CARDIOVASCULAR JOURNAL OF AFRICA • Vol 24, No 3, April 2013
76
AFRICA
Decreased vascular contractility induced by hemin is
associated with a reduced rho-kinase activity
BONAVENTURE AWEDE, MARIE-CHRISTINE LEMAIRE, PIERRE BONNET, VERONIQUE EDER
Abstract
Objectives:
In this study, the role of rho-kinase activity in
the modulation of vascular contractility induced by hemin, a
heme oxygenase inducer, was investigated.
Methods:
Aortic rings from Wistar rats were incubated in
physiological saline solution (PSS) containing hemin at 10
-4
M for six hours then contracted with phenylephrine, and a
dose-response curve was established. The effect of Y-27632, a
rho-kinase inhibitor, on the relaxation of the pre-contracted
aortic rings was then studied.
Results:
Incubation of the aortic rings in hemin induced an
increased expression of heme oxygenase 1 (HO-1). A reduc-
tion in the contractile force of aortic rings incubated in
hemin was observed in response to phenylephrine. Y-27632
at a concentration of 10
-6
M induced a 36% relaxation of the
control aortic rings but only a 20% relaxation in aortic rings
treated with hemin.
Conclusion:
These data suggest that the decreased vascular
contractility induced by hemin could, in part, result from an
inhibition of rho-kinase activity.
Keywords:
haeme oxygenase, vascular contractility, rho-kinase
Submitted 31/12/09, accepted 18/1/13
Cardiovasc J Afr
2013;
24
: 78–81
DOI: 10.5830/CVJA-2013-005
Carbon monoxide (CO), like nitric oxide (NO), has been shown
to decrease vascular contractility. This gas is endogenously
produced by the breakdown of heme into biliverdin, iron and
CO, and the reaction is catalysed by heme oxygenase. Heme
oxygenase (HO) exists in three isoforms: an inductive form,
HO-1, and two constitutive forms, HO-2 and HO-3.
1
Hemin
is one of the components that induces expression of HO-1
in vascular tissues both
in vitro
and
in vivo
. Hemin is also a
substrate of heme oxygenase.
In vitro
induction of HO-1, which results in CO production,
decreased both animal and human arterial contractility.
2,3
Long-
term
in vivo
administration of hemin to spontaneous hypertensive
rats (SHR) has been shown to normalise arterial pressure.
4
If smooth muscle contraction is regulated by the cytosolic
calcium concentration, which induces activation of myosin
light-chain kinase and then phosphorylation of the regulatory
myosin light chain, it is also regulated by the calcium sensitivity
of myofilaments. This mechanism is partly achieved by the
inhibition of myosin light-chain phosphatase, and the small
GTPase rho and its target rho-associated kinase participate in this
inhibition.
5,6
Increased activity of rho-kinase has been observed
in many models of arterial hypertension, and administration of
inhibitors of rho-kinase activity has been shown to lower blood
pressure.
7-10
Activation of rho-kinase is also involved in many
other cardiovascular disorders.
11-13
Previous studies have shown that the effects of carbon
monoxide or heme oxygenase 1 induction on vascular
contractility or blood pressure occur via activation of soluble
guanylate cyclase, which results in the production of cyclic GMP
and via activation of the potassium current.
3,4,14-16
The possible
involvement of rho-kinase has never been investigated
in vitro
.
In this study, we hypothetised that induction of heme
oxygenase could result in decreased activation of rho-kinase.
Therefore the effect of inhibition of rho-kinase on the relaxation
of pre-contracted aortic rings was investigated following
incubation in hemin.
Methods
All animal experimental protocols were performed with the
approval of the regional ethics committee (CREEA no CL2007-
013). Twelve-week-old rats used in this study were housed
at 21°C with 12-hour light/dark cycles. They were fed with
standard laboratory food and had unlimited access to drinking
water. Hemin solution was prepared by dissolving hemin (Fluka,
France) in a phosphate buffer solution (PBS) pH 12 and adjusted
to pH 7.4. Animals were anesthetised by an intraperitoneal
injection of sodium pentobarbital (100 mg/kg of body weight).
Contraction of isolated artery rings
The rats were euthanised and the thoracic aorta was dissected.
Transverse ring sections were isolated and the endothelium
was destroyed by rubbing the intimal surface with forceps. The
rings were then suspended between two wires connected to the
bottom of an organ bath and to an isometric force transducer
that allowed the recording of the tension developed by each ring.
In all experiments, the rings were stretched against a 3-g
pre-load in the organ bath, which was filled with physiological
saline solution (PSS) at 37°C, containing (in mM): NaCl 138.6;
KCl 5.4; CaCl
2
1.8; MgCl
2
1.2; NaH
2
PO
4
0.33; HEPES 10 and
glucose 11. The pH was adjusted to 7.4 using NaOH.
After one hour of rest (equilibrium), the rings were
pre-contracted with a PSS solution containing 80 mM K
+
(K80)
in order to provide maximal contractile amplitude of tone.
This was used as a reference and to check tissue viability. A
relaxation response to acetylcholine was used to confirm the
absence of endothelium.
LAB.P.ART.-EA3852, Faculty of Medicine, University of
Tours, 10 bis Boulevard Tonnellé, Tours, France
BONAVENTURE AWEDE, MD, PhD
MARIE-CHRISTINE LEMAIRE, MD, PhD
PIERRE BONNET, MD, PhD
VERONIQUE EDER, MD, PhD
Unité de Physiologie, Faculté des Sciences de la Santé,
Université d’Abomey-Calavi, Bénin
BONAVENTURE AWEDE, MD, PhD,
