Nitric oxide regulation of the central aortae of the toad Bufo marinus occurs independently of the endothelium
School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria, Australia 3217
* Author for correspondence (e-mail: brsb{at}deakin.edu.au)
Accepted 24 June 2002
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Summary |
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Key words: nitric oxide, endothelial nitric oxide synthase, neural nitric oxide synthase, soluble guanylyl cyclase, vasodilation, aorta, cane toad, Bufo marinus
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Introduction |
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In mammals, the vasodilatory effect of NO is mediated via an
intracellular soluble guanylyl cyclase (soluble GC) located in the cytoplasm
of the vascular smooth muscle cells
(Chinkers and Garbers, 1991;
Schmidt et al., 1993
;
Lucas et al., 2000
). Once
activated, the soluble GC catalyses the conversion of guanosine triphosphate
(GTP) to cyclic guanosine monophosphate (cGMP). The subsequent increase in
cGMP concentration activates cGMP-dependent protein kinase, which lowers the
intracellular calcium in vascular smooth muscle cells to mediate a
vasodilatory response (Lucas et al.,
2000
).
Compared with mammals, there have only been a few studies in non-mammalian
vertebrates that have suggested that an endothelial NO system exists in the
vasculature of birds (Hasegawa and
Nishimura, 1991), reptiles
(Knight and Burnstock, 1993
),
and amphibians (Rumbaut et al.,
1995
; Knight and Burnstock,
1996
). The presence of an endothelial NO system in blood vessels
of fish remains controversial because of studies proposing both its existence
(Mustafa and Agnisola, 1998
;
Fritsche et al., 2000
) and
non-existence (Olson and Villa,
1991
; Kågström and
Holmgren, 1997
; Evans and
Gunderson, 1998
). Miller and Vanhoutte
(1986
) were the first to
demonstrate, in amphibians, that the vasodilatory effect of applied
acetylcholine in the vasculature of the American bullfrog Rana
catesbeiana is endothelium-dependent. In addition, the effect of
acetylcholine was reversed by methylene blue, which suggested that the
vasodilation was occurring via activation of a soluble GC. However,
it was the study by Knight and Burnstock
(1996
) that indicated that an
endothelial NO system is present in amphibians. These authors showed that in
pre-constricted aortic arches of the leopard frog Rana pipiens,
applied acetylcholine caused a vasodilatory response that was abolished or
greatly reduced by L-NAME (a NOS inhibitor), or when the endothelium was
removed. Further evidence for an endothelial NO system was found in the
mesenteric capillaries of R. pipiens, in which a NOS inhibitor
(L-NMMA) decreased the hydraulic conductivity compared to control levels
(Rumbaut et al., 1995
).
Interestingly, studies investigating the presence of a NO system in the vasculature of amphibians have primarily used in vitro organ bath experiments. There has, however, been no histochemical or immunohistochemical evidence in the literature to suggest the presence of NOS in the endothelium or perivascular nerves of amphibians. Therefore, the present study examined the mechanisms involved in NO-mediated vasodilation in the aortae of the toad Bufo marinus using physiological and anatomical approaches. The use of NADPH diaphorase histochemistry and immunohistochemistry using specific antibodies to eNOS and nNOS found no evidence to support the presence of an endothelial NO system, but it did show the presence of neural NOS immunoreactivity in the perivascular nerves. Concurrent physiological studies have provided further evidence that toad aortae contain a neural NO system rather than an endothelial NO system.
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Materials and methods |
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In vitro organ bath physiology
The lateral aortae and dorsal aorta were excised from toads and placed in
Mackenzie's balanced salt solution (115 mmol l-1 NaCl, 3.2 mmol
l-1 KCl, 20 mmol l-1 NaHCO3, 3.1 mmol
l-1 NaH2PO4, 1.4 mmol l-1
MgSO4, 16.7 mmol l-1 D-[+]glucose and 1.3 mmol
l-1 CaCl2, pH 7.2). Individual rings of approximately
4-5 mm in length were mounted horizontally between two hooks for the
measurement of isometric force, and placed in an organ bath. The rings were
bathed in 15 ml of Mackenzie's solution, which was maintained at 22°C and
aerated with 95% O2 and 5% CO2. The force transducer
(Grass-FT03) was linked to a MacLabTM data collection system and a
Macintosh computer, which recorded data for further analysis. An initial
tension of 0.5 g was applied to the blood vessels and they were allowed to
equilibrate for 30 min. In some experiments, the endothelium was removed by
rubbing with a toothpick, and the extent of removal was determined using NADPH
histochemistry. Prior to administering various vasodilatory substances, each
vessel was pre-constricted with endothelin-1 (10-8 mol
l-1), and vasoconstriction was allowed to reach its maximum. A
previous study has shown that 10-8 mol l-1
endothelin-1 elicits an appropriate vasoconstriction for studies of
vasodilatory mechanisms (Minerds and
Donald, 2001). The extent of vasodilation was determined by
scoring the degree of relaxation as a ratio, where dilation to
pre-constriction levels were set at 100%. For experiments, matched controls
were used from the same animal for comparison of drug effects. Data are
expressed as mean ± S.E.M. of five or more experiments, and statistical
analysis was performed with independent t-tests using the SPSS (9.0)
statistical package; P
0.05 was considered significant.
NADPH diaphorase histochemistry
The lateral aortae and dorsal aorta from toads and the descending aorta
from rats were dissected free and immersed in phosphate-buffered saline (PBS,
0.01 mol l-1 phosphate buffer and 0.15 mol l-1 NaCl, pH
7.4) at 4°C. Each vessel was opened and pinned out endothelium side up on
dental wax, prior to fixing for 1 h in 4% formaldehyde (pH 7.4) at 4°C.
The blood vessels were washed in 0.01 mol l-1 PBS (3x 10 min)
and removed from the dental wax. Blood vessels were stained in a NADPH
diaphorase mixture containing 1 mg ml-1 ß-NADPH, 0.25 mg
ml-1 nitroblue tetrazolium (NBT), 1% Triton X-100 in 0.1 mol
l-1 Tris buffer, pH 8, for times ranging from 15-60 min at
25°C. This mixture was kept in the dark, as it is light sensitive. The
vessels were then washed in 0.01 mol l-1 PBS and mounted on slides
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). Blood vessels were observed under a light microscope
(Olympus) and were photographed with a digital colour system (Spot 35 Camera
System). Control experiments were performed on the myenteric plexus of both
rats and B. marinus because previous studies have demonstrated that
neurons in the mammalian and amphibian myenteric plexuses showed positive
NADPH diaphorase staining (Wilhelm et al.,
1998; Li et al.,
1993
, respectively). The descending aorta of rats was used as a
control to demonstrate the presence of NOS in the vascular endothelium.
Immunohistochemistry
Blood vessels from toad and rat were fixed as described above. The blood
vessels were unpinned, washed in 0.01 mol l-1 PBS (3x 10
min), incubated in DMSO (3x 10 min) and washed in 0.01 mol
l-1 PBS (5x 2 min). The blood vessels were then incubated in
a polyclonal antibody raised against mouse endothelial NOS (1:1000;
O'Brien et al., 1995) or a
polyclonal antibody raised against sheep neural NOS (1:4000;
Anderson et al., 1995
) for 24 h
at room temperature in a humid box. The following day, tissues were washed in
0.01 mol l-1 PBS (3x 10 min) to remove any excess antibody
and incubated in a fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse IgG or FITC-conjugated goat anti-sheep IgG (1:200) (Zymed
Labratories, San Francisco, USA) for 3-4 h at room temperature in a humid box.
The blood vessels were then washed in 0.01 mol l-1 PBS (3x 10
min) and mounted in buffered glycerol. Blood vessels were observed under a
fluorescence microscope (Zeiss) using a FITC filter and photographed as
above.
Materials
Sodium nitroprusside (SNP), frog atrial natriuretic peptide (fANP),
acetylcholine, N-nitro-L-arginine (L-NNA),
atropine, hexamethonium, ß-nicotinamide adenine dinucleotide phosphate,
reduced form (ß-NADPH), NBT and Triton X-100 were obtained from Sigma (St
Louis, USA). Endothelin-1 (ET-1) was purchased from Auspep (Melbourne,
Australia), oxadiazole quinoxalin-1 (ODQ) and
L-N5-(1-imino-3-butenyl)-ornithine (vinyl-L-NIO) were
obtained from Alexis (San Diego, USA), and the NOS antibodies were obtained
from Chemicon (Melbourne, Australia).
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Results |
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In the lateral and dorsal aortae, the NO donor, SNP (10-4 mol
l-1), induced an average vasodilation of 66±4.48% and
85.6±3.25%, respectively (N=5,
Fig. 1). In addition, applied
acetylcholine (10-5 mol l-1) produced an average
vasodilatory response of 33±3.8% and 35.5±4%, respectively
(N=5, Fig. 2). The
addition of the soluble GC inhibitor, ODQ (10-5 mol
l-1), completely abolished the vasodilatory effect of SNP
(10-4 mol l-1) and applied acetylcholine
(10-5 mol l-1) in both aortae
(Fig. 3; N=5);
acetylcholine now caused a vasoconstriction. Subsequently, frog ANP
(10-8 mol l-1), which mediates vasodilation via
a particulate guanylyl cyclase (Minerds
and Donald, 2001), caused a vasodilation in the presence of ODQ
(not shown). This indicates that the vasodilatory effect of SNP and
acetylcholine is mediated solely via a soluble GC.
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In the presence of the NOS inhibitor L-NNA (10-4 mol l-1), the vasodilatory effect of applied acetylcholine was abolished in both aortae. However, as expected, SNP, which mediates vasodilation by releasing NO, produced a potent vasodilatory response (Fig. 4; N=5).
|
In endothelium-denuded lateral aortae, applied acetylcholine
(10-5 mol l-1) caused a vasodilation of 23.9±6.7%
compared to 29.4±9.2% in control vessels with an intact endothelium
(P=0.26, N=5; Fig.
5); a similar effect was found in the dorsal aorta (denuded;
28.7±1.9%; control 31.8±2.6%, P=0.35, N=5;
Fig. 5). These data show that
the removal of the endothelium had no significant effect on
acetylcholine-mediated vasodilation in the lateral and dorsal aortae. In the
lateral aortae incubated with vinyl-L-NIO (10-4 mol
l-1), a specific nNOS inhibitor
(Babu and Griffith, 1998),
applied acetylcholine (10-5 mol l-1) caused a
vasodilation of 21.1±5.9%, compared to 35.1±6.2% in control
vessels without vinyl-L-NIO (P<0.05, N=5;
Fig. 6); a similar effect was
found in the dorsal aorta (vinyl-L-NIO; 23.8±1.9%; control
38.9±4.9%, P<0.05, N=6;
Fig. 6). These data indicate
that vinyl-L-NIO significantly reduced the vasodilatory effect of applied
acetylcholine in both aortae.
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In both aortae, acetylcholine-mediated vasodilation was abolished in the presence of the muscarinic receptor antagonist, atropine (10-6 mol l-1), but the nicotinic receptor antagonist, hexamethonium (3x 10-5 mol l-1), had no effect (N=5, results not shown).
NADPH diaphorase histochemistry
In the lateral aortae and dorsal aorta, similar patterns of staining were
observed following NADPH diaphorase histochemistry (N=5). No specific
staining was observed in the endothelium of the toad aortae
(Fig. 7A). In contrast, the
endothelial cells of the rat aorta showed intense, perinuclear staining,
indicating the presence of NOS (Fig.
7C); the NADPH-diaphorase staining pattern in the rat aorta was
similar to that previously reported
(O'Brien et al., 1995). The
absence of specific NADPH-diaphorase staining in the toad endothelium was
found in all preparations incubated for 15, 20, 30 or 60 min. However,
NADPH-diaphorase staining was observed in the perivascular nerve fibres of the
outer layers of the wall of both aortae
(Fig. 8A,C). Specific staining
was observed in nerve bundles and single, varicose nerve fibres. There was no
distinct pattern in the distribution of nerve terminals in either blood
vessel, although it appeared that the lateral aorta contained more
NADPH-diaphorase positive nerves than the dorsal aorta.
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Endothelial NOS and neural NOS immunohistochemistry
To specifically identify the type of NOS present in the endothelium and
nerve fibres, eNOS and nNOS antibodies were used (N=3). Both
antibodies revealed identical patterns of staining in comparison to the NADPH
diaphorase histochemistry (Fig.
7B). In the toad aortae, eNOS immunoreactivity was absent in the
endothelial cells, but it was clearly present in the rat aorta
(Fig. 7D). Neural NOS
immunoreactivity was observed in the perivascular nerve fibres of the lateral
and dorsal aortae of toad (Fig.
8B,D). The nNOS immunoreactive fibres were also NADPH-diapharose
positive.
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Discussion |
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In mammals, it is well documented that acetylcholine mediates vasodilation
by indirectly activating NOS to produce NO (see
Moncada et al., 1991). Many
studies have shown that mammalian blood vessels are regulated by two types of
NO systems: the endothelial NO system, which is the predominant NO system, and
the neural NO system, which has only been identified in blood vessels that
supply the cerebral, pelvic and enteric regions
(Young et al., 2000
).
Accordingly, it can be difficult to determine the specific source of NO and,
prior to the recent development of gene knockout and drugs that selectively
block eNOS and nNOS, the only way that neurally derived NO could be
distinguished from endothelially derived NO was by examining vasodilation with
and without the endothelium (Young et al.,
2000
). In mammals, nerve fibres containing nNOS that innervate
blood vessels of the head and pelvic region have been identified as
parasympathetic neurons, but in mesenteric arteries nNOS is found in sensory
neurones (Young et al.,
2000
).
In comparison with mammals, NO regulation in the amphibian vasculature is
less well understood, although it has been proposed that an endothelial NO
system does exist (Knight and Burnstock,
1996). In the leopard frog R. pipiens, Knight and
Burnstock (1996
) demonstrated
that acetylcholine induced vasodilation. In addition, these authors showed
that the presence of L-NAME abolished the vasodilation caused by
acetylcholine, suggesting that acetylcholine mediates vasodilation
via NOS. This was also the case in the present study in which the
acetylcholine-mediated vasodilation of the aortae of the toad was abolished in
the presence of L-NNA, thus establishing the existence of NOS. To determine if
NOS was located in the endothelium, experiments were performed on
endothelium-denuded blood vessels. To date, this technique has been the
predominant method for determining whether or not the endothelium plays a role
in NO-mediated vasodilation in lower vertebrates
(Miller and Vanhoutte, 1986
;
Olson and Villa, 1991
;
Hasegawa and Nishimura, 1991
;
Knight and Burnstock, 1993
;
Knight and Burnstock, 1996
;
Evans and Gunderson, 1998
).
Knight and Burnstock (1996
)
demonstrated that damage to the endothelial layer abolished the
acetylcholine-mediated vasodilation in R. pipiens and, therefore,
proposed that acetylcholine was likely to be mediating vasodilation
via an endothelial NO system. This is in contrast to our findings in
the toad, which showed no significant difference between
acetylcholine-mediated vasodilation in the lateral and dorsal aortae with or
without the endothelium.
Although the physiological evidence suggested an endothelial NO system was
absent in the blood vessels of the toad, it was important to anatomically
support this finding. To date, there appears to be a lack of studies aimed at
detecting the presence of NOS in the vasculature of lower vertebrates using
anatomical methods. NADPH diaphorase histochemistry and immunohistochemistry
are two anatomical methods that have been widely used to identify the presence
of NOS in the mammalian vasculature
(Beesley, 1995); however, this
is the first study to use such techniques on the amphibian vasculature. The
results showed an absence of NADPH diaphorase staining in the endothelial
cells of the toad lateral aortae and dorsal aorta. In contrast, the
endothelium of the rat aorta (used as a control) showed perinuclear staining,
as previously demonstrated by O'Brien et al.
(1995
). This finding suggests
that toad aortae lack an eNOS and, therefore, do not contain an endothelial NO
system. Importantly, the results of the NADPH diaphorase histochemistry were
supported immunohistochemically by the use of a mammalian eNOS antibody. In
contrast to the perinuclear staining in the aorta of rat, the vascular
endothelium of the toad was devoid of any immunofluorescence. It was assumed
that the mammalian eNOS antibody would cross-react with eNOS if it was present
in the toad because Fritsche et al.
(2000
) demonstrated
cross-reactivity in the zebrafish Danio rerio using a mammalian eNOS
antibody.
Interestingly, NADPH diaphorase staining and nNOS immunoreactivity were
observed in the perivascular nerve terminals innervating the lateral aortae
and dorsal aorta of the toad. As mentioned above, blood vessels in the
cerebral, pelvic and enteric regions of mammals are innervated with nerve
fibres containing nNOS, but they have yet to be located within the systemic
vasculature. The only other non-mammalian study to demonstrate the presence of
nNOS positive nerves fibres in the vasculature was that in the estuarine
crocodile Crocodylus porosus
(Axelsson et al., 2001).
The presence of a neural NO system in the toad was verified by a
significant decrease in the acetylcholine-mediated vasodilation in the
presence of the nNOS inhibitor vinyl-L-NIO. This finding provides evidence
that nNOS is responsible for inducing vasodilation in the central vasculature
of B. marinus. In the mammalian cerebral vasculature, NO generated by
nNOS is an important regulator of vascular tone
(Meng et al., 1998).
Experiments on endothelium-denuded cerebral arteries show that NOS inhibitors
abolish or dramatically reduce vasodilations mediated by transmural electrical
stimulation, which suggests that nerves innervating cerebral arteries produce
NO (Toda and Okamura, 1990
).
More recently, eNOS knockout mice have been used to examine the role of nNOS
in the cerebral vasculature. For example, Meng et al.
(1998
) demonstrated that the
nNOS inhibitor 7-NI attenuated acetylcholine-mediated vasodilation in cerebral
blood vessels that do not express eNOS. Whether a neural NO system is the
primary regulator of cerebral arteries or it is just compensating for the
absence of an endothelial NO system remains to be elucidated. However, the
absence of an endothelial NO system in the toad suggests that a nNOS system is
the only source of NO that could elicit vasodilation.
The presence or absence of an endothelial NO system in fish and amphibians
is controversial. A study by Fritsche et al.
(2000) provided evidence for
the presence of an endothelial NO system in developing zebrafish. They showed
that the diameter of the dorsal artery and vein is decreased by L-NAME, which
indicates that there is a basal release of NO that is contributing to vascular
tone. In addition, they showed the presence of eNOS immunoreactivity using an
antibody to mammalian eNOS. Further evidence for an endothelial NO system in
fish was proposed by Mustafa and Agnisola
(1998
), who showed that NO
production and L-arginine-mediated vasodilation in the trout coronary
vasculature were endothelium dependent. However, several studies in fish could
not demonstrate the presence of an endothelial nitric oxide system
(Olson and Villa, 1991
;
Evans and Gunderson, 1998
;
Kågström and Holmgren,
1997
). In fact, these studies provide evidence for a prostaglandin
as the mediator of endothelium-dependent vasodilation in fish. In amphibians,
the present study has shown that eNOS is absent in the aortae of the toad and
that acetylcholine-dependent vasodilation is mediated by nNOS. The fact that
the findings of the current study differ from that of Knight and Burnstock
(1996
) in R. pipiens
cannot be explained and is unlikely to be as a result of phylogeny. The
present study provides a more comprehensive investigation into vascular NO
regulation because it provides both physiological and anatomical evidence for
an absence of an eNOS system. Further studies are required to characterise the
mechanisms by which NO contributes to the vascular regulation of non-mammalian
vertebrates.
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Acknowledgments |
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