CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 6, November/December 2011
320
AFRICA
have also become established as therapeutic drugs for angina
pectoris, together with
β
-adrenoceptor antagonists and nitrates.
8
Ca
2+
channel antagonists have several features that may relate
to myocardial protection during ischaemia and reperfusion. The
main effect is reduction in oxygen demand due to a decrease in
heart rate and myocardial contractility.
8
Interference with neutro-
phil mobilisation and activation may protect against the produc-
tion of free radicals and the release of proteolytic enzymes.
9
A
direct protective effect may also be produced by interference
with ischaemia-induced intracellular Ca
2+
overload.
10,11
Dihydropyridine Ca
2+
channel blockers were reported to
protect the endothelial function of renal resistance arteries in
hypertensive rats
12
and the mesenteric arteries of rats in circula-
tory shock.
13
Endothelial function is important for the preserva-
tion of the organ function against ischaemic or hypertensive
stress.
14,15
Many studies have reported that Ca
2+
channel blockers
such as amlodipine, nifedipine and benidipine increase NO
production.
16,17
Mebudipine is a new calcium channel blocker with a dihy-
dropyridine structure that has a comparable pharmalogical effect
while offering some advantages, such as a longer biological
half-life to reach peak effect and vasoselectivity.
18,19
There are
no reports on the cardioprotective activity of mebudipine and it
seems that it may attenuate endothelial dysfunction and increase
the production of NO in ischaemic hearts. Therefore, this study
was designed to examine the effect of mebudipine on cardiac
function and the activity of the myocardial nitric oxide system
following ischaemia–repefusion injury in isolated rat hearts.
Methods
Forty male Wistar rats (250–300 g) were obtained from the labo-
ratory animal house at Tabriz University of Medical Sciences.
They were housed in an animal room at 22–24ºC and given free
access to commercial rat chow and tap water. All the experi-
mental procedures used, as well as rat care and handling were
in accordance with guidelines provided by the Experimental
Animal Laboratory and approved by the Animal Care and Ethics
Committee of the Tabriz University of Medical Sciences. The
animals were randomly divided into four groups (
n
=
10): a
sham group (without ischaemia), control group (ischaemia with-
out drug), drug group (ischaemia with drug) and vehicle group
(ischaemia with ethanol: 0.01%).
Longendorff protocol
All animals were anaesthetised intraperitoneally with sodium
pentobarbital (60 mg/kg) and heparinised with sodium heparin
(300 IU intraperitoneally). After opening the chest cavity, the
hearts were quickly excised and immersed in ice-cold Krebs-
Henseliet (K-H) solution. Then the aortae were cannulated
and the hearts were retrogradely perfused via the aorta in a
Longendorff apparatus with K-H solution containing (in mM):
118 NaCl, 4.8 KCl, 1.2 MgSO
4
, 1.0 KH
2
PO
4
, 27.2 NaHCO
3
, 10
glucose and 1.25 CaCl
2
. A mixture of 95% O
2
and 5% CO
2
was
bubbled through the perfusate. A thermostatically controlled
water circulator (Satchwell Sunvic, UK) maintained the perfu-
sate and bath temperatures at 37°C.
The hearts were perfused at a constant mean pressure of 75–80
mmHg. During the stabilisation period, a latex balloon (Harvard)
attached to the end of a piece of stiff polyethylene tubing was
inserted into the left ventricle through the mitral valve. The
balloon and tubing were connected to a pressure transducer
and filled with normal saline to produce a left ventricular end-
diastolic pressure (LVEDP) of 5–10 mmHg at baseline, and the
balloon volume was maintained constant throughout the experi-
ment. The LVEDP, LV peak systolic pressure (LVSP) and the
peak rates of positive and negative changes in LV pressure (
±
dp/
dt) were measured with a Power Lab System (ADInstruments,
Australia). The LV developed pressure (LVDP) was calculated
as follows:
LVDP
=
LVSP – LVEDP (mmHg).
The haemodynamic data were recorded continuously on a
computer using a Powerlab system. The heart rate (HR)
was calculated using a bioelectric amplifier (ADInstruments,
Australia) from the electrocardiogram that recorded via two
electrodes attached to the apex and the right ventricle of the heart
and one reference electrode.
Ischaemia–reperfusion protocols
The hearts were allowed to equilibrate for 20 min prior to each
study. For the ischaemic control group, the hearts were perfused
with the K-H solution for 20 min, and then global ischaemia was
conducted by interrupting the aortic flow for 30 min, followed by
reperfusion with K-H solution for up to 120 min. In the drug and
vehicle groups, before ischaemia, the hearts were perfused with
mebudipine (0.1 nm) or an ethanol-enriched solution (0.01%) for
25 min, respectively.
Several experimental studies have proven Ca antagonists to
be cardioprotective when applied in a concentration that does
not produce a negative inotropic or chronotropic effect.
20-22
Mebudipine was therefore applied throughout the study at a
concentration of 0.1 nm, which did not cause a negative inotropic
or chronotropical effect.
Biochemical measurements
During the first 10 min of the reperfusion period, the coronary
effluent was sampled for lactate dehyrogenase (LDH) and
myocardial creatin kinase (CK-MB) measurement. The concen-
tration of LDH and CK in the coronary effluent was measured
using related kits (Parsazmoon, Iran) and expressed as units per
litre. NO production (nmol/g protein) in the heart homogenates
was determined by measuring the total nitrite and nitrate concen-
tration (NO metabolites), using the Griess method.
23
Deproteinised heart homogenates were used for determina-
tion of NO metabolite concentrations (NO
x
). Briefly, 100
μ
l of
supernatant was applied to a microtitre plate well; 100
μ
l vana-
dium (III) chloride (8 mg/ml) was added to each well (for reduc-
tion of nitrate to nitrite) and this was followed by the addition of
the Griess reagents, 50 μl sulfanilamide (2%) and 50
μ
l N-(1-
naphthyl) ethylendiamine dihydrochloride (0.1%). After 30 min
incubation at 37ºC, the absorbance was read at 540 nm using an
ELISA reader (Lab System, Fanland). The concentration of NO
x
in the heart homogenates was determined from standard linear
curves established from 0–150
μ
mol/l sodium nitrite.
Statistical analyses
All numerical data are expressed as mean
±
SEM. Data on
cardiac function were subjected to a two-way analysis of vari-
