Journal of Biomedical Science
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Research
Effect of the effluent released from the canine internal mammary
artery after intraluminal and extraluminal perfusion of
acetylcholine and adenosine diphosphate
Nilce Mitiko Matsuda1, Paul J Pearson2, Hartzell V Schaff2,
Carlos E Piccinato1, Alfredo J Rodrigues1 and Paulo Roberto Barbosa Evora*1
Address: 1Department of Surgery and Anatomy, Ribeirão Preto Faculty of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil and
2Division of Cardiovascular Surgery, Mayo Clinic Foundation, Rochester, Minnesota, USA
Email: Nilce Mitiko Matsuda - nmmatsuda@uol.com.br; Paul J Pearson - paulpe@prevea.com; Hartzell V Schaff - schaff@mayo.edu;
Carlos E Piccinato - cepiccin@fmrp.usp.br; Alfredo J Rodrigues - alfredo@fmrp.usp.br; Paulo Roberto
Barbosa Evora* - prbevora@netsite.com.br
* Corresponding author
Published: 5 May 2009
Journal of Biomedical Science 2009, 16:45
doi:10.1186/1423-0127-16-45
Received: 18 December 2008
Accepted: 5 May 2009
This article is available from: http://www.jbiomedsci.com/content/16/1/45
© 2009 Matsuda et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an
organ chamber containing physiological salt solution (95% O2/5% CO2, pH = 7.4, 37°C). Segments
were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity
of the effluent from the perfused internal mammary arteries was bioassayed by measuring the
decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had
been contracted with prostaglandin F2α (2 × 10-6 M). Intraluminal perfusion of adenosine
diphosphate (10-5 M) induced significant increase in relaxant activity in the effluent from the
perfused blood vessel. However, when adenosine diphosphate (10-5 M) was added extraluminally
to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast,
acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused
internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and
extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10-5 M) can
be inhibited by site-specific administration of atropine (10-5 M). These experiments indicate that
certain agonists can induce the release of EDRF only by binding to intravascular receptors while
other agonists can induce endothelium-dependent vasodilatation by acting on neural side
receptors.
Background
Accumulated evidence indicates that both the perivascular
nerves located in the adventitia layer and endothelial cells
control the tone of vascular smooth muscle [1,2]. Luminal
release of the endothelium-derived relaxing factor (EDRF)
or the endothelium-derived nitric oxide (EDNO) from the
endothelium stimulated by acetylcholine has been extensively demonstrated [1,3-5].
Previous studies have been described that the coronary
arteries are supplied by cholinergic nerves that modulate
a non-adrenergic and non-cholinergic relaxation in iso-
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Journal of Biomedical Science 2009, 16:45
lated small coronary arteries suggesting that acetylcholine
is also able to stimulate inhibitory non-adrenergic and
non-cholinergic mediators released from the perivascular
neural receptor [6-8].
Even though some authors have demonstrated that certain agonists induce the release of EDRF only by binding
to endothelium side receptors [1,3-5], others have demonstrated inhibitory mediators released by agonists acting
on perivascular nerves located in the adventitia layer [68]. Therefore, the purpose of our work was to determine
the biologic activity of the effluent released from the
canine internal mammary artery after intraluminal and
extraluminal perfusion of acetylcholine and adenosine
diphosphate. The biologic activity of the effluent released
from the canine internal mammary artery was bioassayed
on the coronary artery from which the endothelium had
been previously removed and pre-contracted with prostaglandin.
http://www.jbiomedsci.com/content/16/1/45
tower, and an adjacent aerated tower contained control
solution plus prostaglandin F2α (2 × 10-6 M).
Relaxations of the coronary artery endothelium-free ring
were examined during contraction caused by prostaglandin F2α (2 × 10-6 M). The experimental protocol was
designed that the coronary artery endothelium-free ring
was bathed with the solution released from the canine
internal mammary artery. The time to record the relaxation was almost instantaneous. Once the relaxation
reached its maximum, the coronary artery endotheliumfree ring was perfused again with the effluent released
from the canine internal mammary artery to recover to the
prior level of the contraction. This procedure was repeated
two, three times to confirm the relaxation (to rule out possible artifact). When the antagonists were added, the coronary artery endothelium-free ring was exposed to the
compound for at least 15 minutes before changing the
perfusion to the effluent released from the canine internal
mammary artery.
Materials and methods
Tissue
According to the procedures and the handling of the animals approved by the Institutional Animal Care and Use
Committee of the Mayo Foundation, mongrel dogs (25 to
30 Kg) of either sex were anesthetized with intravenously
injected pentobarbital sodium (30 mg/kg bolus injection;
Fort Dodge Laboratories, Fort Dodge, IA) and exsanguinated. The beating heart, internal mammary artery was
excised and immersed in cold oxygenated physiologic salt
solution with the following composition: NaCl, 118.3
mmol/L; KCl, 4.7 mmol/L; MgSO4, 1.2 mmol/L; KH2PO4,
1.22 mmol/L; CaCl2, 2.5 mmol/L; NaHCO3, 25.0 mmol/
L; and glucose, 11.1 mmol/L.
Bioassay experiments
The internal mammary artery was cleaned of connective
tissue, with care taken not to touch the intimal surface.
The biologic activity from the perfused of the internal
mammary artery was bioassayed on the coronary artery
ring from which the endothelium had been removed
mechanically [9]. The internal mammary artery was perfused at a constant flow (5 ml/min), with the control solution (physiologic salt solution aerated with 95% O2/5%
CO2 at 37°C). There was a transient delay of 1 second
before the fluid reached the bioassay ring, which was suspended below the donor segment. The tension developed
in the coronary bioassay ring was recorded with a force
transducer (Grass FT03; Grass Instrument Company,
Quincy, MA). The rings first were superfused for 60 minutes with control solution that passed through a stainless
steel cannula (direct superfusion). During this time, the
vessel was stretched progressively in a stepwise manner to
its optimum length-tension relation (approximately 1.0
g). Control perfusion was provided from an aerated
Drugs
The following drugs were used: acetylcholine chloride,
atropine sulphate, pirenzepine, 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium
(McN-A343), adenosine diphosphate, prostaglandin F2α (2 × 10-6
M), obtained from the Sigma Chemical Company (St.
Louis, MO), and NG-monomethyl-arginine (L-NMMA),
obtained from Calbiochem Corp (La Jolla, CA). All drugs
were prepared daily with distilled water. The concentrations were expressed as the final molar concentration in
the organ chamber.
Statistics
All data are expressed as mean ± SEM. In all experiments,
n referred to the number of animals from which blood
vessels were harvested. For bioassay experiments, relaxations were expressed as the percentage change in tension
from the contraction of the bioassay ring in response to
prostaglandin F2α (2 × 10-6 M). Statistical evaluation of the
data was performed by analysis of variance and Student's
t test for either paired or unpaired observations. Values
were considered statistically significant when p < 0.05.
Relaxations were expressed as a percentage of reduction of
the steady state tension developed after equilibration
period tension of individual preparations.
Results
The effluent released from the canine internal mammary
artery produced relaxation of the coronary artery endothelium-free ring pre-contracted with prostaglandin when
stimulated by both intraluminal and extraluminal perfusion of acetylcholine (10-5 M) but only by intraluminal
perfusion of adenosine diphosphate (10-5 M) and McN-A343 (10-5 M, Figure 1, n = 6; p < 0.05).
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Figureof1the effluent from canine internal mammary artery on coronary artery endothelium-free ring
Effect
Effect of the effluent from canine internal mammary artery on coronary artery endothelium-free ring. Relaxation of the coronary artery endothelium-free ring induced by the effluent released from canine internal mammary artery stimulated by intraluminal and extraluminal perfusion of acetylcholine (10-5 M), intraluminal and extraluminal adenosine diphosphate
(10-5 M) and intraluminal and extraluminal McN-A-343 (10-5 M). Values represent mean ± SEM; n = 6. Relaxation magnitude is
expressed as % of initial tonus. * p < 0.05.
The relaxation of the coronary artery endothelium-free
ring pre-contracted with prostaglandin caused by the
effluent released from the canine internal mammary
artery stimulated by intraluminal perfusion of acetylcholine (10-5 M) was inhibited only by intraluminal treatment with atropine (10-5 M, Figure 2, n = 6; p < 0.05),
pirenzepine (10-5 M, Figure 3, n = 6; p < 0.05) and L-NNA
(10-4 M, Figure 4, n = 6; p < 0.05). On the other hand, the
relaxation stimulated by extraluminal perfusion of acetylcholine (10-5 M) was inhibited by both the intraluminal
and extraluminal treatment with atropine (10-5 M, Figure
2, n = 6; p < 0.05), pirenzepine (10-5 M, Figure 3, n = 6; p
< 0.05) and L-NNA (10-4 M, Figure 4, n = 6; p < 0.05).
Figureof2the effluent from canine internal mammary artery on coronary artery endothelium-free ring
Effect
Effect of the effluent from canine internal mammary artery on coronary artery endothelium-free ring. Relaxation of the coronary artery endothelium-free ring induced by the effluent released from canine internal mammary artery stimulated by intraluminal and extraluminal perfusion of acetylcholine (10-5 M) before (control) and after intraluminal and
extraluminal atropine (10-5 M). Values represent mean ± SEM; n = 6. Relaxation magnitude is expressed as % of initial tonus. *
p < 0.05.
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Figureof3the effluent from canine internal mammary artery on coronary artery endothelium-free ring
Effect
Effect of the effluent from canine internal mammary artery on coronary artery endothelium-free ring. Relaxation of the coronary artery endothelium-free ring induced by the effluent released from canine internal mammary artery stimulated by intraluminal and extraluminal perfusion of acetylcholine (10-5 M) before (control) and after intraluminal and
extraluminal pirenzepine (10-5 M). Values represent mean ± SEM; n = 6. Relaxation magnitude is expressed as % of initial tonus.
* p < 0.05.
Discussion
Vascular smooth muscle tissue is surrounded internally by
the endothelium cells layer and externally by the adventitia layer containing sympathetic, parasympathetic and
sensorial nerves [1,2,10]. It has been proposed also that
NO is a messenger mediating vascular smooth muscle
relaxation released by the activation of endothelium by
acetylcholine and NANC mediators released from nerves
[3-8]. Our results also suggest that acetylcholine increased
both nerve- and endothelium-dependent EDNO release
since acetylcholine produced significant increase in the
relaxant activity of the effluent of the perfused internal
mammary artery with both intraluminal and extraluminal
Figureof4the effluent from canine internal mammary artery on coronary artery endothelium-free ring
Effect
Effect of the effluent from canine internal mammary artery on coronary artery endothelium-free ring. Relaxation of the coronary artery endothelium-free ring induced by the effluent released from canine internal mammary artery stimulated by intraluminal and extraluminal perfusion of acetylcholine (10-5 M) before (control) and after intraluminal and
extraluminal L-NMMA (10-4 M). Values represent mean ± SEM; n = 6. Relaxation magnitude is expressed as % of initial tonus. *
p < 0.05.
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perfusion and a NO-synthase inhibitor L-NMMA inhibited both.
Competing interests
Even though it has been demonstrated that both NO and
ATP can be released from NANC nerves [11], the inhibitory response in circular smooth muscle of chicken anterior mesenteric artery was caused only by NO released
from endothelium cells stimulated by neuronally ATP and
not by NO or ATP released directly from perivascular
NANC nerves [12]. In our experiments, intraluminal perfusion of adenosine diphosphate induced significant
increase in relaxant activity in the effluent from the perfused blood vessel while extraluminal perfusion of adenosine diphosphate caused no change in relaxant activity of
the effluent, suggesting that purinergic receptor related to
EDNO release is present only on endothelium cells of
canine internal mammary artery.
Authors' contributions
It has been demonstrated that muscarinic receptors mediate diverse effects on the vasculature and three major subtypes of receptors are present in endothelium cells,
nervous tissue and also smooth muscle cells [13-15].
While M1 receptors contract canine venous smooth muscle tissue, M3 receptors contract porcine and bovine coronary arteries and rabbit aorta smooth muscle [13,14]. And
also both M1 and M3 receptors mediate EDRF-dependent
relaxant responses in canine coronary artery and rabbit
aorta respectively [14,15]. In our experiments, acetylcholine seemed to act on the endothelium cells and nerves of
the canine internal mammary artery by different muscarinic receptor since atropine inhibited EDRF release by
both intraluminal and extraluminal perfusion of acetylcholine whereas McN-A-343 stimulated EDRF release
only by intraluminal perfusion.
Intraluminal release of EDRF was stimulated by acetylcholine, McN-A-343 and adenosine diphosphate while extraluminal release of EDRF was stimulated only by
acetylcholine. And both intraluminal and extraluminal
perfusion of acetylcholine were inhibited by intraluminal
but not by extraluminal perfusion of a NO-synthase
inhibitor L-NMMA, suggesting that extraluminal perfusion of acetylcholine stimulated muscarinic receptor on
nerves while intraluminal perfusion of acetylcholine stimulated muscarinic receptor on endothelium cells and both
adventitia layer and endothelial cells activation stimulated EDNO release only from endothelium.
These experiments indicate that certain agonists can
induce EDRF release from canine internal mammary
artery only by binding on the endothelium surface receptors (direct effect), while other agonists can induce EDRFdependent vasodilatation by acting on the adventitia surface receptors (indirect effect).
The authors declare that they have no competing interests.
NMM has been involved in analysis and interpretation of
data, drafting the manuscript and acquisition of funding
to prepare the manuscript. PJP helped to design the study
and collecting data. HVS helped to design the study and
collecting data. CEP helped to design the study. AJR
helped to design the study. PRBE participated in the
design of the study, collecting data and revising the manuscript and has given final approval of the version to be
published.
Acknowledgements
This manuscript was supported by grants from Fundação de Amparo a
Pesquisas do Estado de São Paulo (FAPESP 2006/50084-2) and Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq 474531/
2008-2) to NM Matsuda.
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