Cardiovascular Journal of Africa: Vol 26 No 1 (January/February 2015)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 1, January/February 2015 AFRICA 5
other human tissues such as the heart, stomach, placenta and
mammary epithelium.10-14 In addition to its essential roles in
feeding behaviour and energy balance,11,12 leptin also plays an
important role in many different peripheral processes, including
haematopoietic, nociception, reproduction, immunity, wound
healing, bone remodelling and cognitive functions.13
The internal mammary artery (IMA) is the most commonly
used vessel in coronary artery grafting to bypass stenosed
coronary arteries. Morever its patency rate is longer lasting than
the saphenous vein (SV). The IMA has a dynamic vascular bed
therefore several vasoactive substances may cause contractile or
dilatory responses in the IMA.
Protein kinase C (PKC) is a family of serine/threonine protein
kinases. It plays a critical role in the pathogenesis of many heart
diseases.15-17
Although it has been documented that leptin has a
vasodilatatory effect,18,19 the cellular mechanism of this effect is
not well documented. The aim of this study was to investigate
the possible involvement of PKC-mediated mechanism(s) in the
vasorelaxatory effects of sodium nitroprusside (SNP) and leptin
on norepinephrine pre-contracted excised human IMA.
Methods
Informed consent was obtained from the patients and the
Clinical Research Ethics Committee of Firat University Medical
Faculty (Elazig, Turkey) approved the use of discarded human
IMA segments in this study. Segments of the left IMA were
collected from 20 patients undergoing CABG. Demographic and
clinical characteristics of these patients are given in Table 1.
The IMA were carefully cleaned of loose connective tissue
and cut into rings (about 2–3 mm long). The preparations were
placed in an isolated tissue bath containing Krebs-Henseleit
(KH) solution (composition in mM: NaCl 118, KCl 4.7, MgSO4
1.2, CaCl2 1.25, KH2PO4 1.2, NaHCO3 25, glucose 11, EDTA
0.03) at 37°C and pH 7.4, constantly bubbled with a mixture of
95% O2 and 5% CO2. Contractile activities were recorded using
a physiological force transducer (FDT05, Commat Ltd, Ankara,
Turkey) recorded by MP150WS for Windows (Biopac Systems
Inc, CA, USA).
At the beginning of the experiments, the resting tension of
the IMA vessels was adjusted to 1 g and they were allowed to
equilibrate under this resting tension for 120 min. Following a
stabilisation period, cumulative concentrations of norepinephrine
(NE) (10-9–10-4M) and SNP (10-9–10-4M) were applied to the organ
bath to determine the concentration for a maximum response.
Leptin, NE and SNP were obtained from Sigma (St Louis,
MO, USA). The PKC inhibitor, chelerythrine chloride, was
obtained from Tocris Bioscience. Each stock solution was
diluted to the required concentration immediately before bath
application.
Statistical analysis
Data are presented as mean ± SD. The effects of leptin (1 μM) on
contractile activity were evaluated using the unpaired Student’s
t -test. For all analyses, p < 0.05 was regarded as significant.
Results
The effects of leptin (1μM) and SNP (10-9–10-4M) on the NE
concentration (10 -9–10-4M) that evoked maximal contractile
responses in human IMA rings were studied. The ability of
the PKC inhibitor, chelerythrine chloride, to modulate the
contractile activity to leptin was also examined.
Firstly we tested the effects of leptin on basal tension.
Treatment with leptin (1 μM) did not cause any significant change
in basal tension of the IMA rings (data not shown). Cumulative
concentrations of NE elicited dose-dependent contraction of the
IMA rings. This contractile response was repeatable without any
significant run-down (data not shown).
In different protocols, the effects of leptin on dose-dependent
contractile responses to cumulatively added NE (10-9–10-4M) were
observed. The contractile responses to NE were significantly
attenuated by the addition of leptin (1 μM, Fig. 1A, p < 0.05,
n = 20). Cumulatively added SNP-induced vasodilatation (10-9–
10-4M) was also significantly attenuated by leptin (1 μM) (Fig. 2,
p < 0.05, n = 20).
Furthermore, as can be seen in Fig. 1B, 10 μM chelerythrine
chloride caused a significant attenuation of vasodilatator
response to leptin (Fig. 2, p < 0.05). PKC-mediated signalling
pathways were probably involved in the leptin-induced vasoactive
responses in the human IMA rings.
Discussion
In the present study, we examined the effects NE, SNP and leptin
in isolated human IMA rings. In agreement with the literature
and from our clinical results, application of NE to IMA rings
caused a dose-dependent contraction. Subsequent application
of SNP caused a dose-dependent relaxation. Addition of leptin
interrupted the endothelium-independent relaxatory effect of
SNP, attenuating its relaxatory effect. Leptin alone did not cause
any change in the basal tension of the IMA segments but caused
significant relaxation of the NE-induced contractile activity.
This is the first study to show that leptin provided a relaxatory
effect on the NE-induced contraction of isolated IMA segments,
and this effect was PKC dependent.
Table 1. Some clinical features of 20 patients undergoing CABG
Clinical features Mean ± SD, n (%)
Age 66.5 ± 8.0
Weight 73.2 ± 8.5
Body mass index 27.8 ± 2.6
Gender
Male 12 (60)
Female 8 (40)
Smoking 9 (45)
Diseases
Hypertension 17 (85)
Heart failure 3 (15)
Diabetes 10 (50)
Medication
Organic nitrates 0 (0)
Aspirin 20 (100)
Beta-blockers 14 (70)
Angiotensin inhibitors 9 (45)
Calcium channel blockers 5 (25)
Hypolipidaemics 13 (65)