CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 1, January/February 2011
10
AFRICA
were exposed to GSNO (SNO-proteins). However, the amount of
nitrite measured was not significantly different in the two rings
after GSNO exposure, showing once again that the NO storage
was the same in the rings with and without endothelium.
Effect of ODQ in GSNO-treated aortic rings
To determine the involvement of guanylyl cyclase in the hypo-
responsiveness induced by GSNO and the relaxant effect of
NAC, we studied the effect of ODQ. Aortic segments were
exposed to 1-
µ
M ODQ for 30 min and then stimulated with a
cumulative concentration of NE. When the contractile response
reached a plateau, cumulative concentration-effect curves for
NAC were constructed. Our data show that ODQ (1
µ
M, 30
min) increased the tension of both control and GSNO-treated
intact rings (Fig. 5A, Table 1) by reversing the NO-induced
hypo-responsiveness to NE. By contrast, there was no effect in
the denuded control rings, but complete abolishment of the loss
of reactivity to NE in the GSNO-treated denuded rings (Fig.
5C, Table 1). NAC-evoked relaxations in GSNO-treated rings
with and without endothelium were abolished by ODQ, which
Fig. 5. The GSNO-induced hypo-reactivity and associated relaxation mechanisms involved the NO/cGMP pathway. (A)
shows that the guanylate cyclase inhibitor, ODQ (1
µ
M, 30 min) had no effect in denuded control rings, but completely
abolished the loss of reactivity to NE in GSNO-treated denuded rings. By contrast, (C) indicates that ODQ increased
the tension of both controls and GSNO-treated intact rings by reversing the NO-induced hypo-responsiveness to NE.
(B) and (D) demonstrate that soluble guanylyl cyclase mediated the relaxant effect of NAC in the controls and GSNO-
treated aortic rings with or without endothelium. Results are means
±
SEM of five experiments; ns: not significant; *
p
<
0.05; **
p
<
0.01 in comparison with respective controls.
A
B
–9.5 –9.0 –8.5 –8.0 –7.5 –7.0 –6.5
log [NE], M
0
20
40
60
80
–4
–3
–2
Relaxation (%)
log [NAC], M
2.5
2.0
1.5
1.0
0.5
0.0
Contraction (g)
Without endothelium
C
D
–9.5 –9.0 –8.5 –8.0 –7.5 –7.0 –6.5
log [NE], M
0
20
40
60
80
–4
–3
–2
Relaxation (%)
log [NAC], M
2.5
2.0
1.5
1.0
0.5
0.0
Contraction (g)
With endothelium
Control
GSNO
Control
ODQ
GSNO
GSNO/ODQ
ns
ns
Fig. 4. Quantification of nitrosothiols in rat thoracic aorta.
Rat aortic rings were subjected to the S-nitrosylation
assays and incubated with 100
µ
M GSNO for 30 min
(SNO-proteins) or solvent (SH-proteins). Nitrite formation
was determined with the Saville-Griess assay after selec-
tive displacement of the NO group from nitrosothiols with
0.5 mM mercuric chloride (Hg
2
+
). Data are means
±
SEM of
three independent experiments.
20
15
10
5
0
– –
+ +
– –
+ +
[Nitrites] –
µ
M
SH-protein
SNO-protein
ns
ns
Hg
2
+
With endothelium
Without endothelium
