced vascular relaxation
(Bennett et al., 1994
; Li and Rand, 1996
). The authors suggested that
inhibitors like proadifen do not inhibit P450 isoform(s) present in
blood vessels or that another enzyme such as NADPH-cytochrome P450
reductase is capable of metabolizing glyceryl trinitrate to NO. The
present data lead us to propose a similar hypothesis for the
mechanism(s) underlying endothelium-independent relaxation elicited by
compounds with a C=NOH function.
It should be stressed that due to the above-mentioned differences in the mechanisms of action of L-NOHA in different blood vessels, the data obtained here in the isolated rat aorta should not be extrapolated to other vessels. Further studies are required to evaluate the involvement of formation of NO from compounds with a C=NOH function in the effects of these compounds in different vascular beds and in in vivo conditions.
In conclusion, the above results indicate that compounds bearing a
C=NOH function, including L-NOHA and especially
non-
-amino acid-substituted benzamidoximes can act as efficient NO
synthase-independent activators of the cGMP pathway in the rat aorta.
They can help identifying a novel pathway for endothelium-independent
NO formation in vessels. In addition, they might provide valuable
surrogates for impaired endothelial NO activity in vascular diseases
with endothelial injury.
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Acknowledgments |
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We are grateful to Dr B. Lutz-Bucher for the generous gift of 125I-cGMP and antibodies against cGMP.
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Footnotes |
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Accepted for publication July 17, 2002.
Received for publication May 15, 2002.
This investigation was partially supported by Barrande Grant 00967ZD. P.V., P.B., and K.C. were the recipients of fellowships provided by the French Embassy in Prague.
Primary laboratory of origin: Pharmacology and Physico-Chemistry, Centre National de la Recherche Scientifique (Unité Mixte Recherche 7034) and University Louis Pasteur (Strasbourg, France).
DOI: 10.1124/jpet.102.038612
Address correspondence to: Jean-Claude Stoclet, Pharmacologie et Physico-Chimie, Université Louis Pasteur, Faculté de Pharmacie, BP 24, F-67401 Illkirch Cedex, France. E-mail: stoclet{at}aspirine.u-strasbg.fr
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Abbreviations |
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L-NOHA, N
-hydroxy-L-arginine;
P450, cytochrome P450;
PTIO, 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide;
ODQ, 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one;
L-NAME, N
-nitro-L-arginine methyl
ester;
IBMX, isobutylmethylxanthine;
ClBZA, 4-chlorobenzamidoxime;
NO2BZA, 4-nitrobenzamidoxime;
HXBZA, 4-n-(hexyloxy)benzamidoxime;
MXBZA, 4-methoxybenzamidoxime;
ClBK, 4-chloroacetophenone oxime;
DMSO, dimethyl sulfoxide.
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528-534[CrossRef][Medline].This article has been cited by other articles:
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