CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 1, January/February 2011
AFRICA
9
stores after NOS blockade.
Our study also shows that the acetylcholine-induced relaxa-
tion was not affected by the addition of GSNO to rings with
intact endothelium (Fig. 2B). By contrast, rings without endothe-
lium did not display vasorelaxation to acetylcholine even after
GSNO exposure, suggesting that only endothelium-derived NO
and not those from exogenous sources account for vasorelaxation
to acetylcholine.
Formation and quantification of S-nitrosothiols
In order to confirm the ability to store exogenous NO in the
vascular wall, we proceeded to characterise the S-nitrosylated
residues following exposure of rat aortic smooth muscle cells
to GSNO. As indicated in Fig. 3, S-nitrosylated residues were
detected using polyclonal antibodies directed against the S-NO
moiety. A weak staining was observed in the rings treated with
mercuric chloride or NAC (which selectively cleaves NO bound
from S-NO) after exposure to GSNO. This was similar to what
was observed in the control, which was not exposed to GSNO.
These data clearly indicate the presence of nitrosothiols as a stor-
age form of NO in the vasculature.
However, quantification of nitrosothiols with the Saville-
Griess reaction (Fig. 4) has shown that in aortic rings both
with and without endothelium and not exposed to GSNO
(SH-proteins), NO released by Hg
2+
was not significantly elevat-
ed. By contrast, the NO mobilised by the same conditions and
transformed to nitrite was significantly higher when the rings
B 0
20
40
60
80
100
–9
–8
–7
–6
–5
Relaxation (%)
log [Ach], M
GSNO
Control
GSNO
Control
Without endothelium
With endothelium
Fig. 2. Blunted GSNO-induced hypo-responsiveness to contractile agonist in aortic rings with endothelium was asso-
ciated with an endogenous NO production which only accounted for vasorelaxation to acetylcholine. (A) shows a
decrease of vascular tone after GSNO exposure to rings with endothelium in the presence of the nitric oxide synthase
(NOS) inhibitor, N-nitro-L-arginine methylester (L-NAME, 300
µ
M). (B) indicates that acetylcholine-induced relaxation
was not affected by GSNO exposure in rings with and without endothelium and did not display vasorelaxation to
acetylcholine even after GSNO exposure. Results are means
±
SEM of four to six experiments; ns: not significant; **
p
<
0.01 in comparison with respective controls.
A 3.0
2.5
2.0
1.5
1.0
0.5
0.0
–9.5
–8.5
–7.5
–6.5
Contraction (g)
log [NE], M
ns
L-Name E/GSNO
L-Name
With endothelium
GSNO
ns
ns
Fig. 3. Nitrosothiol formation as a storage form of NO in the vasculature. (A) shows immunostaining of S-nitrosylated
residues in rat aortic smooth muscle cells (control) and after
in vitro
exposure to GSNO (100
µ
M, 30 min) or after
GSNO exposure and treatment with mercuric chloride (GNSO
+
Hg
2
+
) or N-acetyl cysteine (GSNO
+
NAC). (B) indicates
corresponding cumulative data shown as means
±
SEM of three different experiments. **
p
<
0.01 in comparison with
respective controls.
Control
GSNO
GSNO
+
NAC
GSNO
+
Hg
2
+
A
1000
800
600
400
200
0
Control
GSNO GSNO
+
NAC
GSNO
+
Hg2
+
Relative floorescence
(% to control)
B
