Pharmacology & Pharmacy, 2011, 2, 141-145
doi:10.4236/pp.2011.23019 Published Online July 2011 (http://www.scirp.org/journal/pp)
141
Inhibitory Effect of Fentanyl on
Phenylephrine-Induced Contraction on Rabbit
Aorta
Sevda Sasmaz1, Ayse Saide Sahin2, Ipek Duman2*
1Departments of Anesthesia and Pharmacology, Selcuk University, Konya, Turkey; Ipek Duman, 2Department of Pharmacology,
Selcuk University, Meram Faculty of Medicine, Konya, Turkey.
Received January 1st, 2011; revised April 10th, 2011; accepted May 4th, 2011.
ABSTRACT
This in vitro study was designed to assess the effects of fentanyl on isolated rabbit thoracic aorta rings contracted with
phenylephrine. Methods included contraction of aorta rings with phenylephrine (10–5 M) and recording the changes
after increasing concentrations of fentanyl (10–9 M – 10–5 M). Similar experiments were done after incubation with Nω-
nitro-L-arginine methyl ester (10–4 M), indomethacin (10–5 M), naloxone (10–5 M), ouabain (10–5 M), TEA (10–4 M) and
glibenclamide (10–5 M). It was revealed that, fentanyl causes relaxation in rabbit aorta rings precontracted with
phenylephrine. Removal of endothelium significantly reduces the relaxant response to fentanyl. Nitric oxide synthase
inhibitor L-NAME, K+ channel blocker glibenclamide and Na+-K+ -ATPase inhibitor ouabain inhibits the relaxant ef-
fect of fentanyl in endothelium intact aorta rings. These results suggest that fentanyl causes dose dependent vasodilata-
tion in the rabbit aorta via activation of KATP channels and Na+-K+ -ATPase, and nitric oxide released from endothe-
lium.
Keywords: Fentanyl, Nitric Oxide, Potassium Channels, Rabbit, Vascular Smooth Muscle
1. Introduction
Fentanyl is a synthetic opioid which is commonly used
for cardiac and vascular anesthesia in very high doses 1,
2. Opioids can have marked effects upon the circulation
in animals, and these effects can be mediated by either
alteration in autonomic nervous system activity or by
direct actions on blood vessels 1. Studies regarding the
effects of fentanyl on different vascular structures, and
the mechanisms involved in these actions revealed con-
flicting results 3-5. While some reports claim that fen-
tanyl acts as an alpha-blocker 6, others demonstrated that
fentanyl attenuates norepinephrine and epinephrine-me-
diated contractions independent of opioid receptors and
endothelial function 7. Thus it would appear that dif-
ferent mechanisms might be involved in fentanyl’s vas-
cular effects, depending on the species and the blood
vessel. To best of our knowledge, the effect of fentanyl
on rabbit aorta has not been studied. This in vitro study
was designed to assess the effects and the possible
mechanisms of these effects of fentanyl on isolated rab-
bit thoracic aorta rings contracted with phenylephrine.
2. Materials and Methods
With approval from our Institutional Animal Care and
Use Committee, male New Zealand rabbits (3 - 4 kg;
Selcuk University Experimental Medicine Research and
Application Centre) were anesthetized with ether and
were sacrificed by rapid exsanguination.
2.1. Sample Collection and Blood Vessel
Preparation
The thoracic aorta was removed and placed in warm
Krebs-Henseleit Solution (KHS). The excess fat and
connective tissue were removed from the vessel and cut
into spiral strips 10 - 15 mm in length. The strips were
suspended between two stainless steel hooks in organ
baths (10 ml) containing Krebs-Henseleit buffer main-
tained at 37˚C. One hook was anchored onto the organ
bath and the other was connected to a movable trans-
ducer (Model FT 03, Grass Instrument Co. MA, USA)
and a polygraph (Model 7, Grass Instrument Co. MA,
USA) for measurement and recording of changes in iso-
metric tension.

142 Inhibitory Effect of Fentanyl on Phenylephrine-Induced Contraction on Rabbit Aorta
2.2. Experimental Protocol
Strips were aerated with a gas mixture of 95% O2, 5%
CO2 throughout the experiment. Strips were initially
placed under a resting tension of 1.5 g and were allowed
to equilibrate for one hour. During this period the bath
solution was changed every 10 minutes and the resting
tension was readjusted to the 1.5 g level.
Some protocols were conducted with endothelium in-
tact artery strips while some protocols were conducted
with endothelium denuded artery strips. Endothelium
removal was done by gently denuding the endothelium
of the aorta with cotton swabs. The integrity of the en-
dothelium were assessed by pre-contracting the rings
with phenylephrine (10–5 M), then adding acetylcholine
(10–6 M) before each experiment. This confirmed re-
moval of a major portion of the endothelium because
endothelium-denuded vessels contracted in response to
acetylcholine, whereas endothelium intact vessels re-
laxed.
After an equilibrium period, the following procedures
were conducted:
Protocol 1: to assess the effect of the endothelium, ef-
fect of cumulative fentanyl on phenylephrine induced
contractions in aorta strips with intact endothelium (n = 8)
and denuded endothelium (n = 8) were studied. In aorta
strips with and without endothelium, after a resting pe-
riod, and the maximal contractile response to 10–5 M
phenylephrine had been recorded, increasing concentra-
tions of fentanyl (10–9 M – 10–5 M) were added cumula-
tively to the bath and the concentration-response curves
were obtained.
Protocol 2: to determine whether the vasorelaxant ef-
fect of fentanyl is mediated by nitric oxide (NO) or
prostanoids, endothelium intact aorta strips precontracted
with phenylephrine (10–5 M) were incubated for 20 min-
utes with a NO synthase inhibitor, Nω-nitro-L-arginine
methyl ester (l-NAME) (10–4 M) (n = 8) and a
cyclooxygenase inhibitor, indomethacin (10–5M) (n = 8)
After this incubation, concentration-response curves
were obtained to cumulative doses of fentanyl (10–9 M –
10–5 M).
Protocol 3: to evaluate the role of opioid receptors in
the effect of fentanyl, the endothelium intact aorta strips
precontracted with phenylephrine (10–5 M) were incu-
bated for 20 minutes with naloxone (10–5 M) (n = 8).
After this incubation, concentration-response curves
were obtained to cumulative doses of fentanyl (10–9 M –
10–5 M)
Protocol 4: to determine whether the vasorelaxant ef-
fect of fentanyl is mediated by K+ channels, the endothe-
lium intact aorta strips were preincubated with K+ chan-
nel blockers glibenclamide (10–6 M) (n = 8) and tetra-
ethylammonium (TEA,) (10–4 M) (n = 8) after maximal
contractile response to phenylephrine (10–5 M), fentanyl
(10–9 – 10–5 M) was added 20 minutes later, and concen-
tration-response curves were recorded.
Protocol 5: endothelium intact aorta strips precontracted
with phenylephrine (10–5 M) were incubated for 20 min-
utes with Na+-K+ -ATPase inhibitor, ouabain (10–5 M) (n
= 8) before fentanyl (10–9 – 10–5 M) was added, and
dose-response curves were recorded.
Modified Krebs-Henseleit solution was prepared in the
laboratory with compositions of (in mM) NaCl 119; KCl
4.7; MgSO4 1.5; KH2PO4 1.2; CaCl2 2.5; NaHCO3 25;
glucose 11. Fentanyl was obtained from Abbott, (Abbott
Park, IL, USA) and phenylephrine, Nω-nitro-L-arginine
methyl ester (L-NAME), indomethacin, ouabain, naloxone,
glibenclamide and tetraethyl ammonium (TEA); were
purchased from Sigma Chemical Co. (St. Louis, MO,
USA). All agents were dissolved in distilled water.
2.3. Data and Statistical Analysis
The effects of fentanyl are expressed as the percentages
of the control contractile response elicited at 10–5 M of
phenylephrine. pIC50 values (the negative logarithm of
concentration eliciting 50% of the maximal relaxation)
and Emax values (the percentage of the maximal response
of the tissue) were calculated. Results are expressed as
means ±SD. Results were evaluated using Student t test.
A p value of <0.05 was considered significant.
3. Results
Phenylephrine (10–5 M) produced contraction in rabbit
aorta strips. Responses to phenylephrine were reproduci-
ble, and time-dependent changes were not observed. Fen-
tanyl (10–9 M – 10–5 M) administered to endothelium
intact preparations at the plateau of a maximal contrac-
tion to phenylephrine (10–5 M), induced relaxation in a
concentration-dependent manner (Figures 1 and 2). Re-
moval of endothelium significantly reduced the relaxant
response to fentanyl when compared with the endothe-
lium intact rabbit aorta strips (Table 1). Preincubation of
the rabbit aorta strips with L-NAME (10–4 M), signifi-
cantly reduced the relaxant response to fentanyl when
compared with the control group (p < 0.05). Similarly,
the relaxant responses of aorta strips to fentanyl were
also inhibited significantly by glibenclamide (10-5 M),
and ouabain (10–5 M) (p < 0.05) (Figure 1). Prein- cuba-
tion of the strips with naloxone (10–5 M), and with indo-
methacin (10–5 M) or preincubation with TEA (10–4 M)
did not influence relaxant responses to fentanyl in intact
rabbit aorta strips (p > 0.05) (Figure 2). The effects of
endothelium removal and various pre-treatment agents
used on the Emax and pIC50 values for fentanyl are pre-
sented in Table 1.
C opyright © 2011 SciRes. PP

143 Inhibitory Effect of Fentanyl on Phenylephrine-Induced Contraction on Rabbit Aorta
Figure 1. Concentration-response curves for fentanyl (10–9
– 10–5 M) on phenylephrine-induced constriction of rabbit
aorta strips after incubation with l-NAME (10–4 M), gliben-
clamide (10–6 M) and ouabain (10–5 M). Data expressed as
the percentage of the control contractile response elicited at
10–5 M of phenylephrine. mean ±SD (n = 8 for each group).
Table 1. The Emax (the percentage of the maximal response
of the tissue) and pIC50 (the negative logarithm of concen-
tration eliciting 50% of the maximal relaxation) values for
fentanyl (10–9 – 10–5 M) induced relaxation in endothelium
intact and endothelium denuded rabbit aorta strips and
after various pre-treatment agents.
E
max (%) pIC50
Endothelium intact (n = 8) 72.8 ± 5.2 7.4 ± 0.19
Endothelium denuded
(n = 8) 36.4 ± 4.3* 5.9 ± 0.12*
L-NAME (10-4 M) (n = 8) 41.8 ± 4.2* 6.8 ± 0.11*
Indomethacin (10-5 M) (n
= 8) 62.3 ± 4.8 7.6 ± 0.13
TEA (10-4 M) (n = 8) 65.4 ± 5.3 7.2 ± 0.17
Glibenclamide (10-6 M)
(n = 8) 39.2 ± 3.8* 5.7 ± 0.14*
Ouabain (10-5 M) (n = 8) 51.9 ± 4.2* 6.2 ± 0.13*
Naloxone (10-5 M) (n = 8) 70.2 ± 5.1 7.3 ± 0.15
Statistical significance of p < 0.05, as compared with the control group.
Figure 2. Concentration-response curves for fentanyl (10–9
– 10–5 M) on phenylephrine-induced constriction of rabbit
aorta strips after incubation with indomethacin (10–5 M),
TEA (10–4 M) and naloxone (10–5 M). Data expressed as the
percentage of the control contractile response elicited at
10–5 M of phenylephrine. mean ±SD (n = 8 for each group).
4. Discussions
Results of this in vitro study show that fentanyl causes
concentration-dependent relaxation in rabbit aorta rings
precontracted with phenylephrine. Removal of the endo-
thelium does not totally block but significantly reduces
the relaxant response to fentanyl. Nitric oxide synthase
inhibitor L-NAME, K+ channel blocker glibenclamide
and Na+-K+ -ATPase inhibitor ouabain inhibits the re-
laxant effect of fentanyl in endothelium intact aorta
rings.
The present results imply that inhibitory effect of fen-
tanyl on phenylephrine-induced contractions in rabbit
aorta strips has both endothelium dependent and endo-
thelium independent components. Endothelium releases
autacoids such as NO and prostacyclin 8,9. These
autacoids could modulate the response of relaxant agents.
In the present study, fentanyl-induced relaxation re-
sponses decreased significantly in the presence of L-

144 Inhibitory Effect of Fentanyl on Phenylephrine-Induced Contraction on Rabbit Aorta
NAME, which is an inhibitor of nitric oxide synthase
enzyme. This finding suggests that NO released from
endothelium may play a partial role in fentanyl-caused
arterial dilation. On the other hand, the block of prostacy-
clin synthesis with indomethacin did not change these
responses. These results suggest that the relaxant effects
of fentanyl on rabbit aorta are partially mediated by en-
dothelium derived NO rather than prostacyclin. Previous
research on the effect of fentanyl on vascular reactivity
on different species and vessels revealed different results.
In studies conducted on human radial artery and porcine
coronary artery, it was shown that the relaxant effect of
fentanyl is independent of the endothelium 3,10. Simi-
larly, in another study conducted on human saphenous
veins the vasorelaxant effect of fentanyl was not reversed
by NO and prostacyclin synthase blockade 11.
Kaye et al. demonstrated that fentanyl has potent va-
sodepressor activity in the pulmonary vascular bed of the
cat and this response may be mediated opiate receptor
sensitive pathways 12. To determine if vascular re-
sponses were mediated by an opioid receptor, dose-re-
sponse studies for fentanyl were repeated in the presence
of naloxone, an opioid-receptor antagonist. Similar to
Sohn et al. 13 who studied the effects alfentanil, an
opioid with a similar structure to fentanyl on phenyle-
phrine-induced contractions in rat aorta, our results also
show that, in the presence of naloxone, fentanyl’s effect
was unchanged, indicating that vasodilatation was not
mediated by opioid receptors in rabbit aorta.
Potassium (K+) channels play an important role in the
regulation of vascular smooth muscle cell membrane
excitability and tonus 14. The activation of K+ chan-
nels in the vascular smooth muscles hyperpolarizes the
cell membranes and closes voltage dependent Ca2+ chan-
nels. These actions decrease intracellular Ca2+ and cause
vascular smooth muscle relaxation. On the contrary, the
inhibition of the channels produces membrane depolari-
zation and vascular smooth muscles contraction. In the
present study, glibenclamide, ATP-sensitive K+ channel
blocker, and TEA, Ca2+-activated K+ channel blocker,
were used to characterize the mechanism of fentanyl-
induced relaxation in rabbit aorta. Relaxant activity of
fentanyl in rabbit aorta was not blocked by TEA, but was
antagonized by glibenclamide, reflecting some role of
the hyperpolarizing ATP-sensitive K-channels in the
mechanism of action of the drug. In their study on human
saphenous veins, Sahin et al. found that the addition of
glibenclamide (ATP sensitive K+ canal blockers) and
TEA (K+ canal blockers activated by Ca++) suppress the
fentanyl induced relaxation responses 12.
Previous studies suggest that the sarcolemmal Na+-K+
-ATPase plays an essential role in the maintenance of the
vascular smooth muscle tone 15-17. In smooth muscles,
this pump can directly contribute to the cell resting
membrane potential by actively pumping more sodium
ions out than potassium ions into the cells 18. Mem-
brane depolarization in response to inhibition of Na+-K+
-ATPase caused Ca++ channels to open and/or in- creased
Ca2+ influx through Na+-Ca2+ exchange mecha- nism. It
has been suggested that normal Na+-K+ -ATPase activity
is necessary for mediating vasorelaxant effects of some
drugs 19,20. In rabbit aorta strips, the relaxant effect of
fentanyl was inhibited by ouabain. Such partial inhibition
has also been obtained with ouabain in human saphenous
vein 12.
Results of this study provides evidence that increasing
doses of fentanyl result in concentration-dependent re-
laxation in the rabbit aorta. Both endothelium-dependent
and endothelium-independent mechanisms are involved
in the relaxation activation of KATP channels and Na+-K+
-ATPase, and nitric oxide released from the endothelium
are responsible for the vasodilation caused by fentanyl.
Further research will be needed to elucidate the exact
pathways of fentanyl-induced vasoreactivity in blood
vessels.
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