CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 1, January/February 2011
8
AFRICA
by rubbing the intimal surface of the rings with forceps.
Changes in isometric tension of isolated arteries were
assessed in organ chambers. The rings were allowed to equili-
brate for 60 min before experiments were carried out, while the
resting tension was adjusted, as required. Rings from various
types of arteries were first exposed to GSNO (1 µM) or solvent
for 30 min. After a 60-min washout period for drug removal,
they were pre-contracted with norepinephrine (NE). Once
the contraction reached a steady-state level, NAC was added.
Parallel experiments were performed using N
w
-nitro-L-arginine
methylester (L-NAME, an inhibitor of NO synthase), 1H-[1,2,4]
oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, a selective inhibitor
of guanylyl cyclase), and tetraethylammonium (TEA, as a non-
selective blocker of potassium channels).
For the characterisation of S-nitrosothiols, rat aortic smooth
cells (RASMCs) were cultured in Labtek
®
chamber slides to
confluence and then exposed to 100
µ
M S-nitrosoglutathion for
30 min. They were washed three times, then treated with HgCl
2
(0.5 mM) or NAC (0.1 mM) and washed again. The cells were
then fixed for one hour in 4% paraformaldehyde in PBS (0.1
M, pH 7.4) for one hour. They were then incubated for at least
three hours at room temperature with a primary polyclonal anti-
body directed against S-nitrosothiols residues [1/100 diluted in
a solution of PBS-Triton 0.5% (v/w)], followed by a secondary
anti-rabbit IgG antibody coupled with fluorescein (Alexa Fluor
®
488) diluted 1/200 in PBS-Triton. The preparations were then
observed by confocal microscopy (Bio-Rad 1024 MRC
®
) with
an epifluorescence at 40
×
magnification.
To confirm and quantify the formation of nitrosylated
protein, rat thoracic aorta (with and without endothelium) were
first exposed to 100
µ
M GSNO for one hour at 37°C in organ
chambers, followed by several washouts. The aortic rings were
transferred into 24-well tissue-culture plates containing 0.2 ml of
Krebs solution. The content of S-nitrosothiols (S-NO) was then
measured using the Saville-Griess assay, involving diazotation of
sulfanilamide and subsequent coupling with N-(1-naphtyl)ethyl-
enediamine, after selective displacement of the NO group from
the nitrosothiols by Hg
2+
(0.5 mM). During the whole experimen-
tal period, the samples were protected from light.
Unless otherwise indicated, drugs were purchased from Sigma
Chemical Co or Aldrich (Saint Quentin-Fallavier, France). Rabbit
polyclonal antibodies directed against conjugated NO-cysteine
were obtained as previously described.
13
Alexa Fluor 488 was
purchased from Molecular Probes (Leiden, the Netherlands).
Horseradish peroxidase-labelled antibody (goat anti-rabbit IgG)
was purchased from Diagnostic Pasteur (Paris, France). NAC
(Fluimucil) was a generous gift from Zambon Laboratory
(Antibes, France). Sodium pentobarbital was purchased from
Sanofi Santé Animale (Libourne, France). GSNO was prepared
as previously described.
14
Statistical analysis
Values are expressed as means
±
SEM. Statistical evaluation was
performed with the Student’s
t
-test for paired data or ANOVA,
followed by Fischer’s protected least-significant difference test
where appropriate. Values of
p
<
0.05 were considered statisti-
cally significant.
Results
Effect of GSNO exposure on rat aorta
In endothelium-denuded rings from rat aorta pre-exposed to
GSNO (1
µ
M, 30 min), the contractile response to NE (1 nM
to 30
µ
M) was diminished in comparison to the controls (Fig.
1A). However, in rings with endothelium pre-exposed to GSNO,
the effect of the contractile agonists was not affected (Fig. 1A).
In both rings, NAC (0.1–10 mM) exerted a relaxant effect (Fig.
1B). The relaxing effects of NAC demonstrated the existence of
releasable NO stores in the vessels with as well as those with-
out endothelium. However, the vascular endothelium masked
the existence of these stores because only the vessels without
endothelium showed the hypo-responsiveness characteristic,
indicating the presence of these stores.
As expected, in rings with endothelium in the presence of the
NO-synthase inhibitor L-NAME (300
µ
M), when added to NE
15 min before the contractile response, we observed the same
decrease in vascular tone as seen in the rings without endothe-
lium after GSNO addition (Fig. 2A). These data indicate that
NO storage remains an effective mechanism of formation of NO
A
B
3
2
1
0
–9.0 –8.5 –8.0 –7.5 –7.0
Contraction (g)
log [NE], M
ns
0
20
40
60
80
100
–4
–3
–2
Relaxation (%)
log [NAC], M
GSNO
Control
GSNO
Control
Without endothelium With endothelium
Fig. 1. Effect of contractile and relaxant agonists in aortic rings pre-exposed to GSNO. (A) Pre-exposure to GSNO
(1
µ
M, 30 min) was associated with a decrease in contractile response to norepinephrine, NE (1 nM to 30
µ
M) only
in rat thoracic aortic rings without endothelium. (B) The low-molecular weight thiol NAC, which selectively cleaves
NO bound from nitrosothiols (S-NO) after GSNO exposure, exerted a relaxant effect in rings both with and without
endothelium. Results are means
±
SEM of four to eight experiments; ns: not significant; *
p
<
0.05; **
p
<
0.01, in
comparison with respective controls.
