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SA JOURNAL OF DIABETES & VASCULAR DISEASE
RESEARCH ARTICLE
VOLUME 14 NUMBER 1 • JULY 2017
25
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
5mg/kg intraperitoneal, respectively). A thoracotomywas 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 NaHCO
3
; 4.7
KCl; 1.2 KH
2
PO
4
; 1.2 MgSO
4
·7H
2
O; 2.5 CaCl
2
; 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% O
2
and 5% CO
2
) 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 pD
2
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 (E
max
) 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 (pD
2
value)].
There was significant decrease in maximum contractile response
(Emax) to Clo in the control group due to the presence of
pioglitazone and/or losartan (Fig 5). In the absence of pioglitazone
and losartan (control), Clo induced contraction. In the presence of
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 ± 7.2
a
Blood glucose
level (mg/dl)
120.3 ± 6.6 371.7 ± 18.1
b
177.6 ± 15.4 395.4 ± 14.1
b
Systolic blood
pressure
(mmHg)
96.4 ± 2.9 155.2 ± 5.2
c
187.9 ± 3.9
c
161.5 ± 7.1
c
Values are expressed as mean ± SEM.
a
p
< 0.05, compared to control group.
b
p
< 0.05, compared to control group. Blood
glucose levels > 250 mg/dl
(13.88 mmol/l) indicated diabetes.
c
p
< 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 from Wistar rats
Control
group
pD
2
(
n
= 15)
HT group
pD
2
(
n
= 7)
DM group
pD
2
(
n
= 19)
HT+DM
group
pD
2
(
n
= 12)
Control
7.26 ± 0.08
7.53 ± 0.04 7.29 ± 0.07 7.27 ± 0.07
Pioglitazone 6.80 ± 0.08
a
7.04 ± 0.07
a
7.10 ± 0.06
a
7.23 ± 0.07
Losartan
6.76 ± 0.10
b
6.95 ± 0.13
b
7.03 ± 0.06
b
7.13 ± 0.10
Pioglitazone
+ losartan
6.61 ± 0.08
c
6.81 ± 0.08
c,d
6.97 ± 0.05
c
6.97 ± 0.09
c,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:
a
Cont vs pio (
p
< 0.001);
b
Cont vs los (
p
< 0.001);
c
Cont vs
pio+los (
p
< 0.001).
HT group:
a
Cont vs pio (
p
< 0.001);
b
Cont vs los (
p
< 0.001);
c
Cont vs
pio+los (
p
< 0.001);
d
Pio vs pio+los (
p
= 0.046).
DM group:
a
Cont vs pio (
p
= 0.037);
b
Cont vs los (
p
= 0.005);
c
Cont vs
pio+los (
p
= 0.001).
HT + DM group:
c
Cont vs pio+los (
p
= 0.013);
d
Pio vs pio+los (
p
= 0.030).