CARDIOVASCULAR JOURNAL OF AFRICA • Vol 24, No 3, April 2013
AFRICA
77
The vascular rings were then contracted with a cumulative
dose of phenylephrine (10
-9
to 10
-6
M). After washing the rings to
suppress the effects of phenylephrine, the rings were incubated
in PSS containing hemin at a concentration of 10
-4
M. After
six hours of incubation, the rings were contracted again with a
cumulative dose of phenylephrine.
Relaxation effect of Y-27632
Relaxation induced by Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-
4-(1-aminoethyl)-cyclohexanecarboxamide] (Tocris, France), a
rho-kinase inhibitor, was tested on either the control rings or
rings incubated in hemin following contraction induced by
phenylephrine at 10
-6
M. Y-27632 was used at a concentration
of 3
×
10
-7
M. Results were expressed as a percentage of the
magnitude of contraction with 10
-6
M of phenylephrine.
All the data were collected with a computerised data-
acquisition system using Genie software (Adventech, USA). All
the analyses were carried out using Origin 6 software (Microcal
Software, Northampton, MA, USA).
Immunohistochemistry
Aortic rings incubated in either PSS or PSS with hemin at 10
-4
M were embedded in ‘optimal cutting temperature compound’
(Tissue-Tek
R
OCT compound, Sakura Finetek, France) and then
frozen in liquid nitrogen. Transverse sections of 7 µm were
made using a cryostat and mounted on a microscope slide.
Immunohistochemical analysis of HO-1 was performed.
Sections were first incubated with a rabbit polyclonal anti-rat
HO-1 antibody at room temperature for four hours. The antigen–
antibody reaction was detected using a molecular probe Alexa
fluor dye goat anti-rabbit secondary antibody (Interchim, France).
The positive reaction was visualised under confocal microscopy.
Statistical analysis
Results are expressed as means
±
SEM. Differences between
control and hemin groups were compared using Student’s
t
-test
and the level of statistical significance was set at
p
<
0.05.
Results
Cumulative concentrations of phenylephrine induced a
concentration-dependent increase in the contraction of aortic
rings. As shown in Fig. 1, incubation of aortic rings in hemin
solution induced a decrease of the contractile force of the aortic
rings at all concentrations of phenylephrine from 3
×
10
-8
to
10
-6
M.
The application of Y-27632, a specific and potent rho-kinase
inhibitor, induced a relaxation in the isolated aortic rings. In the
control aortic rings, the magnitude of the relaxation at 3
×
10
-7
M of Y-27632 was 36% of the contraction induced by 10
-6
M of
phenylephrine. In aortic rings treated with hemin, the relaxation
induced by Y-27632 was reduced to 20% of the contraction
induced by phenylephrine (Fig. 2).
Immunohistochemical study showed expression of HO-1
in both control and hemin-treated aortic rings. As shown in
Fig. 3, six-hour incubation of aortic rings in hemin resulted in an
increased expression of HO-1.
Discussion
The main findings of this study were that the decreased
contractility of the aortic rings induced by hemin was associated
with a reduced effect of Y-27632, a rho-kinase inhibitor, on the
relaxation of aortic rings pre-contracted with phenylephrine.
The reduced vascular smooth muscle contractile force induced
by hemin found in this study was associated with an increased
expression of heme oxygenase HO-1. This is in agreement with
data previously published on rat and human vessels.
3
The change in contractile force produced was observed after
six hours of incubation in hemin, not after four hours, as was
observed in human vessels. The four-hour duration in our study
was without significant effect on the phenylephrine-induced
contraction of aortic rings.
The hemin effect on vascular contractility occurs via
mechanisms involving heme oxygenase, as it has been shown
that these effects were suppressed by inhibition of HO-1
activity.
3,4,15
Moreover, it has been shown that vascular effects of
heme oxygenase and CO are mediated by activation of soluble
guanylate cyclase, with increased production of cyclic GMP and
activation of potassium channels.
3,4,15
Fig. 2. Relaxation effect of Y-27632, a rhoA-kinase inhibi-
tor, on aortic ring contraction induced by phenylephrine
at 10
-6
M. The clear column represents control aortic rings
and shaded column aortic rings incubated in hemin.
Values are expressed as percentage of the magnitude
of contraction induced by 10
-6
M of phenylephrine. **
Significantly different from control (
p
< 0.01.)
50
40
30
20
10
0
Control,
n
=
12
Hemin,
n
=
10
Relaxation (% of contraction
with phenyleprhine)
**
Fig. 1. Cumulative dose effect of phenylephrine on the
contraction of control aortic rings (triangle) and aortic
rings incubated in hemin solution 10
-4
M for six hours
(square). Values are expressed as percentage of magni-
tude of contraction induced by 10
-6
M of phenylephrine
(PE) before the six-hour incubation. * Significantly differ-
ent from control (
p
< 0.05.)
120
100
80
60
40
20
0
1,00E–09 1,00E–08 3,00E–08 1,00E–07 3,00E–07 1,00E–06
Force (% maximal contraction with PE)
Phenylephrine concentration (mol/l)
Hemin,
n
=
7
Control,
n
=
9
*
*
*
*
