Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 13, 2010). doi:10.1152/ajpregu.00148.2009
Nitric oxide control of toad mesenteric vasculature
Page 1
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,
3217
Running title: Nitric oxide control of toad mesenteric vasculature
*Corresponding author: Department of Pharmacology, College of Medicine, University of
Tennessee Health Science Centre, Memphis, TN 38163
Tel.: (901)-448-6010; Fax: (901)-448-7300
Email address: [email protected]
Copyright © 2010 by the American Physiological Society.
Nitric oxide control of toad mesenteric vasculature
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Abstract
This study determined the role of nitric oxide (NO) in neurogenic vasodilation in
mesenteric resistance arteries of the toad, Bufo marinus. Nitric oxide synthase (NOS) was
anatomically demonstrated only in perivascular nerves but not the endothelium.
Acetylcholine (ACh) and nicotine caused tetrodotoxin (TTX)-sensitive neurogenic
vasodilation of mesenteric arteries. The ACh-induced vasodilation was endotheliumindependent and was mediated by the NO/soluble guanylyl cyclase (GC) signalling pathway,
as the vasodilation was blocked by the soluble GC inhibitor, 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ), and the NOS inhibitors, Nω-Nitro-L-arginine methyl ester (LNAME) and Nω-Nitro-L-arginine (L-NNA). Furthermore, the ACh-induced vasodilation was
significantly decreased by the more selective neural NOS (nNOS) inhibitor, N5-(1-Imino-3butenyl)-L-ornithine (vinyl-L-NIO). The nicotine-induced vasodilation was endotheliumindependent and mediated by NO and calcitonin gene-related peptide (CGRP), as pretreatment of mesenteric arteries with a combination of L-NNA 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
either calcium-activated potassium (KCa) or voltage-gated potassium (KV) 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.
Keywords: amphibian; autonomic nervous system; nitric oxide synthase; endothelium
Nitric oxide control of toad mesenteric vasculature
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Introduction
In the mammalian mesenteric vasculature, it is well-established that the endothelium
releases numerous vasoactive molecules such as NO, prostaglandins, endothelium-derived
hyperpolarising 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). Nitric oxide is
clearly important in maintaining vasodilator tone of mesenteric arteries as inhibition of 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 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 staining and nNOS-immunoreactivity (nNOS-IR) have been demonstrated in
perivascular nerves (34, 35, 41, 49), but, isoform-specific localisation of NOS within the
amphibian mesenteric vasculature has not been performed. There is evidence in amphibians
that NO is involved in capillary fluid regulation as inhibition of NOS decreased 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 production of NO within the mesenteric circulation of
amphibians, it was assumed, based on the mammalian paradigm, that the capillary
endothelium was the source of NO (55).
To date, the majority of physiological studies in amphibians on NO control of the
circulation have focussed on large conductance blood vessels. Recently, we provided
Nitric oxide control of toad mesenteric vasculature
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evidence that the systemic (8, 9) and pulmonary (29) arteries of the toad, B. marinus, lack an
endothelial NO system, and instead, NO control is provided by nitrergic nerves. In addition,
we showed that toad cutaneous arteries are similarly regulated by NO released from nitrergic
nerves (29). However, it is unknown if an endothelial or neural NO signalling 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 if 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 endothelial and/or nNOS, and to investigate potential intracellular
mechanism(s) of NO signalling.
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 sex and a 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 ºC and were not fed during captivity (up to 1
month), but had ad libitum access to water. Prior to experimentation, animals were sacrificed
by decapitation, followed by pithing of the spinal cord. Sprague Dawley rats, Rattus
norvegicus, of both sex 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 with ad libitum access to water and rat chow. Prior to
experimentation, rats were euthanised with carbon dioxide overdose, according to standard
operating procedures at Deakin University.
Nitric oxide control of toad mesenteric vasculature
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NADPH-diaphorase histochemistry. The small intestine and attached mesentery or isolated
mesenteric arteries were dissected free and placed in ice-cold PBS. Following this, the
mesentery attached to the small intestine was stretched and pinned out on dental wax. All
tissues were fixed for 1 h in 4 % formaldehyde (pH 7.4) at 4 ºC, and then washed in PBS (3 x
10 min). Isolated mesenteric arteries were cryoprotected overnight in a solution containing
PBS, 30 % sucrose and 0.1 % sodium azide, placed into moulds containing OCT TissueTek®,
and frozen at -80 °C. Twelve µm sections 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 then stained in a NADPH-diaphorase mixture containing 1 mg/ml
β-NADPH, 0.25 mg/ml nitroblue tetrazolium, 1 % Triton X-100 in 0.1 mol l-1 TRIS buffer
(pH 8) for 15 min at 37 °C. This mixture was kept in the dark as it is light sensitive. Tissues
were again washed in PBS (3 x 5 min) and covered in buffered glycerol (0.5 mol l-1 Na2CO3
added dropwise to 0.5 mol l-1 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; see 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).
Neural NOS immunohistochemistry. Wholemounts 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 a nNOS antibody (polyclonal sheep, 1:4000, 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 in PBS (3 x 10 min) to remove
any excess primary antibody, and were incubated with FITC-conjugated donkey anti-sheep
Nitric oxide control of toad mesenteric vasculature
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IgG (1:200) for 2-3 h at room temperature in a humid box. Tissues were washed again in
PBS (3 x 10 min), mounted in buffered glycerol and observed and photographed using a point
scanning laser confocal microscope (LSM 510 META; Zeiss). Immunohistochemical
controls were performed by omission of primary antibody.
Dual-wire myography. After sacrifice, mesenteric arteries (internal diameter approximately
200 µm) 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, 20mM NaHCO3, 3.1mM 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 rings of 2-3 mm, mounted horizontally between two pieces of 40 µm wire,
which 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 were
maintained at 22 °C and aerated with 95 % O2 and 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 they were left to equilibrate for at least 30 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), mesenteric arteries were pre-constricted with the
prostaglandin H2-analogue, U-46619 (10-6 mol l-1), and vasoconstriction was allowed to reach
its maximum. The extent of vasodilation was determined as a percentage of the initial U46619-induced vasoconstriction. For experiments, matched controls were used from the same
animal for comparison of drug effects. In some experiments, the endothelium was removed
from the artery by rubbing with a wire, which was verified with standard haematoxylin and
eosin staining.
Nitric oxide control of toad mesenteric vasculature
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Statistical analysis. Data are expressed as mean + one standard error (SE) of a minimum of
five experiments, and statistical analysis was performed with paired samples t-tests using the
SPSS (14.0) statistical package; a P value ≤ 0.05 was considered significant.
Materials. Sodium nitroprusside (SNP), ACh, atropine, L-NAME, L-NNA, indomethacin,
esculetin, guanethidine monosulfate, clotrimazole, levcromakalim, β-NADPH, reduced form,
nitroblue tetrazolium and Triton X-100 were obtained from Sigma (St. Louis, USA). Rat
atrial natriuretic peptide (rANP) and CGRP(8-37) were purchased from Auspep (Melbourne,
Australia), and nicotine was purchased from BDH chemicals (Poole, UK). 3morpholinosydnonimine (SIN-1), ODQ, vinyl-L-NIO and glibenclamide were obtained from
Alexis® Biochemicals (San Diego, USA) and U-46619 and L-N6-(1-Iminoethyl)lysine (L-NIL)
were purchased from Cayman Chemical (Ann Arbor, USA). Tetrodotoxin was purchased
from Alomone labs (Jerusalem, Israel). The nNOS and FITC-conjugated donkey anti-sheep
IgG antibodies were obtained from Chemicon (Melbourne, Australia).
Results
Presence and distribution of NOS in the mesenteric circulation. No specific NADPHdiaphorase 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 fibres located throughout the mesentery (Fig. 1C, N = 3). Specifically,
NADPH-diaphorase positive varicose perivascular nerve fibres were observed running
parallel to mesenteric blood vessels (Fig. 1D). Neural NOS-IR was observed in single
varicose fibres in the outer media and adventitia of the blood vessels (Fig. 1E, F, N = 3). No
Nitric oxide control of toad mesenteric vasculature
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specific immunoreactivity was observed in tissues that were incubated in secondary antibody
only (results not shown).
Dual-wire myography. The physiology data are summarised in Table 1.
NO/cGMP signalling in toad mesenteric arteries. The NO donors, SNP (10-4 mol l-1; Fig. 2A)
and SIN-1 (10-4 mol l-1; Fig. 2B), mediated vasodilation of mesenteric arteries, indicating the
presence of a NO receptor. The SNP and SIN-1 vasodilations were blocked following preincubation with the soluble GC inhibitor, ODQ (10-5 mol l-1; Fig. 2C); generally, addition of
ODQ to the bath caused a constriction after application to the bath. Subsequent addition of
rANP (10-8 mol l-1), 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-1), a known activator
of nNOS in toad large systemic arteries (8), caused a vasodilation (Fig. 3A); this response was
blocked by pre-incubation with the muscarinic receptor antagonist, atropine (10-6 mol l-1;
Table 1). The ACh-induced vasodilation was completely blocked following pre-incubation of
mesenteric arteries with ODQ (10-5 mol l-1; Fig. 3B); subsequent addition of rANP (10-8 mol l1
) caused vasodilation (Fig. 3B). Pre-incubation of mesenteric arteries with the non-specific
NOS inhibitors, L-NAME (10-4 mol l-1; Fig. 3C), and L-NNA (10-4 mol l-1; Table 1), also
inhibited the ACh-induced vasodilation, but SNP (10-4 mol l-1), 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-1), a selective nNOS
inhibitor in mammalian NO signalling systems, caused a significant decrease in the ACh-
Nitric oxide control of toad mesenteric vasculature
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induced vasodilation (Fig. 3E), but inhibition of inducible NOS with L-NIL (10-5 mol l-1) had
no effect (Table 1).
Pre-incubation of the mesenteric arteries with a combination of indomethacin (10-5 mol l-1)
and esculetin (10-5 mol l-1), inhibitors of cyclooxygenase and lipoxygenase, respectively, had
no effect on the ACh-induced vasodilation (Table 1). Blockade of voltage-gated sodium
channels with TTX (10-6 mol l-1) significantly decreased the ACh-induced vasodilation (Table
1) and antagonism of sympathetic neurotransmission with guanethidine (10-6 mol l-1) had no
effect on the vasodilation (Table 1). Pre-incubation with clotrimazole (10-5 mol l-1), a nonspecific potassium channel inhibitor, caused a significant decrease in ACh-induced
vasodilation of the mesenteric arteries (Fig. 4), but the K+ATP channel inhibitor, glibenclamide
(10-5 mol l-1), did not affect the vasodilation following addition of ACh (10-5 mol l-1; Table 1).
Interestingly, levcromakalim (10-5 mol l-1), a K+ATP channel opener, did not affect vascular
tone.
Nicotine-induced vasodilation. Nicotine has previously been used to activate nNOS in toad
large arteries (17, 29). Nicotine (10-4 mol l-1) caused vasodilation of the mesenteric arteries
(Fig. 5A), which was significantly decreased following pre-incubation with ODQ (10-5 mol l-1;
Fig. 5B). Subsequent addition of SNP (10-4 mol l-1) to the ODQ-treated vessels was without
effect, but rANP (10-8 mol l-1) mediated vasodilation. Inhibition of NOS with L-NNA (10-4
mol l-1) also significantly decreased the vasodilation (Fig. 5C). A combination of L-NNA (104
mol l-1) and the CGRP receptor antagonist, CGRP(8-37) (10-6 mol l-1), completely blocked the
response to nicotine (10-4 mol l-1); in fact, a constriction was now observed (Fig. 5D). SNP
(10-4 mol l-1) caused vasodilation of the L-NNA/CGRP(8-37)-treated mesenteric arteries.
Finally, the nicotine-induced vasodilation was not affected by disruption of the endothelium
(Fig. 5E).
Nitric oxide control of toad mesenteric vasculature
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Inhibition of the cyclooxygenase and lipoxygenase pathways with a combination of
indomethacin (10-5 mol l-1) and esculetin (10-5 mol l-1), respectively, did not affect the
vasodilation (Table 1). Pre-incubation of the mesenteric arteries with TTX (10-6 mol l-1)
significantly decreased the nicotine-induced vasodilation (Table 1), and blockade of
sympathetic neurotransmission with guanethidine (10-6 mol l-1) caused a significant increase
in the vasodilation (Fig. 6). The nicotine-induced vasodilation was not affected following
pre-incubation with clotrimazole (10-5 mol l-1; Table 1) or glibenclamide (10-5 mol l-1; Table
1), respectively.
Discussion
Nitric oxide signals via activation of soluble GC and generation of the second messenger,
cyclic GMP, which in blood vessels generally mediates vasodilation (56). Intriguingly, NOmediated 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). Nitric oxide-mediated vasodilation is always found in amphibian blood vessels as
the NO donor, SNP, is a generic vasodilator (8, 9, 29, 57). The presence of a NO/cyclic GMP
signalling system was confirmed in toad mesenteric arteries as both 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, was investigated using NADPH-diaphorase histochemistry
and immunohistochemistry. No positive NADPH staining was 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 EST analyses have
demonstrated that eNOS is apparently not present in fish but first appears in amphibians
(Xenopus laevis see e.g. 14, accession no. AW765292; Ambystona tigrinum, see e.g. 53,
Nitric oxide control of toad mesenteric vasculature
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accession no. CN053573). Recently, our laboratory has sequenced a full-length eNOS cDNA
from the western clawed 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), 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 very similar pattern to that of
NADPH-positive 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 has been found to cause the release of NO from perivascular nitrergic
nerves in toad systemic vasculature (8, 9), presumably by neuronal signalling that activates
nNOS. In toad mesenteric arteries, applied 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 non-specific NOS inhibitors, L-NAME and L-NNA. Given the
presence of nitrergic nerves in the mesenteric arteries and the fact that endothelial removal
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 pre-incubation 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
Nitric oxide control of toad mesenteric vasculature
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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 CGRPcontaining nerves (39), but 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 vessels (17, 29). In the mesenteric arteries, nicotine caused an
endothelium-independent vasodilation because it was unaffected by pre-treatment 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, nicotineinduced vasodilation of large conductance vessels and the cutaneous artery of toad was
entirely caused by NO (17, 29). Subsequently, pre-incubation 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 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, as both CGRP and SP are colocalised in the nerves (39). A similar
situation was observed in the mammalian carotid arterial bed in which CGRP and SP are
colocalised in sensory nerves (67), but nicotine-induced vasodilation was mediated by the
release of CGRP rather than SP (51).
Nitric oxide control of toad mesenteric vasculature
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To determine if the vasodilatory responses induced by ACh and nicotine in toad mesenteric
arteries involved neurogenic mechanisms, TTX was used to inhibit voltage-gated sodium
channels. Pre-incubation 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
conducting arteries is also TTX-sensitive (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 (see 65), thus it
is not possible to explain why the nicotine effects in the toad vasculature show variable TTX
sensitivity.
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 a NO-mediated vasodilation by phentolamine and guanethidine (64). Thus, in
mammalian vascular beds, nicotine-induced vasoconstriction is dominant and vasodilation is
observed following blockade of vasoconstriction (18). However, in toad mesenteric arteries,
the dominant effect of nicotine is NO/CGRP-mediated vasodilation, and vasoconstriction is
only revealed following inhibition of the vasodilator signalling 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, NO-mediated 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
Nitric oxide control of toad mesenteric vasculature
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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 endotheliumdenuded 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 nicotineinduced NO vasodilation is a direct effect that is not dependent on interaction with adrenergic
nerves because pre-treatment of the arteries with guanethidine did not attenuate the NO
vasodilation as it does in mammals; in fact, a larger vasodilation was observed.
The current 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 signalling 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 signalling
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 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, both 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
Nitric oxide control of toad mesenteric vasculature
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vertebrates in which NO derived from nNOS and endothelial prostaglandins are the primary
vasodilators prior to the evolution of endothelial NO signalling 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 potassium channels (43). In mammalian mesenteric arteries, it has been shown
that NO-mediated vasodilation is caused, in part, by opening of KCa (11, 30, 38, 52, 62) and
KV (27, 68) channels. In toad, clotrimazole, an inhibitor of large conductance KCa (BKCa)
channels, intermediate conductance KCa (IKCa) channels and KV channels (see [58]),
significantly decreased the ACh-induced vasodilation that is attributed to NO. While the
exact potassium channel(s) involved in contributing to 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 both 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 cyclic GMPdependent, as has been previously reported in mammalian mesenteric arteries (11, 27, 69).
Nitric oxide released from nitrergic nerves following addition of nicotine would
presumably mediate vasodilation in the same manner as that released by ACh stimulation, but
pre-incubation 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
Nitric oxide control of toad mesenteric vasculature
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release of two vasodilators, NO and CGRP, whereby CGRP signals via a different
intracellular mechanism than NO. For example, CGRP has been shown to dilate mammalian
mesenteric arteries independently of both 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 K+ATP channels has also been reported to be involved in both NO- (21, 40)
and CGRP- (7, 42) mediated vasodilation of the mammalian mesenteric artery. However, in
toad mesenteric arteries, inhibition of K+ATP channels with glibenclamide had no effect on the
vasodilation induced by either ACh or nicotine. In fact, levcromakalim, a K+ATP channel
opener, had no effect on vascular tone, suggesting that toad mesenteric arteries may lack
K+ATP channels. Interestingly, levcromakalim caused vasodilation of the toad pulmonary and
cutaneous arteries (29), which demonstrates a heterogenous distribution of K+ATP 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 and
consequently blood pressure. In fishes, the blood vessels of cyclostomes and chondrichthyans
have few adrenergic nerves, and intriguingly, NO-mediated vasodilation is not apparent in
these vertebrate classes (47). In contrast, the first vertebrate class in which blood vessels have
an extensive sympathetic innervation is the teleost fishes (16), and NO vasodilation first
appears in this group (47). In fishes, the eNOS gene or cDNA cannot be found in any genome
or EST database but nNOS is ubiquitous in vertebrates and is the key NOS in the evolution of
NO control of the vasculature. This study, in combination with our previous research, has
Nitric oxide control of toad mesenteric vasculature
Page 17
demonstrated the critical role of nNOS in NO-mediated vasodilation of blood vessels of eel
and toad (8, 9, 28, 29). It appears that nNOS expression in vertebrate perivascular nerves
occurred in parallel with the development of sympathetic innervation, thus providing a
mechanism for rapid vasodilation to oppose sympathetic vasoconstriction. The next step in
understanding the evolution of NO vasodilation is to utilise the fish and amphibians genomes
in combination with physiology to delineate the co-evolution of the cellular mechanisms
underlying vasodilation, such as the role of K+ channels; K+ channels have been implicated in
cGMP-mediated vasodilation in fish (15), amphibians (this study, 29) and birds (45), but the
types of channels have only been deduced from pharmacological experiments.
Acknowledgements
Brett Jennings was supported by a Deakin University Postgraduate Award.
Nitric oxide control of toad mesenteric vasculature
Page 18
References
[1] Afsharimani B, Moezi L, Sadeghipour H, Rahimzadeh-Rofouyi B, Nobakht M,
Sanatkar M, Ghahremani MH, Dehpour AR. Effect of chronic lithium administration
on endothelium-dependent relaxation of rat mesenteric bed: role of nitric oxide. Can J
Physiol Pharmacol 85: 1038-1046, 2007.
[2] Ahlner J, Ljusegren ME, Grundström N, Axelsson KL. Role of nitric oxide and cyclic
GMP as mediators of endothelium-independent neurogenic relaxation in bovine
mesenteric artery. Circ Res 68: 756-762, 1991.
[3] Anderson CR, Furness JB, Woodman HL, Edwards SL, Crack PJ, Smith AI.
Characterisation of neurons with nitric oxide synthase immunoreactivity that project to
prevertebral ganglia. J Auton Nerv Syst 52: 107-116, 1995.
[4] Arnhold S, Antoine D, Bläser H, Bloch W, Andressen C, Addicks K. Nitric oxide
decreases microvascular permeability in bradykinin stimulated and nonstimulated
conditions. J Cardiovasc Pharmacol 33: 938-947, 1999.
[5] Babu BR, Griffith OW. N5-(1-Imino-3-butenyl)-L-ornithine: A neuronal isoform
selective mechanism-based inactivator of nitric oxide synthase. J Biol Chem 273: 88828889, 1998.
[6] Beesley JE. Histochemical methods for detecting nitric oxide synthase. Histochem J 27:
757-769, 1995.
[7] Brayden JE, Quayle JM, Standen NB, Nelson MT. Role of potassium channels in the
vascular responses to endogenous and pharmacological vasodilators. Blood Vessels 28:
147-153, 1991.
[8] Broughton BRS, Donald JA. Nitric oxide regulation of the central aortae of the toad
Bufo marinus occurs independently of the endothelium. J Exp Biol 205: 3093-3100, 2002.
Nitric oxide control of toad mesenteric vasculature
Page 19
[9] Broughton BRS, Donald JA. Nitric oxide control of large veins in the toad Bufo marinus.
J Comp Physiol B 175: 157-166, 2005.
[10] Busse R, Edwards G, Félétou M, Fleming I, Vanhoutte PM, Weston AH. EDHF:
bringing the concepts together. Trends Pharmacol Sci 23: 374-380, 2002.
[11] Carrier GO, Fuchs LC, Winecoff AP, Giulumian AD, White RE. Nitrovasodilators
relax mesenteric microvessels by cGMP-induced stimulation of Ca-activated K channels.
Am J Physiol Heart Circ Physiol 273: H76-H84, 1997.
[12] Chataigneau T, Félétou M, Huang PL, Fishman MC, Duhault J, Vanhoutte PM.
Acetylcholine-induced relaxation in blood vessels from endothelial nitric oxide synthase
knockout mice. Br J Pharmacol 126: 219-226, 1999.
[13] Chlopicki S, Kozlovski VI, Lorkowska B, Drelicharz L, Gebska A. Compensation of
endothelium-dependent responses in coronary circulation of eNOS-deficient mice. J
Cardiovasc Pharmacol 46: 115-123, 2005.
[14] Clifton S, Johnson SL, Blumberg B, Song J, Hillier L, Pape D, Martin J, Wylie T,
Underwood K, Theising B, Bowers Y, Person B, Gibbons M, Harvey N, Ritter E,
Jackson Y, McCann R, Waterston R, Wilson R. Accession no. AW765292,
Washington University Xenopus EST project, 1999.
[15] Dombkowski RA, Whitfield NL, Motterlini R, Gao Y, Olson KR. Effects of carbon
monoxide on trout and lamprey vessels. Am J Physiol Regul Integr Comp Physiol 296:
R141-R149, 2009.
[16] Donald JA. Autonomic nervous system. In: The physiology of fishes, edited by Evans
DH. Boca Raton, FL: CRC Press, 1998, p. 407-439.
[17] Donald JA, Broughton BRS. Nitric oxide control of lower vertebrate blood vessels by
vasomotor nerves. Comp Biochem Physiol A 142: 188-197, 2005.
Nitric oxide control of toad mesenteric vasculature
Page 20
[18] El-Mas MM, El-gowilly SM, Gohar EY, Ghazal A-RM. Pharmacological
characterization of cellular mechanisms of the renal vasodilatory effect of nicotine in rats.
Eur J Pharmacol 588: 294-300, 2008.
[19] Evans DH, Gunderson MP. A prostaglandin, not NO, mediates endothelium-dependent
dilation in ventral aorta of shark (Squalus acanthias). Am J Physiol Regul Integr Comp
Physiol 274: R1050-R1057, 1998.
[20] Gardiner SM, Compton AM, Bennett T, Palmer RMJ, Moncada S. Control of
regional blood flow by endothelium-derived nitric oxide. Hypertension 15: 486-492, 1990.
[21] Garland CJ, McPherson GA. Evidence that nitric oxide does not mediate the
hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery. Br J
Pharmacol 105: 429-435, 1992.
[22] Hatanaka Y, Hobara N, Honghua J, Akiyama S, Nawa H, Kobayashi Y, Takayama
F, Gomita Y, Kawasaki H. Neuronal nitric-oxide synthase inhibition facilitates
adrenergic neurotransmission in rat mesenteric resistance arteries. J Pharmacol Exp Ther
316: 490-497, 2006.
[23] He P, Liu B, Curry FE. Effect of nitric oxide synthase inhibitors on endothelial [Ca2+]i
and microvessel permeability. Am J Physiol Heart Circ Physiol 272: H176-H185, 1997.
[24] He P, Zeng M, Curry FE. Effect of nitric oxide synthase inhibitors on basal
microvessel permeability and endothelial cell [Ca2+]i. Am J Physiol Heart Circ Physiol
273: H747-H755, 1997.
[25] Huang A, Sun D, Shesely EG, Levee EM, Koller A, Kaley G. Neuronal NOSdependent dilation to flow in coronary arteries of male eNOS-KO mice. Am J Phsyiol
Heart Circ Physiol 282: H429-H436, 2002.
Nitric oxide control of toad mesenteric vasculature
Page 21
[26] Hutri-Kähönen N, Kähönen M, Jolma P, Wu X, Sand J, Nordback I, Ylitalo P,
Arvola P, Pörsti I. Control of mesenteric arterial tone in vitro in humans and rats.
Naunyn-Schmiedeberg’s Arch Physiol 359: 322-330, 1999.
[27] Irvine JC, Favaloro JL, Kemp-Harper BK. NO– activates soluble guanylate cyclase
and KV channels to vasodilate resistance arteries. Hypertension 41: 1301-1307, 2003.
[28] Jennings BL, Broughton BRS, Donald JA. Nitric oxide control of the dorsal aorta and
the intestinal vein of the Australian short-finned eel Anguilla australis. J Exp Biol 207:
1295-1303, 2004.
[29] Jennings BL, Donald JA. Neurally-derived nitric oxide regulates vascular tone in
pulmonary and cutaneous arteries of the toad, Bufo marinus. Am J Physiol Regul Integr
Comp Physiol 295: R1640-R1646, 2008.
[30] Khan SA, Mathews WR, Meisheri KD. Role of calcium-activated K+ channels in
vasodilation induced by nitroglycerine, acetylcholine and nitric oxide. J Pharmacol Exp
Ther 267: 1327-1335, 1993.
[31] Leckström A, Ahlner J, Grundström N, Axelsson KL. Involvement of nitric oxide
and peptides in the inhibitory non-adrenergic, non-cholinergic (NANC) response in bovine
mesenteric artery. Pharmacol Toxicol 72: 194-198, 1993.
[32] Lee TJF, Zhang W, Sarwinski S. Presynaptic β2-adrenoceptors mediate nicotineinduced NOergic neurogenic dilation in porcine basilar arteries. Am J Physiol Heart Circ
Physiol 279: H808-H816, 2000.
[33] Lei S, Mulvany MJ, Nyborg NC. Characterization of the CGRP receptor and
mechanisms of action in rat mesenteric small arteries. Pharmacol Toxicol 74: 130-135,
1994.
Nitric oxide control of toad mesenteric vasculature
Page 22
[34] Li ZS, Furness JB, Young HM, Campbell G. Nitric oxide synthase immunoreactivity
and NADPH diaphorase enzyme activity in neurons of the gastrointestinal tract of the
toad, Bufo marinus. Arch Histol Cytol 55: 333-350, 1992.
[35] Li ZS, Murphy S, Furness JB, Young HM, Campbell G. Relationships between nitric
oxide synthase, vasoactive intestinal peptide and substance P immunoreactivites in neurons
of the amphibian small intestine. J Auton Nerv Syst 44: 197-206, 1993.
[36] Meng W, Ayata C, Waeber C, Huang PL, Moskowitz MA. Neuronal NOS-cGMPdependent Ach-induced relaxation in pial arterioles of endothelial NOS knockout mice.
Am J Physiol Heart Circ Physiol 274: H411-H415, 1998.
[37] Meng W, Ma J, Ayata C, Hara H, Huang PL, Fishman MC, Moskowitz MA. ACh
dilates pial arterioles in endothelial and neuronal knockout mice by NO-dependent
mechanisms. Am J Physiol Heart Circ Physiol 271: H1145-H1150, 1996.
[38] Mistry DK, Garland CJ. Nitric oxide (NO)-induced activation of large conductance
Ca2+-dependent K+ channels (BKCa) in smooth muscle cells isolated form the rat
mesenteric artery. Br J Pharmacol 124: 1131-1140, 1998.
[39] Morris JL, Gibbins IL, Campbell G, Murphy R, Furness JB, Costa M. Innervation
of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptidecontaining neurons. Cell Tissue Res 243: 171-184, 1986.
[40] Murphy ME, Brayden JE. Nitric oxide hyperpolarizes rabbit mesenteric arteries via
ATP-sensitive potassium channels. J Physiol 486: 47-58, 1995.
[41] Murphy S, Li Z-S, Furness JB, Campbell G. Projections of nitric oxide synthase- and
peptide-containing neurons in the small and large intestines of the toad (Bufo marinus). J
Auton Nerv Syst 46: 75-92, 1993.
Nitric oxide control of toad mesenteric vasculature
Page 23
[42] Nelson MT, Huang Y, Brayden JE, Hescheler J, Standen NB. Arterial dilations in
response to calcitonin gene-related peptide involve activation of K+ channels. Nature 344:
770-773, 1990.
[43] Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in
arterial smooth muscle. Am J Physiol Cell Physiol 268: C799-C822, 1995.
[44] Nguyen LS, Villablanca AC, Rutledge JC. Substance P increases microvascular
permeability via nitric oxide-mediated convective pathways. Am J Physiol Regul Integr
Comp Physiol 268: R1060-R1068, 1995.
[45] Nishimura H, Yang Y, Hubert C, Gasc J-M, Ruijtenbeek K, De Mey J, Struijker
Boudier HAJ, Corvol P. Maturation-dependent changes of angiotensin receptor
expression in fowl. Am J Physiol Regul Integr Comp Physiol 285: R231-R242, 2003.
[46] Okamura T, Ayajiki K, Uchiyama M, Kagami K, Toda N. Mechanisms underlying
constrictor and dilator responses to perivascular nerve stimulation in canine lingual arteries.
Eur J Pharmacol 354: 43-50, 1998.
[47] Olson KR, Donald JA. Nervous control of circulation - The role of gasotransmitters,
NO, CO, and H2S. Acta Histochem 111: 244-256, 2009.
[48] Olson KR, Villa J. Evidence against nonprostanoid endothelium-derived relaxing
factor(s) in trout vessels. Am J Physiol Regul Integr Comp Physiol 260: R925-R933, 1991.
[49] Olsson C. Distribution and effects of PACAP, VIP, nitric oxide and GABA in the gut of
the African clawed frog Xenopus laevis. J Exp Biol 205: 1123-1134, 2002.
[50] Osborne PB. The innervation of the gastrointestinal tract and mesenteric arterial bed of
the toad Bufo marinus. PhD Thesis, University of Melbourne, Australia, 1991.
[51] Perren MJ, Connor HE, Beattie DT. NK1 and CGRP receptor-mediated dilatation of
the carotid arterial bed of the anaesthetized rabbit. Neuropeptides 30: 141-148, 1996.
Nitric oxide control of toad mesenteric vasculature
Page 24
[52] Plane F, Sampson LJ, Smith JJ, Garland CJ. Relaxation to authentic nitric oxide and
SIN-1 in rat isolated mesenteric arteries: variable role for smooth muscle hyperpolarization.
Br J Pharmacol 133: 665-672, 2001.
[53] Putta S, Smith JJ, Walker JA, Rondet M, Weisrock DW, Monaghan J, Samuels
AK, Kump K, King DC, Maness NJ, Habermann B, Tanaka E, Bryant SV, Gardiner
DM, Parichy DM, Voss SR. From biomedicine to natural history research: EST resources
for ambystomatid salamanders. BMC Genomics 5: 54, 2004.
[54] Rumbaut RE, Huxley VH. Similar permeability responses to nitric oxide synthase
inhibitors of venules from three animal species. Microvasc Res 64: 21-31, 2002.
[55] Rumbaut RE, McKay MK, Huxley VH. Capillary hydraulic conductivity is decreased
by nitric oxide synthase inhibition. Am J Physiol Heart Circ Physiol 268: H1856-H1861,
1995.
[56] Schmidt HHHW, Lohmann SM, Walter U. The nitric oxide and cGMP signal
transduction system: regulation and mechanism of action. Biochim Biophys Acta 1178:
153-175, 1993.
[57] Schwerte T, Printz E, Fritsche R. Vascular control in larval Xenopus laevis: the role of
endothelial-derived factors. J Exp Biol 205: 225-232, 2002.
[58] Shah MM, Miscony Z, Javadzadeh-Tabatabaie M, Ganellin CR, Haylett DG.
Clotrimazole analogues: effective blockers of the slow afterhyperpolarization in cultured
rat hippocampal pyramidal neurones. Br J Pharmacol 132: 889-898, 2001.
[59] Si M-L, Lee TJF. Presynaptic α7-nicotinic acetylcholine receptors mediate nicotineinduced nitric oxidergic neurogenic vasodilation in porcine basilar arteries. J Pharnacol
Exp Ther 298: 122-128, 2001.
Nitric oxide control of toad mesenteric vasculature
Page 25
[60] Sun D, Huang A, Smith CJ, Stackpole CJ, Connetta JA, Shesely EG, Koller A,
Kaley G. Enhanced release of prostaglandins contributes to flow-induced arteriolar
dilation in eNOS knockout mice. Circ Res 85: 288-293, 1999.
[61] Takenaga M, Kawasaki H, Wada A, Eto T. Calcitonin gene-related peptide mediates
acetylcholine-induced endothelium-independent vasodilation in mesenteric resistance
blood vessels of the rat. Circ Res 76: 935-941, 1995.
[62] Tanaka Y, Tang G, Takizawa K, Otsuka K, Eghbali M, Song M, Nishimaru K,
Shigenbou K, Koike K, Stefani E, Toro L. KV channels contribute to nitric oxide- and
atrial natriuretic peptide-induced relaxation of a rat conduit artery. J Pharmacol Exp Ther
317: 341-354, 2006.
[63] Toda N, Okamura T. Modification by L-NG-monomethyl arginine (L-NMMA) of the
response to nerve stimulation in isolated dog mesenteric and cerebral arteries. Jpn J
Pharmacol 52: 170-173, 1990.
[64] Toda N, Okamura T. Mechanism of neurally induced monkey mesenteric artery
relaxation and contraction. Hypertension 19: 161-166, 1992.
[65] Toda N, Okamura T. The pharmacology of nitric oxide in the peripheral nervous
system of blood vessels. Pharmacol Rev 55: 271-324, 2003.
[66] Tsuchiya K, Urabe M, Yamamoto R, Asada Y, Lee TJ-F. Effects of Nω-nitro-Larginine and capsaicin in neurogenic vasomotor responses in isolated mesenteric arteries of
the monkey. J Pharm Pharmacol 46: 155-157, 1994.
[67] Uddman R, Edvinsson L, Ekman R, Kingman T, McCulloch J. Innervation of the
feline cerebral vasculature by nerve fibres containing calcitonin gene-related peptide:
Trigeminal origin and co-existence with substance P. Neurosci Lett 62: 131-136, 1985.
Nitric oxide control of toad mesenteric vasculature
Page 26
[68] White RE, Owen MP, Stallone JN. Testosterone-induced vasorelaxation of rat
mesenteric microvasculature is K+ channel- and nitric oxide-dependent but estrogenindependent. FASEB J 21: 972.10, 2007.
[69] Winecoff AP, Jones LF, Carrier GO. Nitrovasodilators promote relaxation and
activation of large-conductance, calcium-dependent K channels in microvessels of rat
mesentery. FASEB J 9: A32, 1995.
[70] Zhang W, Edvinsson L, Lee TJ-F. Mechanism of nicotine-induced relaxation in the
porcine basilar artery. J Pharmacol Exp Ther 284: 790-797, 1998.
[71] Zheng Z, Shimamura K, Anthony TL, Travagli RA, Kreulen DL. Nitric oxide is a
sensory nerve neurotransmitter in the mesenteric artery of guinea pig. J Auton Nerv Syst
67: 137-144, 1997.
Nitric oxide control of toad mesenteric vasculature
Page 27
Table 1: Summary of the vasodilatory responses of toad mesenteric arteries to various
treatments associated with the NO signalling system. Values are mean percentages of the
initial constriction of the vessels ± SE of at least 5 experiments. * denotes significant
difference.
Treatment
SNP
SIN-1
Control
Control
Atropine
Control
ODQ*
Control
L-NAME
Control
L-NNA*
Control
+ CGRP(8-37)
Control
Endothelium denuded
L-NNA
Control
Vinyl-L-NIO*
Control
L-NIL
Control
Indomethacin/Esculetin
Control
TTX*
Control
Guanethidine*
Control
Clotrimazole*
Control
Glibenclamide
49 ± 7
34 ± 2
ACh
45 ± 3
41 ± 6
No response
44 ± 4
No response
34 ± 7
No response
44 ± 7
No response
Not tested
44 ± 8
39 ± 7
P = 0.71
44 ± 2
19 ± 1
P < 0.05
47 ± 4
44 ± 4
P = 0.48
48 ± 1
47 ± 6
P = 0.87
47 ± 7
34 ± 9
P < 0.05
44 ± 5
46 ± 6
P = 0.39
44 ± 1
28 ± 4
P < 0.05
45 ± 8
50 ± 6
P = 0.70
Nicotine
58 ± 6
Not tested
55 ± 7
17 ± 4
P < 0.05
Not tested
53 ± 3
19 ± 5
P < 0.05
55 ± 12
Constriction
57 ± 6
56 ± 6
P = 0.97
Not tested
Not tested
54 ± 6
55 ± 10
P = 0.99
55 ± 4
25 ± 6
P < 0.05
63 ± 4
78 ± 4
P < 0.05
60 ± 8
54 ± 9
P = 0.75
58 ± 6
57 ± 8
P = 0.86
Nitric oxide control of toad mesenteric vasculature
Page 28
Figure Legends
Fig. 1. Photomicrographs showing sectioned preparations of toad (A, F) and rat (B)
mesenteric arteries, and wholemount preparations of toad mesentery (C-E), following
processing for NADPH-diaphorase histochemistry (A-D) and nNOS immunohistochemistry
(E, F). No punctate NOS-positive staining was observed in endothelial cells of toad
mesenteric arteries (A, arrow). In contrast, in the rat artery (B), punctate, NOS-positive
staining (arrowhead) occurred around the nuclei (arrow) of the endothelial cells. Positive
NADPH-diaphorase staining and nNOS-IR was observed in perivascular nerve fibres (C-E,
arrows) and nerve bundles (C, arrowhead) throughout the mesentery. In addition, nerve fibres
displaying nNOS-IR were observed in the adventitia and media of the blood vessels (F,
arrows). Scale bars, (A, B, 10 µm; C-F, 50 µm).
Fig. 2. Representative tension recordings showing the vasodilatory effect of the NO donor
compounds, SNP (10-4 mol l-1; A) and SIN-1 (10-4 mol l-1; B), and the effect of the soluble GC
inhibitor, ODQ (10-5 mol l-1; C), on toad mesenteric artery. The artery was pre-incubated with
ODQ for approximately 10 min prior to pre-constriction with U-46619 (10-6 mol l-1). ODQ
completely blocked the vasodilations induced by SNP and SIN-1; subsequent addition of
rANP (10-8 mol l-1), which mediates vasodilation via a particulate GC, caused a marked
vasodilation (C). Addition of ODQ caused an initial vasoconstriction prior to pre-constriction.
Fig. 3. Representative tension recordings showing the vasodilatory effect of ACh (10-5 mol l-1;
A) on the mesenteric artery that is completely blocked in the presence of ODQ (10-5 mol l-1;
B), and the NOS inhibitor, L-NAME (10-4 mol l-1; C), but unaffected following endothelial
disruption (D), and significantly decreased in the presence of the more selective nNOS
inhibitor, vinyl-L-NIO (E). In B, C and E, blood vessels were pre-incubated with the
Nitric oxide control of toad mesenteric vasculature
Page 29
inhibitors for approximately 10 min, and in D the endothelium was disrupted prior to preconstriction with U-46619 (10-6 mol l-1). In B, initial application of ODQ to the bath caused a
vasoconstriction prior to the addition of U-46619 and subsequent addition of rANP (10-8 mol
l-1) caused vasodilation. In C, subsequent addition of SNP (10-4 mol l-1), which acts
independently of NOS, also mediated vasodilation.
Fig. 4. Representative tension recordings showing the vasodilatory effect of ACh (10-5 mol l-1;
A) on the mesenteric artery, which is significantly decreased in the presence of the nonspecific potassium channel inhibitor, clotrimazole (10-5 mol l-1; B). The artery was preincubated with clotrimazole for approximately 10 min prior to pre-constriction with U-46619
(10-6 mol l-1).
Fig. 5. Representative tension recordings showing the vasodilatory effect of nicotine (10-4
mol l-1; A) on the mesenteric artery, which is significantly decreased in the presence of ODQ
(10-5 mol l-1; B) and L-NNA (10-4 mol l-1; C), and completely blocked in the presence of a
combination of L-NNA (10-4 mol l-1) and the CGRP receptor antagonist, CGRP(8-37) (10-6 mol
l-1; D). The response to nicotine was unaffected by disruption of the endothelium (E). In B,
C and D, the arteries were pre-incubated with the inhibitors for approximately 10 min prior to
pre-constriction with U-46619 (10-6 mol l-1). In B, addition of SNP (10-4 mol l-1) was without
effect, but subsequent addition of rANP (10-8 mol l-1) caused vasodilation. In C, subsequent
addition of SNP (10-4 mol l-1) caused vasodilation, and in D, nicotine actually caused a
constriction, but, addition of SNP (10-4 mol l-1) still caused vasodilation.
Fig. 6. Representative tension recordings showing the vasodilatory effect of nicotine (10-4
mol l-1; A) on the mesenteric artery, which is significantly increased in the presence of the
Nitric oxide control of toad mesenteric vasculature
Page 30
sympathetic neurotransmission inhibitor, guanethidine (10-6 mol l-1; B). The artery was preincubated with guanethidine for approximately 30 min prior to pre-constriction with U-46619
(10-6 mol l-1).
Nitric oxide control of toad mesenteric vasculature
Figure 1
A B
C D
E F
Nitric oxide control of toad mesenteric vasculature
Figure 2
A
SNP
U-46619
2 min 2 mN
B
SIN -1
U-46619
2 min 2 mN
SNP
C
rANP
SIN-1
U-46619
ODQ
Nitric oxide control of toad mesenteric vasculature
Figure 3
A
ACh
U-46619
B
rANP
U-46619
ACh
ODQ
C
4 min 2 mN
U-46619
ACh
SNP
L-NAME
D
ACh
Endothelium Denuded Artery
U-46619
E U-46619
U-46619
ACh
Vinyl-L-NIO
4 min 2 mN
Nitric oxide control of toad mesenteric vasculature
Figure 4
A
ACh
4 min 2 mN
U-46619
B
U-46619
Clotrimazole
ACh
Nitric oxide control of toad mesenteric vasculature
Figure 5
A
Nicotine
U-46619
rANP
B
U-46619
Nicotine
SNP
ODQ
4 min 2 mN
C
U-46619
Nicotine
SNP
L-NNA
D
U-46619
SNP
Nicotine
L-NNA/CGRP(8-37)
4 min 2 mN
E
Nicotine
Endothelium Denuded Artery
U-46619
Nitric oxide control of toad mesenteric vasculature
Figure 6
A
Nicotine
U-46619
B
5 min 2 mN
U-46619
Nicotine
Guanethidine