Cardiovascular Journal of Africa: Vol 27 No 3 (May/June 2016)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 3, May/June 2016 AFRICA 165
drinking water in the hypertensive groups) ad libitum. Diabetes
was induced by a single intraperitoneal injection of 50 mg/kg
STZ in the DM group. Hypertension was induced by giving
L-NAME (50 mg/kg) in the drinking water for three weeks in
the HT group. Hypertension plus diabetes were induced by a
single intraperitoneal injection of 50 mg/kg STZ and providing
L-NAME (50 mg/kg) in the drinking water for three weeks in the
HT + DM group.
Body weights of the treated groups were measured at weekly
intervals. In vitro experiments were started three weeks after
the drug injections. Systolic blood pressure (SBP) of the rats
was measured before the in vitro experiments using the tail-cuff
method. Blood was obtained from a tail vein in conscious rats.
At least five readings were done at every session and the mean
of four values was used to obtain the SBP of each rat. Glucose
concentrations were determined using an International Medical
Equipment Diabetes Care (IME-DC) blood glucose meter
(Oberkotzau, Germany).
Preparation of aortic rings and in vitro experiments
The rats were anaesthetised with ketamine and xylasine (50
and 5 mg/kg intraperitoneal, respectively). A thoracotomy
was performed and the thoracic aorta was removed from the
diaphragm to the heart. The aorta was then placed in ice-cold
Krebs’ solution where it was cleaned and any adhering fat was
removed. The composition of the Krebs’ solution (mmol/l)
was 118.0 NaCl; 25.0 NaHCO3; 4.7 KCl; 1.2 KH2PO4; 1.2
MgSO4·7H2O; 2.5 CaCl2; and 10.1 glucose.
The aorta was then cut into small rings (4–5 mm in width).
The rings were suspended horizontally between two stainless
steel wires and mounted in a 20-ml organ bath filled with
warmed (37°C) and oxygenated (95% O2 and 5% CO2) Krebs’
solution. One end of the ring was connected to a force transducer
(MAY FDT 05, Commat Ltd. Ankara, Turkey). The rings were
equilibrated for 60 min under a resting tension of 2 g with the
bath fluids being changed every 15 min. Measurement of the
isometric force was recorded on a data-acquisition system (MP
36, Biopac Systems, Inc).
After the equilibration period, the rings were sub-maximally
contracted with Phe (10-7 M), and the cumulative concentration–
response curve to acetylcholine (10 -9–10-5 M) was then obtained
to test their contractile capacity. Intact vessels failing to achieve
at least 60% relaxation to acetylcholine were assumed to be
damaged and were discarded. Cumulative responses to Phe
(10-9–10 -5 M) and Clo (10-9–10-5 M) were recorded in the aortic
rings in the absence (control) and presence of pioglitazone (10
μM) and/or losartan (10 μM), which was added to the bathing
solution 15 min prior to the contractile responses of Phe or Clo.
Pioglitazone hydrochloride was obtained as a gift sample
from Sandoz (Istanbul, Turkey). Streptozotocin, phenylephrine,
clonidine, L-NAME and the other chemicals were purchased
from Sigma Chemicals. Losartan potassium was purchased from
Fluka China (Interlab, Izmir, Turkey).
Statistical analysis
The results are expressed as mean ± SEM. Statistical evaluation
of the data was performed by analysis of variance (ANOVA) and
the Student’s t-test. Results were considered significant when p <
0.05. The agonist pD2 value (–log EC50 ) was calculated from the
concentration–response curve by non-linear regression analysis
of the curve, using a base-fitting program (Prism, Graphpad).
Results
STZ-injected animals developed diabetes in the DM and HT +
DM groups. In the HT + DM group, five rats died in the first
week after the STZ injection. The body weights, blood glucose
levels and SBP are shown in Table 1.
There was a significant increase in blood glucose levels in the
STZ-injected groups (DM and HT + DM groups). The daily
intake of L-NAME was calculated from the daily water intake
and was approximately 21–23 mg/kg/day for the HT and HT
+ DM groups. There was a significant increase in SBP in the
L-NAME-treated groups (HT and HT + DM groups) Table 1.
Phe induced a concentration-dependent contractile response
in the aortic rings from all four groups. These curves are shown
in Figs 1–4. There was no significant change in maximum
contractile response (Emax) to Phe in all groups due to the
presence of pioglitazone and/or losartan; these drugs shifted
the contractile response to Phe to the right. The sensitivity of
the aortic rings to Phe was however decreased in the presence
of pioglitazone and/or losartan in all groups [Table 2 (pD2
value)].
There was significant decrease in maximum contractile
response (Emax) to Clo in the control group due to the presence
Table 1. Body weight, blood glucose levels and systolic blood
pressure before the in vitro experiments
Parameters
Control
group
(n = 15)
DM group
(n = 20)
HT group
(n = 20)
HT+DM
group
(n = 15)
Body weight (g) 275.1 ± 6.1 279.1 ± 5.9 309.4 ± 9.5 201.1 ± 7.2a
Blood glucose
level (mg/dl) 120.3 ± 6.6 371.7 ± 18.1b 177.6 ± 15.4 395.4 ± 14.1 b
Systolic blood
pressure (mmHg) 96.4 ± 2.9 155.2 ± 5.2c 187.9 ± 3.9c 161.5 ±7.1c
Values are expressed as mean ± SEM.
a p < 0.05, compared to control group.
bp < 0.05, compared to control group. Blood glucose levels > 250 mg/dl
(13.88 mmol/l) indicated diabetes.
cp < 0.05, compared to control group.
Table 2. Acute effects of pioglitazone and losartan on
vascular sensitivity (pD2) to pheylephrine in segments
of thoracic aorta fromWistar rats
Control group
pD2 (n = 15)
HT group
pD2 ( n = 7)
DM group
pD2 ( n = 19)
HT+DM group
pD2 ( n = 12)
Control 7.26 ± 0.08 7.53 ± 0.04 7.29 ± 0.07 7.27 ± 0.07
Pioglitazone 6.80 ± 0.08a 7.04 ± 0.07a 7.10 ± 0.06a 7.23 ± 0.07
Losartan 6.76 ± 0.10b 6.95 ± 0.13b 7.03 ± 0.06b 7.13 ± 0.10
Pioglitazone
+ losartan
6.61 ± 0.08c 6.81 ± 0.08c,d 6.97 ± 0.05c 6.97 ± 0.09c,d
n is the number of aortic segments in each group. Values are expressed as
mean ± SEM.
Cont: control, Pio: pioglitazone, Los: losartan, Pio+Los: pioglitazone +
losartan.
Control group: aCont vs pio (p < 0.001); bCont vs los (p < 0.001); c Cont vs
pio +los (p <0.001).
HT group: aCont vs pio (p < 0.001); bCont vs los (p < 0.001); cCont vs
pio +los (p <0.001); dPio vs pio+los (p = 0.046).
DM group: a Cont vs pio (p =0.037); bCont vs los (p = 0.005); cCont vs
pio +los (p =0.001).
HT + DM group: c Cont vs pio+los (p = 0.013); dPio vs pio+los (p = 0.030).