Am J Physiol Regul Integr Comp Physiol 298: R767–R775, 2010.
First published January 13, 2010; doi:10.1152/ajpregu.00148.2009.
Mechanisms of nitric oxide-mediated, neurogenic vasodilation in mesenteric
resistance arteries of toad Bufo marinus
Brett L. Jennings and John A. Donald
School of Life and Environmental Sciences, Deakin University, Geelong, Victoria, Australia
Submitted 12 March 2009; accepted in final form 7 January 2010
amphibian; autonomic nervous system; nitric oxide synthase; endothelium
IN THE MAMMALIAN MESENTERIC vasculature, it is well established
that the endothelium releases numerous vasoactive molecules,
such as nitric oxide (NO), prostaglandins, endothelium-derived
hyperpolarizing factor (EDHF), and endothelium-derived contracting factors, which contribute to the regulation of vascular
tone (26). In addition, mesenteric arteries are innervated by
sympathetic vasoconstrictor and primary sensory vasodilator
nerves (71). NO is clearly important in maintaining the vasodilator tone of mesenteric arteries, inasmuch as inhibition of
NO synthase (NOS) causes vasoconstriction and increases
blood pressure (20). Endothelial NOS (eNOS) is the predominant isoform responsible for NO generation in mesenteric
arteries, but there is also evidence that neural NOS (nNOS) in
perivascular nitrergic nerves generates NO to provide neurally
derived vasodilation (2, 31, 63, 66).
In contrast to mammals, much less is known about the role
of NO in the regulation of mesenteric vascular tone in amphibians. In the small intestine, NADPH-diaphorase histochemical
Address for reprint requests and other correspondence: B. L. Jennings, Dept.
of Pharmacology, College of Medicine, Univ. of Tennessee Health Science
Centre, Memphis, TN 38163 (e-mail: [email protected]).
http://www.ajpregu.org
staining and nNOS immunoreactivity (nNOS-IR) have been
demonstrated in perivascular nerves (34, 35, 41, 49), but
isoform-specific localization of NOS within the amphibian
mesenteric vasculature has not been performed. There is evidence in amphibians that NO is involved in capillary fluid
regulation, inasmuch as inhibition of NOS has been reported to
decrease hydraulic conductivity (55) and capillary permeability
(23, 24, 54) in the mesenteric circulation of the leopard frog
Rana pipiens. Furthermore, substance P (SP) increased microvascular permeability in R. pipiens by increasing NO production
(44). Although there is no definitive evidence establishing the site
of NO production within the mesenteric circulation of amphibians, it was assumed, on the basis of the mammalian paradigm, that
the capillary endothelium was the source of NO (55).
The majority of physiological studies in amphibians on NO
control of the circulation have focused on large-conductance
blood vessels. Recently, we provided evidence that the systemic (8, 9) and pulmonary (29) arteries of the toad Bufo
marinus lack an endothelial NO system and, instead, NO
control is provided by nitergic nerves. In addition, we showed
that toad cutaneous arteries are similarly regulated by NO
released from nitrergic nerves (29). However, it is unknown
whether an endothelial or a neural NO signaling system exists
in small-resistance arteries of the circulation of amphibians. A
comparison between NO regulation of large and small systemic
vessels of B. marinus is essential to establish whether NO plays
a role in regulating blood pressure and tissue blood flow.
Therefore, the aim of this study was to determine whether
mesenteric resistance arteries of B. marinus are regulated by
NO generated by eNOS and/or nNOS and to investigate potential intracellular mechanism(s) of NO signaling.
METHODS
Animals. All experiments complied with Australian law on the use
of animals for experimentation and were approved by the Animal
Welfare Committee of Deakin University (approval no. A5/2006).
Toads (B. marinus) of both sexes and mass of 90 –150 g were
purchased from a commercial supplier in Queensland, Australia.
Toads were maintained at the Deakin University Animal House at
20 –25°C and were not fed during captivity (up to 1 mo) but had ad
libitum access to water. Before experimentation, the animals were
killed by decapitation and then pithed. Sprague-Dawley rats (Rattus
norvegicus) of both sexes and a mass of 200 –300 g were obtained
from a breeding colony at the Deakin University Animal House. The
animals were housed in rat boxes and maintained at 23°C on a 12:12-h
light-dark cycle; they had ad libitum access to water and rat chow.
Before experimentation, rats were euthanized with a carbon dioxide
overdose, according to standard operating procedures at Deakin University.
NADPH-diaphorase histochemistry. The small intestine and attached mesentery or isolated mesenteric arteries were dissected free
and placed in ice-cold PBS. Then the mesentery attached to the small
intestine was stretched and pinned out on dental wax. All tissues were
0363-6119/10 $8.00 Copyright © 2010 American Physiological Society
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Jennings BL, Donald JA. Mechanisms of nitric oxide-mediated, neurogenic vasodilation in mesenteric resistance arteries of toad Bufo marinus. Am
J Physiol Regul Integr Comp Physiol 298: R767–R775, 2010. First published
January 13, 2010; doi:10.1152/ajpregu.00148.2009.—This study determined the role of nitric oxide (NO) in neurogenic vasodilation in
mesenteric resistance arteries of the toad Bufo marinus. NO synthase
(NOS) was anatomically demonstrated in perivascular nerves, but not
in the endothelium. ACh and nicotine caused TTX-sensitive neurogenic vasodilation of mesenteric arteries. The ACh-induced vasodilation was endothelium-independent and was mediated by the NO/
soluble guanylyl cyclase signaling pathway, inasmuch as the vasodilation was blocked by the soluble guanylyl cyclase inhibitor 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the NOS inhibitors N␻nitro-L-arginine methyl ester and N␻-nitro-L-arginine. Furthermore,
the ACh-induced vasodilation was significantly decreased by the more
selective neural NOS inhibitor N5-(1-imino-3-butenyl)-L-ornithine.
The nicotine-induced vasodilation was endothelium-independent and
mediated by NO and calcitonin gene-related peptide (CGRP), inasmuch as pretreatment of mesenteric arteries with a combination of
N␻-nitro-L-arginine and the CGRP receptor antagonist CGRP-(8 –37)
blocked the vasodilation. Clotrimazole significantly decreased the
ACh-induced response, providing evidence that a component of the
NO vasodilation involved Ca2⫹-activated K⫹ or voltage-gated K⫹
channels. These data show that NO control of mesenteric resistance
arteries of toad is provided by nitrergic nerves, rather than the
endothelium, and implicate NO as a potentially important regulator of
gut blood flow and peripheral blood pressure.
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domethacin, esculetin, guanethidine monosulfate, clotrimazole, levcromakalim, ␤-NADPH (reduced form), nitro blue tetrazolium, and Triton
X-100 were obtained from Sigma (St. Louis, MO); rat atrial natriuretic peptide (rANP) and calcitonin gene-related peptide (CGRP)(8 –37) from Auspep (Melbourne, Australia); nicotine from BDH
Chemicals (Poole, UK); 3-morpholinosydnonimine (SIN-1), 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), N5-(1-imino-3butenyl)-l-ornithine (vinyl-L-NIO), and glibenclamide from Alexis
Biochemicals (San Diego, CA); U-46619 and L-N6-(1-iminoethyl)lysine (L-NIL) from Cayman Chemical (Ann Arbor, MI); TTX from
Alomone Labs (Jerusalem, Israel); and the nNOS and FITC-conjugated donkey anti-sheep IgG antibodies from Chemicon (Melbourne,
Australia).
RESULTS
Presence and distribution of NOS in the mesenteric circulation.
No specific NADPH-diaphorase staining was evident in the
endothelium of toad mesenteric arteries (Fig. 1A; n ⫽ 3). In
contrast, positive NADPH-diaphorase staining, typical of
eNOS, was observed in the endothelium of rat mesenteric
arteries, as has been previously reported (Fig. 1B) (1, 4). In
contrast to the endothelium of toad, positive NADPH-diaphorase histochemical staining was observed in nerve fibers located throughout the mesentery (Fig. 1C; n ⫽ 3). Specifically,
NADPH-diaphorase-positive varicose perivascular nerve fibers
were observed running parallel to mesenteric blood vessels
(Fig. 1D). nNOS-IR was observed in single varicose fibers in
the outer media and adventitia of the blood vessels (Fig. 1, E
and F; n ⫽ 3). No specific immunoreactivity was observed in
tissues that were incubated in secondary antibody only (results
not shown).
Dual-wire myography. The physiological data are summarized in Table 1.
NO/cGMP signaling in toad mesenteric arteries. The NO
donors SNP (10⫺4 mol/l; Fig. 2A) and SIN-1 (10⫺4 mol/l; Fig. 2B)
mediated vasodilation of mesenteric arteries, indicating the
presence of an NO receptor. The SNP and SIN-1 vasodilations
were blocked after preincubation with the soluble GC inhibitor
ODQ (10⫺5 mol/l; Fig. 2C); generally, addition of ODQ to the
bath caused a constriction. Subsequent addition of rANP (10⫺8
mol/l), which mediates its effects through a particulate GC,
caused vasodilation in the presence of ODQ (Fig. 2C).
ACh-induced vasodilation. In toad mesenteric arteries, ACh
(10⫺5 mol/l), a known activator of nNOS in toad large systemic
arteries (8), caused a vasodilation (Fig. 3A); this response was
blocked by preincubation with the muscarinic receptor antagonist
atropine (10⫺6 mol/l; Table 1). The ACh-induced vasodilation
was completely blocked after preincubation of mesenteric arteries
with ODQ (10⫺5 mol/l; Fig. 3B); subsequent addition of rANP
(10⫺8 mol/l) caused vasodilation (Fig. 3B). Preincubation of
mesenteric arteries with the nonspecific NOS inhibitors L-NAME
(10⫺4 mol/l; Fig. 3C) and L-NNA (10⫺4 mol/l; Table 1) also
inhibited the ACh-induced vasodilation, but SNP (10⫺4 mol/l),
which acts independently of NOS, mediated vasodilation. Removal of the endothelium had no effect on the ACh-induced
vasodilation (Fig. 3D). Preincubation with vinyl-L-NIO (10⫺5
mol/l), a selective nNOS inhibitor in mammalian NO signaling
systems, caused a significant decrease in the ACh-induced vasodilation (Fig. 3E), but inhibition of inducible NOS with L-NIL
(10⫺5 mol/l) had no effect (Table 1).
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fixed for 1 h in 4% formaldehyde (pH 7.4) at 4°C and then washed
three times (10 min each) in PBS. Isolated mesenteric arteries were
cryoprotected overnight in a solution containing PBS, 30% sucrose,
and 0.1% sodium azide, placed into molds containing optimum
cutting temperature (OCT) TissueTek, and frozen at ⫺80°C. Sections
(12 ␮m) were cut on a cryostat (Microm HM 550 OMP), transferred
to silane-subbed slides, and allowed to air dry for 30 min. Wholemount and sectioned preparations were stained in an NADPH-diaphorase mixture containing 1 mg/ml ␤-NADPH, 0.25 mg/ml nitro blue
tetrazolium, and 1% Triton X-100 in 0.1 mol/l Tris buffer (pH 8) for
15 min at 37°C. This mixture, which is light-sensitive, was kept in
darkness. Tissues were again washed three times (5 min each) in PBS
and covered with buffered glycerol (0.5 mol/l Na2CO3 added dropby-drop to 0.5 mol/l NaHCO3 to pH 8.6 combined 1:1 with glycerol).
Tissues were evaluated for NADPH-diaphorase staining (formazan
precipitates) using a light microscope (Zeiss) and photographed with
a digital imaging system (Spot 35 camera system) (6). Mesenteric
arteries from rats were used as a control to demonstrate the presence
of NOS in the vascular endothelium, as has been previously demonstrated (1, 4).
nNOS immunohistochemistry. Whole mounts of mesentery and
mesenteric arteries were fixed for 2 h in 4% paraformaldehyde and
then washed in PBS. Pieces of blood vessel and mesentery were
incubated with an nNOS antibody (polyclonal sheep, 1:4,000 dilution)
(3) overnight at 4°C in a humid box. The nNOS antibody is raised
against an epitope sequence of rat nNOS (aa 1409 –1429) that is 90%
similar to the corresponding region of Xenopus laevis nNOS (accession no. NP_001079155). Tissues were washed three times (10 min
each) in PBS for removal of any excess primary antibody and
incubated with FITC-conjugated donkey anti-sheep IgG (1:200 dilution) for 2–3 h at room temperature in a humid box. Tissues were
washed three times (10 min each) in PBS, mounted in buffered
glycerol, and observed and photographed using a point-scanning
confocal laser microscope (LSM 510 META, Zeiss). Immunohistochemical controls were performed by omission of primary antibody.
Dual-wire myography. After the animals were killed, mesenteric
arteries (⬃200 ␮m ID) were dissected from branches of the superior
mesenteric artery. Arteries were cleaned of any surrounding connective tissue and placed in Mackenzie’s balanced salt solution [115.0
mM NaCl, 3.2 mM KCl, 20 mM NaHCO3, 3.1 mM NaH2PO4·H2O,
1.4 mM MgSO4·7H2O, 16.7 mM D-(⫹)-glucose, and 1.3 mM
CaCl2·2H2O, pH 7.2]. Arteries were cut into individual 2- to 3-mm
rings, which were mounted horizontally between two pieces of 40-␮m
wire, which, in turn, were attached to separate jaws of a dual-wire
myograph (model 410A, Danish Myo Technology). The rings were
bathed in 5 ml of Mackenzie’s balanced salt solution and maintained
at 22°C and aerated with 95% O2-5% CO2. Tension was placed on the
arteries by increasing the distance between the internal wires until
they were flush against the vessel wall, and the arteries were left to
equilibrate for ⱖ30 min. The myograph was linked to a Myo-Interface
system, which was attached to a PowerLab data collection system and
a personal computer.
To determine vasodilator mechanism(s), arteries were preconstricted with the prostaglandin H2 analog U-46619 (10⫺6 mol/l), and
vasoconstriction was allowed to reach its maximum. The extent of
vasodilation was determined as a percentage of the initial U-46619induced vasoconstriction. For experiments, matched controls were
used from the same animal for comparison of drug effects. In some
experiments, the artery was rubbed with a wire for removal of the
endothelium from the artery, which was verified with standard hematoxylin-eosin staining.
Statistical analysis. Values are means ⫾ SE of a minimum of five
experiments. Statistical analysis was performed with paired-samples
t-tests using the SPSS (version 14.0) statistical package. P ⱕ 0.05 was
considered significant.
Materials. Sodium nitroprusside (SNP), ACh, atropine, N␻-nitroL-arginine methyl ester (L-NAME), N␻-nitro-L-arginine (L-NNA), in-
NITRIC OXIDE CONTROL OF TOAD MESENTERIC VASCULATURE
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Preincubation of the mesenteric arteries with a combination of
indomethacin (10⫺5 mol/l) and esculetin (10⫺5 mol/l), inhibitors
of cyclooxygenase and lipoxygenase, respectively, had no effect
on the ACh-induced vasodilation (Table 1). Blockade of voltagegated Na⫹ channels with TTX (10⫺6 mol/l) significantly decreased the ACh-induced vasodilation (Table 1), and antagonism
of sympathetic neurotransmission with guanethidine (10⫺6 mol/l)
had no effect on the vasodilation (Table 1). Preincubation with
clotrimazole (10⫺5 mol/l), a nonspecific K⫹ channel inhibitor,
caused a significant decrease in ACh-induced vasodilation of the
mesenteric arteries (Fig. 4), but the ATP-sensitive K⫹ (KATP)
channel inhibitor glibenclamide (10⫺5 mol/l) did not affect the
vasodilation after addition of ACh (10⫺5 mol/l; Table 1). Interestingly, levcromakalim (10⫺5 mol/l), a KATP channel opener, did
not affect vascular tone.
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Nicotine-induced vasodilation. Nicotine has previously been
used to activate nNOS in toad large arteries (17, 29). Nicotine
(10⫺4 mol/l) caused vasodilation of the mesenteric arteries
(Fig. 5A), which was significantly decreased after preincubation with ODQ (10⫺5 mol/l; Fig. 5B). Subsequent addition of
SNP (10⫺4 mol/l) to the ODQ-treated vessels was without
effect, but rANP (10⫺8 mol/l) mediated vasodilation. Inhibition
of NOS with L-NNA (10⫺4 mol/l) also significantly decreased
the vasodilation (Fig. 5C). A combination of L-NNA (10⫺4 mol/l)
and the CGRP receptor antagonist CGRP-(8 –37) (10⫺6 mol/l)
completely blocked the response to nicotine (10⫺4 mol/l); in fact,
a constriction was observed (Fig. 5D). SNP (10⫺4 mol/l) caused
vasodilation of mesenteric arteries treated with L-NNA ⫹ CGRP(8 –37). Finally, the nicotine-induced vasodilation was not affected by disruption of the endothelium (Fig. 5E).
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Fig. 1. Photomicrographs showing sectioned preparations of toad (A and F) and rat (B) mesenteric arteries
and whole-mount preparations of toad mesentery (C–E)
after processing for NADPH-diaphorase histochemistry
(A–D) and neural nitric oxide synthase (nNOS) immunohistochemistry (E and F). No punctate NOS-positive
staining was observed in endothelial cells of toad mesenteric arteries (A, arrow). In rat artery (B), punctate,
NOS-positive staining (arrowhead) was observed around
nuclei (arrow) of endothelial cells. Positive NADPHdiaphorase staining and nNOS immunoreactivity (nNOSIR) were observed in perivascular nerve fibers (C–E, arrows) and nerve bundles (C, arrowhead) throughout the
mesentery. Nerve fibers displaying nNOS-IR were observed in adventitia and media of blood vessels (F, arrows). Scale bars, 10 ␮m (A and B) and 50 ␮m (C–F).
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NITRIC OXIDE CONTROL OF TOAD MESENTERIC VASCULATURE
Table 1. Summary of the vasodilatory responses of toad
mesenteric arteries to various treatments associated with the
NO signaling system
Treatment
Experiment(s)
Nitric Oxide
49 ⫾ 7
34 ⫾ 2
SNP
SIN-1
significantly decreased the nicotine-induced vasodilation (Table 1), and blockade of sympathetic neurotransmission with
guanethidine (10⫺6 mol/l) caused a significant increase in the
vasodilation (Fig. 6). The nicotine-induced vasodilation was
not affected after preincubation with clotrimazole (10⫺5 mol/l;
Table 1) or glibenclamide (10⫺5 mol/l; Table 1).
DISCUSSION
ACh
45 ⫾ 3
Nicotine
58 ⫾ 6
Control
Atropine
41 ⫾ 6
No response
Not tested
Control
ODQ*
44 ⫾ 4
No response
55 ⫾ 7
17 ⫾ 4
P ⬍ 0.05
Control
34 ⫾ 7
No response
Not tested
L-NAME
Control
L-NNA*
44 ⫾ 7
No response
53 ⫾ 3
19 ⫾ 5
P ⬍ 0.05
Control
L-NNA ⫹ CGRP(8-37)
Not tested
55 ⫾ 12
Constriction
Control
Endothelium denuded
44 ⫾ 8
39 ⫾ 7
P ⫽ 0.71
57 ⫾ 6
56 ⫾ 6
P ⫽ 0.97
Control
Vinyl-L-NIO*
44 ⫾ 2
19 ⫾ 1
P ⬍ 0.05
Not tested
Control
L-NIL
47 ⫾ 4
44 ⫾ 4
P ⫽ 0.48
Not tested
Control
Indomethacin⫹esculetin
48 ⫾ 1
47 ⫾ 6
P ⫽ 0.87
54 ⫾ 6
55 ⫾ 10
P ⫽ 0.99
Control
TTX*
47 ⫾ 7
34 ⫾ 9
P ⬍ 0.05
55 ⫾ 4
25 ⫾ 6
P ⬍ 0.05
Control
Guanethidine*
44 ⫾ 5
46 ⫾ 6
P ⫽ 0.39
63 ⫾ 4
78 ⫾ 4
P ⬍ 0.05
Control
Clotrimazole*
44 ⫾ 1
28 ⫾ 4
P ⬍ 0.05
60 ⫾ 8
54 ⫾ 9
P ⫽ 0.75
Control
Glibenclamide
45 ⫾ 8
50 ⫾ 6
P ⫽ 0.70
58 ⫾ 6
57 ⫾ 8
P ⫽ 0.86
Values are mean ⫾ SE % of the initial constriction of the vessels of at least
5 experiments. NO, nitric oxide; SNP, sodium nitroprusside; SIN-1, 3-morpholinosyndnonimine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one;
L-NAME, N␻-nitro-L-arginine methylester; L-NNA, N␻-nitro-L-arginine;
CGRP, calcitonin gene-related peptide; vinyl-L-NIO, N5-(1-imino-3-butenyl)L-ornithine; L-NIL, N6-(1-iminoethyl)lysine. *Significant difference.
Inhibition of the cyclooxygenase and lipoxygenase pathways with a combination of indomethacin (10⫺5 mol/l) and
esculetin (10⫺5 mol/l) did not affect the vasodilation (Table 1).
Preincubation of the mesenteric arteries with TTX (10⫺6 mol/l)
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NO signals via activation of soluble GC and generation of
the second messenger cGMP, which in blood vessels generally
mediates vasodilation (56). Intriguingly, NO-mediated vasodilation is not ubiquitous in all vertebrates, because it is not
observed in chondrichthyans and lungfishes; the first group in
which it is generally observed is the bony fishes (47). NOmediated vasodilation is always found in amphibian blood
vessels, inasmuch as the NO donor SNP is a generic vasodilator (8, 9, 29, 57). The presence of an NO/cGMP signaling
system was confirmed in toad mesenteric arteries, inasmuch as
SNP and SIN-1 caused vasodilation, which was blocked by the
soluble GC inhibitor ODQ.
The type and location of NOS isoforms and, therefore, the
potential source of endogenous NO in toad mesenteric arteries
were investigated using NADPH-diaphorase histochemistry
and immunohistochemistry. No positive NADPH staining was
Fig. 2. Representative tension recordings showing the vasodilatory effect of the
NO donor compounds sodium nitroprusside (SNP, 10⫺4 mol/l; A) and 3-morpholinosydninimine (SIN-1, 10⫺4 mol/l; B) and the effect of the soluble GC
inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10⫺5 mol/l;
C) on toad mesenteric artery. Artery was preincubated with ODQ for ⬃10 min
before preconstriction with U-46619 (10⫺6 mol/l). ODQ completely blocked
vasodilations induced by SNP and SIN-1; subsequent addition of rat atrial
natriuretic peptide (rANP, 10⫺8 mol/l), which mediates vasodilation via a
particulate guanylyl cyclase, caused a marked vasodilation (C). Addition of
ODQ caused an initial vasoconstriction before preconstriction.
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Fig. 3. Representative tension recordings showing the vasodilatory effect of
ACh (10⫺5 mol/l; A) on the mesenteric artery, which is completely blocked in
the presence of ODQ (10⫺5 mol/l; B) and the NOS inhibitor N␻-nitro-Larginine methyl ester (L-NAME, 10⫺4 mol/l; C) but unaffected after endothelial disruption (D) and significantly decreased in the presence of the more
selective nNOS inhibitor N5-(1-imino-3-butenyl)-l-ornithine (vinyl-L-NIO, E).
In B, C, and E, blood vessels were preincubated with the inhibitors for ⬃10
min; in D, endothelium was disrupted before preconstriction with U-46619
(10⫺6 mol/l). In B, initial application of ODQ to the bath caused a vasoconstriction before addition of U-46619, and subsequent addition of rANP (10⫺8
mol/l) caused vasodilation. In C, subsequent addition of SNP (10⫺4 mol/l),
which acts independently of NOS, also mediated vasodilation.
observed in the endothelium of the mesenteric arteries, which
provides further evidence that amphibian vascular endothelial
cells do not express eNOS/NOS. Recent genomic and expressed sequence tag (EST) analyses have demonstrated that
eNOS is apparently not present in fish but first appears in
amphibians [X. laevis, accession no. AW765292 (Washington
University Xenopus EST project, 1999); Ambystona tigrinum,
accession no. CN053573 (53)]. Recently, our laboratory sequenced a full-length eNOS cDNA from the Western clawed
AJP-Regul Integr Comp Physiol • VOL
Fig. 4. Representative tension recordings showing the vasodilatory effect of
ACh (10⫺5 mol/l; A) on the mesenteric artery, which is significantly decreased
in the presence of the nonspecific K⫹ channel inhibitor clotrimazole (10⫺5
mol/l; B). Artery was preincubated with clotrimazole for ⬃10 min before
preconstriction with U-46619 (10⫺6 mol/l).
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frog Silurana tropicalis (accession no. FJ665981) and a partial
eNOS cDNA from B. marinus (accession no. GU138862).
Furthermore, in S. tropicalis, eNOS mRNA is not expressed in
systemic blood vessels (Trajanovska and Donald, unpublished
observations), further supporting our contention that eNOS or
NO derived from the endothelium does not play a role in
vascular regulation in amphibians. In contrast to the endothelium, positive NADPH staining was always observed in
perivascular nerves of mesenteric blood vessels, and nNOS-IR
nerves showed a pattern very similar to that of NADPHpositive nerves. The presence of nitrergic nerves in toad
mesenteric arteries is consistent with our previous studies in
large systemic blood vessels (8, 9) and the pulmocutaneous
vasculature (29) of this species; the functionality of the nerves
in the mesenteric resistance arteries was determined with
myography.
ACh- and nicotine-induced vasodilation of toad mesenteric
arteries. Previously, ACh was found to cause the release of NO
from perivascular nitrergic nerves in toad systemic vasculature
(8, 9), presumably by neuronal signaling that activates nNOS.
In toad mesenteric arteries, application of ACh also mediated
vasodilation that was solely attributed to NO generation of
cGMP, because it was completely blocked by the soluble GC
inhibitor ODQ and the nonspecific NOS inhibitors L-NAME
and L-NNA. Given the presence of nitrergic nerves in the
mesenteric arteries and the fact that removal of the endothelium had no effect on the ACh-induced vasodilation, it is then
probable that ACh is inducing NO neurotransmission and
subsequent vasodilation. This is further supported by the observation that preincubation of the arteries with the more
selective nNOS inhibitor vinyl-L-NIO (5) significantly decreased the response to ACh. Any role for inducible NOS and
prostaglandin/leukotriene synthesis was discounted by the use
of selective inhibitors that had no effect on the ACh vasodilation. In the mammalian mesenteric vasculature, ACh mediated
an endothelium-dependent vasodilation that was due to NO and
an endothelium-independent vasodilation that was due to ACh
binding to muscarinic receptors on CGRP-containing nerves
causing CGRP release (61). In toad, the mesenteric blood
vessels are innervated by CGRP-containing nerves (39), but
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Fig. 5. Representative tension recordings showing the vasodilatory effect of
nicotine (10⫺4 mol/l; A) on the mesenteric artery, which is significantly
decreased in the presence of ODQ (10⫺5 mol/l; B) and N␻-nitro-L-arginine
(L-NNA, 10⫺4 mol/l; C) and completely blocked in the presence of a combination of L-NNA (10⫺4 mol/l) and the calcitonin gene-related peptide (CGRP)
receptor antagonist CGRP-(8 –37) (10⫺6 mol/l; D). Response to nicotine was
unaffected by disruption of the endothelium (E). In B–D, arteries were
preincubated with the inhibitors for ⬃10 min before preconstriction with
U-46619 (10⫺6 mol/l). In B, addition of SNP (10⫺4 mol/l) was without effect,
but subsequent addition of rANP (10⫺8 mol/l) caused vasodilation. In C, subsequent addition of SNP (10⫺4 mol/l) caused vasodilation; in D, nicotine actually
caused a constriction, but addition of SNP (10⫺4 mol/l) still caused vasodilation.
ACh does not appear to cause a similar release of CGRP (see
below).
In addition to ACh, nicotine also facilitates the release of
NO from perivascular nitrergic nerves in toad conductance
AJP-Regul Integr Comp Physiol • VOL
Fig. 6. Representative tension recordings showing the vasodilatory effect of
nicotine (10⫺4 mol/l; A) on the mesenteric artery, which is significantly
increased in the presence of the sympathetic neurotransmission inhibitor
guanethidine (10⫺6 mol/l; B). Artery was preincubated with guanethidine for
⬃30 min before preconstriction with U-46619 (10⫺6 mol/l).
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vessels (17, 29). In the mesenteric arteries, nicotine caused an
endothelium-independent vasodilation, because it was unaffected by pretreatment with indomethacin ⫹ esculetin or removal of the endothelium. The nicotine-induced vasodilation
was found to have two components. The first component was
mediated by NO, because the majority of the vasodilation was
inhibited by both ODQ and L-NNA. Interestingly, nicotine-induced
vasodilation of large-conductance vessels and the cutaneous artery of
toad was entirely caused by NO (17, 29). Subsequently, preincubation of toad mesenteric arteries with a combination of
L-NNA and the CGRP receptor antagonist CGRP-(8 –37) completely blocked the nicotine-induced vasodilation; in fact, addition of nicotine now caused a vasoconstriction. The evidence
for neurally mediated CGRP vasodilation is supported by
previous data from toad showing that CGRP-containing nerves
occur in mesenteric arteries and that CGRP is a vasodilator of
mesenteric blood vessels (50). In addition, the nicotine-induced
vasodilation of toad pulmonary artery was due to NO and
CGRP (29). It is curious that nicotine appears to specifically
stimulate the release of CGRP, but not SP, from toad mesenteric artery perivascular nerves, inasmuch as CGRP and SP are
colocalized in the nerves (39). A similar situation was observed
in the mammalian carotid arterial bed, in which CGRP and SP
are colocalized in sensory nerves (67), but nicotine-induced
vasodilation was mediated by the release of CGRP, rather than
SP (51).
To determine whether the vasodilatory responses induced by
ACh and nicotine in toad mesenteric arteries involved neurogenic mechanisms, TTX was used to inhibit voltage-gated Na⫹
channels. Preincubation of the mesenteric artery with TTX
significantly decreased the vasodilation caused by ACh and
nicotine, providing further evidence that the NO vasodilation is
due to activation of nitrergic nerves. ACh-induced NO-mediated vasodilation of toad conductance arteries is also TTXsensitive (17), but TTX does not affect nicotine-induced NO
vasodilation in toad systemic and pulmonary arteries (17, 29).
In mammals, the precise cellular pathways activated by nicotine to cause NO vasodilation are unclear (65); thus it is not
possible to explain why the nicotine effects in the toad vasculature show variable TTX sensitivity.
NITRIC OXIDE CONTROL OF TOAD MESENTERIC VASCULATURE
AJP-Regul Integr Comp Physiol • VOL
endothelially derived prostaglandins are the primary vasodilators before the evolution of endothelial NO signaling via eNOS
in the higher vertebrates.
Cellular mechanisms of ACh- and nicotine-induced vasodilation of toad mesenteric arteries. In arterial smooth muscle,
one of the major intracellular facilitators of NO-mediated
vasodilation is K⫹ channels (43). In mammalian mesenteric
arteries, it has been shown that NO-mediated vasodilation is
caused, in part, by opening of Ca2⫹-activated K⫹ (KCa) (11,
30, 38, 52, 62) and voltage-gated K⫹ (KV) (27, 68) channels.
In toad, clotrimazole, an inhibitor of large-conductance KCa
(BKCa) channels, intermediate-conductance KCa (IKCa) channels, and KV channels (58) significantly decreased the AChinduced vasodilation that is attributed to NO. Although the
exact K⫹ channel(s) involved in the NO-induced vasodilation
of toad mesenteric arteries was not elucidated, opening of IKCa
channels can tentatively be discounted as follows. In the
mammalian vasculature, it is widely accepted that opening of
IKCa channels is involved in EDHF-mediated vasodilation
(10). However, in this study, the involvement of EDHF can be
excluded, because the ACh-induced vasodilation is not dependent on an intact endothelium and is solely due to NO. Thus it
would appear that ACh-induced, NO-mediated vasodilation of
toad mesenteric arteries involves the opening of BKCa and/or
KV channels. Since ACh- and NO donor-mediated vasodilations are completely inhibited by ODQ in toad mesenteric
arteries, it is most likely that opening of the ion channel(s) is
cGMP-dependent, as has been previously reported in mammalian mesenteric arteries (11, 27, 69).
NO released from nitrergic nerves after addition of nicotine
would presumably mediate vasodilation in the same manner as
that released by ACh stimulation, but preincubation of the toad
mesenteric artery with clotrimazole did not significantly decrease the nicotine-induced vasodilation. This could be due to
the fact that nicotine causes the release of two vasodilators, NO
and CGRP, whereby CGRP signals via an intracellular mechanism different from NO. For example, CGRP has been shown
to dilate mammalian mesenteric arteries independently of KCa
and KV channels (33). In toad, it is possible CGRP caused a
KCa/KV channel-independent vasodilation that resulted in a net
insignificant effect of clotrimazole, despite the fact that the
NO-mediated vasodilation (as demonstrated with ACh) is inhibited by clotrimazole.
Opening of KATP channels has also been reported to be
involved in NO-mediated (21, 40) and CGRP-mediated (7, 42)
vasodilation of the mammalian mesenteric artery. However, in
toad mesenteric arteries, inhibition of KATP channels with
glibenclamide had no effect on the vasodilation induced by
ACh or nicotine. In fact, levcromakalim, a KATP channel
opener, had no effect on vascular tone, suggesting that toad
mesenteric arteries may lack KATP channels. Interestingly,
levcromakalim caused vasodilation of the toad pulmonary and
cutaneous arteries (29), which demonstrates a heterogenous
distribution of KATP channels, as well as distinct cellular
mechanisms underlying vascular regulation, in toad.
Perspective
The evolution of autonomic nervous system control of the
systemic circulation has focused on the role of sympathetic,
adrenergic vasoconstrictor nerves in maintaining vascular tone
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Previous studies in various mammalian vascular beds have
reported that the initial response to nicotine is a transient
contraction that is reversed to a relaxation in the presence of
various antagonists. For example, in canine lingual arteries,
nicotine-induced vasoconstriction is converted to vasodilation
in the presence of adrenergic and purinergic blockade (46), and
in monkey mesenteric arteries, nicotine-induced vasoconstriction was reversed to an NO-mediated vasodilation by phentolamine and guanethidine (64). Thus, in mammalian vascular
beds, nicotine-induced vasoconstriction is dominant, and vasodilation is observed after blockade of vasoconstriction (18).
However, in toad mesenteric arteries, the dominant effect of
nicotine is NO/CGRP-mediated vasodilation, and vasoconstriction is only revealed after inhibition of the vasodilator
signaling pathways.
Another interesting point of difference between mammals
and toad is the interaction between adrenergic and nitrergic
mechanisms in vascular control. In various mammalian blood
vessels, there is evidence that endothelium-independent, NOmediated vasodilation induced by nicotine is dependent on
perivascular adrenergic nerves, because the vasodilation is
abolished by guanethidine and chemical sympathectomy (18,
32, 59, 70). It is proposed that nicotine binds to receptors on
adrenergic terminals, causing the release of norepinephrine
(NE), which then binds to adrenergic receptors on adjacent
nitrergic nerves to initiate the release of NO. In rat mesenteric
arteries, a different mechanism is found. In these vessels, it is
postulated that NO is acting presynaptically to regulate NE
release, because in endothelium-denuded mesenteric arteries,
inhibition of nNOS augmented the adrenergic vasoconstriction
upon nerve stimulation, which was concluded to be due to
attenuation of NO inhibiting NE release from adrenergic
nerves (22). It appears in toad mesenteric arteries that the
nicotine-induced NO vasodilation is a direct effect that is not
dependent on interaction with adrenergic nerves, because pretreatment of the arteries with guanethidine did not attenuate the
NO vasodilation, as it does in mammals; in fact, a larger
vasodilation was observed.
The present data, in combination with our previous studies,
clearly show that NO control of toad (and teleost fish) blood
vessels is provided by nNOS in nitrergic perivascular nerves
and that an endothelial NO signaling system is absent. In fact,
in fish and amphibians, there is strong evidence that prostaglandins are key endothelial vasodilators, instead of NO (19,
28, 48). Interestingly, NO control of toad blood vessels shows
similarities to the NO signaling phenotype of eNOS knockout
mice. For example, the ACh-induced NO vasodilation of toad
mesenteric arteries resembles the ACh-induced dilation of pial
arterioles from eNOS knockout mice, in which the source of
NO is attributed to activation of nNOS in perivascular nerves.
It was proposed that nNOS compensates for the loss of eNOS
in pial arterioles, inasmuch as evidence for a role for nNOS is not
apparent in wild-type mice (36, 37); a similar finding was
reported in the coronary circulation of eNOS knockout mice
(13). Moreover, in eNOS knockout mice, neurally derived NO
(25) and endothelially derived prostaglandins (12, 60) have
been reported to compensate for the lack of endothelially
derived NO in different vascular beds, including the mesenteric
circulation (12). Thus it appears that the phenotype for vasodilation in eNOS knockout mice shows remarkable similarity
to that of lower vertebrates, where neurally derived NO and
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NITRIC OXIDE CONTROL OF TOAD MESENTERIC VASCULATURE
12.
13.
15.
16.
17.
18.
19.
20.
21.
22.
GRANTS
B. L. Jennings was supported by a Deakin University Postgraduate Award.
23.
DISCLOSURES
No conflicts of interest are declared by the author(s).
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