Angiotensin II-induced inotropism requires an endocardial endothelium-nitric oxide mechanism in the in-vitro heart of Anguilla anguilla
1 Department of Cellular Biology, University of Calabria, 87030, Arcavacata di
Rende, CS, Italy
2 Department of Pharmaco-Biology, University of Calabria, 87030, Arcavacata di
Rende, CS, Italy
3 Zoological Station `A. Dohrn', Villa Comunale, 80121 Naples, Italy
* Author for correspondence (e-mail: tota{at}unical.it)
Accepted 28 April 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: fish heart, angiotensin, nitric oxide, endocardial endothelium, FrankStarling response, European eel, Anguilla anguilla
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both ANG II receptors have been identified in the heart. Cardiac
AT1 is responsible for most of the ANG II-mediated effects on
cardiac performance (i.e. chronotropism and inotropism) and rate of protein
synthesis in isolated myocyte preparations
(Schorb et al., 1993).
AT1 receptors belong to the superfamily of
seven-transmembrane-spanning-domain receptors and are coupled to a classical
second messenger system via G-protein. The main post-receptor signal
transduction pathways include activation of the slow Ca2+ channel
(Freer et al., 1976
),
acceleration of phosphoinositide hydrolysis
(Baker and Aceto, 1989
;
Baker et al., 1989
) and
stimulation of nitric oxide synthase activity
(Paton et al., 2001
).
In fish, RAS components have been identified in teleosts
(Nishimura, 1985;
Olson, 1992
) and elasmobranchs
(Kobayashi and Takei, 1996
).
In bony fish, the RAS is active in multiple effector systems and there are
numerous examples of parallel actions in ANG II-mediated responses in teleosts
and mammals (for references, see Kobayashi
and Takei, 1996
). Several fish ANG II receptors are now being
cloned (Marsigliante et al.,
1996
; Tran van Chuoi et al.,
1999
). An eel angiotensin receptor cDNA sequence in the GenBank
(Accession no. AJ05132; Tran Van Chuoi et
al., 1999
) shows 60% homology with the mammalian AT1
receptor (Russell et al.,
2000
). In the cardiovascular system, species-specific ANG II
effects have been documented in teleosts, most notably in synergy with the
adrenergic system (Oudit and Butler,
1995
; Bernier and Perry,
1999
). ANG II has been found to exert both direct and indirect
(i.e. via cardiac adrenoceptors) stimulatory effects on the heart of
the American eel Anguilla rostrata and of the trout Oncorhynchus
mykiss (Oudit and Butler,
1995
; Bernier et al.,
1999
). Evidence for an intracardiac RAS in teleosts comes from
angiotensin-converting enzyme (ACE) activity in the ventricles of a variety of
species (Lipke and Olson,
1988
). However, apart from these few studies, no data have been
obtained in the fish heart about the direct effects of ANG II, the site(s) of
action of the hormone, either local or systemic, and the signal transduction
mechanisms involved. Eels represent an extraordinary example of flexibility in
hydromineral and cardiovascular regulation and therefore the cardiac role of
ANG II in these organisms may be of particular interest.
The aim of this study was to analyse the role of ANG II in modulating cardiac performance in isolated and perfused working heart preparations of the European eel Anguilla anguilla both under basal (i.e. non-stimulated) conditions and after chemical and mechanical stimuli.
As in a previous study (Imbrogno et
al., 2001), we used juvenile eel hearts with a compact outer
ventricular layer and a poorly developed coronary circulation. With this
system, one may analyse the effects of cardioactive substances such ANG II
without interference from the coronary vasculature and also explore the
involvement of the endocardial endothelium (EE), i.e. the single-cell-thick
lining of the cardiac chambers, in sensing and transducing ANG II stimuli. We
previously reported that in the eel heart the EE acts as an endoluminal
modulator of mechanical performance by way of a nitric oxide (NO)-cGMP
mechanism (Imbrogno et al.,
2001
). We now demonstrate that ANG II exerts a direct suppressive
effect on eel heart performance; this effect involves AT1-like
receptors, Gi/o proteins and the cholinergic system, and occurs
via an EE-NO-cGMP-cGMP-activated protein kinase (PKG) cascade.
Preliminary results of this study have been presented in abstract form
(Imbrogno et al., 2002
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Measurements and calculations
Pressure was measured through T-tubes placed immediately before the input
cannula and after the output cannula, and connected to two MP-20D pressure
transducers (Micron Instruments, Simi Valley, CA, USA) in conjunction with a
Unirecord 7050 (Ugo Basile, Comerio, Italy). Pressure measurements (input and
output) (kPa) were corrected for cannula resistance. Heart rate
(fH) was calculated from pressure recording curves.
Cardiac output was collected over 1 min and weighed; values were corrected for
fluid density and expressed as volume measurements. The afterload (mean aortic
pressure) was calculated as two-thirds diastolic pressure plus one-third
maximum pressure. Stroke volume (cardiac output/heart rate,
VS, in ml kg-1) was used as a measure of
ventricular performance; changes in VS were considered to
be inotropic effects. Cardiac output and VS were
normalized per kg wet body mass. Ventricular stroke work
[WS; mJ g-1; (afterload
preload)xVS/ventricle mass] served as an index of
systolic functionality.
Experimental protocols
Basal conditions
Isolated perfused hearts were allowed to maintain a spontaneous rhythm for
up to 15-20 min. In all experiments the control conditions were a mean output
pressure of approximately 3.00 kPa, with a cardiac output rate set to 10 ml
min-1 kg-1 body mass by appropriately adjusting the
filling pressure. These values are within the physiological range (for
references, see Imbrogno et al.,
2001). Cardiac variables were simultaneously measured during
experiments. To analyse the inotropic effects as distinct from the
chronotropic actions of substances, the preparations were electrically paced.
Hearts that did not stabilize within 20 min from the onset of perfusion were
discarded.
Drug application
After the 15-20 min control period, both spontaneously beating and paced
hearts were perfused for 20 min with Ringer's solution enriched with ANG II at
increasing concentrations to construct cumulative concentrationresponse
curves. We used the homologue teleost octapeptide ANG II
(Oudit and Butler, 1995).
Paced heart preparations were used to test the effects of 10-8 mol l-1 of ANG II in the presence of the ANG II receptor antagonists [CGP42112, Losartan, Candesartan (CV11974)], the specific NOS substrate L-arginine, the NO scavenger haemoglobin, the NOS inhibitors [L-N5(1-iminoethyl)ornithine (L-NIO) and NG-monomethyl-L-arginine (L-NMMA)], the soluble guanylate cyclase (GC) specific inhibitor [1H-(1,2,4)oxadiazole-(4,3-a)quinoxalin-1-one (ODQ)], and after inhibition of protein kinase G (PKG) by KT5823. The effects of ANG II (10-8 mol l-1) were also analysed after pre-treatment with isoproterenol (ISO), phenylephrine, propanolol, phentolamine, sotalol and atropine. In the above-mentioned protocols the hearts were perfused for 20 min with Ringer's solution enriched with the specific drug at the given concentrations before the addition of ANG II.
In another set of experiments the effects of ANG II (10-8 mol l-1) were tested after inhibition of G-proteins by pertussis toxin (PTx); in this case the hearts were pre-incubated for 60 min with PTx (10-11 mol l-1).
The effect of ANG II (10-8 mol l-1) was also studied
after inducing functional damage of the ventricular EE with the detergent
Triton X-100. 0.1 ml of 0.05% Triton X-100 was injected through a needle
inserted into the posterior ventral region of the ventricular wall to avoid
damage to the atrium (for further details, see
Imbrogno et al., 2001). At
this concentration the detergent does not affect the subjacent myocardium, as
assessed by viability tracer and confocal microscopy
(Sys et al., 1997
).
FrankStarling response
To study the interaction between ANG II and the FrankStarling
response, we generated a Starling curve (baseline condition) by varying the
atrial reservoir height to alter the preload on the in-vitro heart.
After baseline assessment, the atrial reservoir height was returned to basal
conditions and a second Starling curve (untreated time-control) was generated.
These time-control curves were compared with Starling curves constructed in
the presence of ANG II.
Statistics
Percentage changes were evaluated as means ±
S.E.M. of percentage changes obtained from
individual experiments. Because each heart acted as its own control, the
statistical significance of differences was assessed using the paired
Student's t-test (P<0.05). We used the Student's
t-test on absolute values for within-group comparisons of the
Starling curves; between-group comparisons were made using two-way analysis of
variance (ANOVA). Significant differences from the time-control group were
detected with Duncan's multiple-range test.
Drugs and chemicals
All the solutions were prepared in double-distilled water [except for ODQ,
which was prepared in ethanol]; dilutions were made in Ringer's solution
immediately before use. ANG II, CGP42112, Losartan, L-arginine,
haemoglobin, L-NIO, L-NMMA, PTx, Triton X-100, ISO,
phenylephrine, propanolol, phentolamine, sotalol and atropine sulphate salt
were purchased from Sigma Chemical Company (St Louis, MO, USA). KT5823 (used
in a darkened perfusion apparatus to prevent degradation) was purchased from
Calbiochem (Milan, Italy). Candesartan (CV11974) was a generous gift from
Takeda Pharmaceutical Company, Ltd. (Osaka, Japan).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of ANG II on basal cardiac performance
A concentrationresponse curve of the effect of ANG II
(10-10 mol l-1 to 10-7 mol l-1) on
spontaneously beating eel heart preparations revealed that
fH was significantly reduced at concentrations as low as
10-9 mol l-1 (Fig.
1). In electrically paced preparations, ANG II (at concentrations
from 10-11 to 10-7 mol l-1) induced a
negative inotropism, as shown by a significant decrease in
VS and WS only at higher
concentrations (10-8 and 10-7 mol l-1)
(Fig. 2). These effects
appeared between 5 and 10 min of exposure of the preparation to ANG II.
|
|
Transducing receptors and G-protein interactions
To identify the receptors involved in the ANG II-dependent inotropic
response we used a classic mammalian AT1 antagonist, losartan, and
CV11974 and CGP42112, two AT1 and AT2 antagonists,
respectively, which are selective toward non-mammalian cardiac
(Cerra et al., 2001) and
non-cardiac (Tierney et al.,
1997
; Hazon et al.,
1997
) ANG II receptors. The negative inotropism induced by ANG II
(10-8 mol l-1) was not affected by either losartan
(10-6 mol l-1) or CGP42112 (10-6 mol
l-1), whereas it was abolished by CV11974 (10-7 mol
l-1) (Fig. 3). These
results implicate an AT1-like receptor in the effects of ANG II on
the heart. The AT1 receptor belongs to the guanine
nucleotide-binding protein (G protein)-coupled receptor superfamily
characterised by seven-transmembrane segments
(Sasaki et al., 1991
). To
examine whether G proteins mediate the inhibitory inotropic action of ANG II
(10-8 mol l-1), the cardiac preparations were
pre-incubated with pertussis toxin (PTx; 10-11 mol l-1),
which uncouples signal transduction between several families of receptors and
Gi or Go proteins (Ai
et al., 1998
and references therein). PTx alone did not influence
mechanical performance (data not shown), whereas pre-treatment with the toxin
abolished the effects of ANG II (Fig.
4), suggesting that they are mediated by the G protein system.
|
|
Involvement of the adrenergic and cholinergic systems
It has been suggested that the cardiovascular effects of ANG II in teleosts
may be mediated by catecholamines (Oudit
and Butler, 1995; Bernier and
Perry, 1999
) or by modulation of cholinergic tone
(Reid, 1992
). We assessed the
relative contributions of cholinergic and adrenergic activation to the cardiac
effects induced by ANG II (10-8 mol l-1) in A.
anguilla. Pre-treatment with atropine (an unspecific muscarinic
antagonist; 10-6 mol l-1) abolished the negative effects
of ANG II on VS and WS
(Fig. 4). In contrast,
treatment with both adrenoceptor antagonists phentolamine (10-8 mol
l-1), propanolol (10-8 mol l-1) and sotalol
(10-7 mol l-1) and agonists phenylephrine
(10-9 mol l-1) and ISO (10-9 mol
l-1), did not modify the ANG II-mediated inotropic response
(Fig. 5).
|
Involvement of an EE-NO-cGMP-PKG signal transduction pathway
Nitric oxide, via activation of GC, is an important modulator of
cardiac performance in the working eel heart in vitro
(Imbrogno et al., 2001). There
is evidence of cross talk between ANG II and nitric oxide synthase (NOS) in
the downstream transduction cascade activated by AT1
(Paton et al., 2001
). To
analyse whether the ANG II response involves a NO-cGMP pathway, the paced
preparations were pre-treated with the natural NOS substrate
L-arginine (10-6 mol l-1), the NO scavenger
haemoglobin (10-6 mol l-1), the NOS inhibitors
L-NIO and L-NMMA (10-5 mol l-1)
and the guanylyl cyclase blocker ODQ (10-5 mol l-1). The
inotropic effect of ANG II (10-8 mol l-1) was
significantly enhanced in the presence of L-arginine, but it was
abolished by haemoglobin, L-NIO, L-NMMA and ODQ
(Fig. 6).
|
cGMP modulates cardiac contractility through several intramyocardial
mechanisms, one of which is via activation of a cGMP-PKG pathway
(Hove-Madsen et al., 1996). We
studied the ANG II inotropic response before and after treatment with a
specific inhibitor of PKG (KT5328, 10-7 mol l-1). This
treatment reduced the inotropic effect of ANG II
(Fig. 7), which suggests that
the NO-cGMP-PKG pathway plays a role in the effects of ANG II on the
heart.
|
The avascular heart of A. anguilla possesses a highly trabeculated
ventricle with an extensive EE surface which, being an important source of NO,
modulates cardiac performance (Imbrogno et
al., 2001). The EE impairment caused by Triton X-100 (0.05%), a
detergent which, at this concentration, damages the EE functionally but not
structurally (see Sys et al.,
1997
), abolished the ANG II (10-8 mol
l-1)-mediated inotropic effects
(Fig. 6), thereby implicating
EE in the transduction of endoluminal ANG II signalling.
ANG II and the FrankStarling response
Intracardiac NO increases the sensitivity of the in-vitro eel
heart to filling pressure changes, i.e. to the FrankStarling response
(preload-induced increases in cardiac output at constant afterload and heart
rate; see Imbrogno et al.,
2001). The influence of ANG II on the FrankStarling
response of the isolated and perfused eel heart was studied by increasing the
preload (see Materials and methods) in the presence and absence of ANG II
(10-8 mol l-1). Two-way ANOVA showed no significant
differences between the FrankStarling curves obtained with and without
ANG II (Fig. 8). To separate
the time factor (i.e. the heart's `memory') of loading stimulation, we
generated baseline and time-control curves. ANOVA showed that the curves were
identical within the limits of experimental error.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of ANG II on basal cardiac performance
Exogenous ANG II exerted direct chronotropic and inotropic effects on the
isolated working heart of freshwater A. anguilla. In spontaneously
beating heart preparations, ANG II induced negative chronotropism, which
became significant at a concentration of 10-9 mol l-1.
Nanomolar concentrations of ANG II had a negative chronotropic effect in
pacemaker cells of rabbit through modulation of the L-type
Ca2+ current (Habuchi et al.,
1995). In electrically paced preparations, ANG II at
10-8 mol l-1 and 10-7 mol l-1
caused a significant decrease in VS and
WS, indicating a direct negative modulation of mechanical
performance. The cardiac effects of ANG II are species-specific in mammals.
For example, ANG II elicits positive inotropism in dogs, cats, rabbits,
chickens and humans, while it has no effect in rats or guinea-pigs
(Ai et al., 1998
, and
references therein). The mechanisms whereby ANG II exerts its effect, however,
are only partially understood (Meulemans
et al., 1990
). Results obtained with mammalian ventricular
myocardia suggest that this wide range of species variations in ANG
II-mediated inotropism is due either to different intracardiac endocrine
stores, e.g. the release of catecholamines responsible for the indirect
positive inotropism of the peptide in dog papillary muscle
(Drimal and Boska, 1973
), or
to different AT receptor patterns, tissue localization and coupling to
divergent facilitatory or inhibitory intracellular signal-transduction
pathways (Nishimura,
2001
).
There are very few studies on the cardiotropic effects of ANG II in fish.
In conscious freshwater Anguilla rostrata, physiological doses of the
homologous peptide increased cardiac output mainly by increasing
VS, a finding that was attributed to positive inotropism
and/or to the FrankStarling mechanism
(Oudit and Butler, 1995).
Using in situ heart preparations, Bernier and Perry
(1999
) observed that rapid
changes in systemic vascular resistance and slower longer-lasting changes in
cardiac output contributed to the ANG II-mediated vasopressor responses in
Oncorhynchus mykiss and A. rostrata. However, in the latter
species, unlike in the trout, changes in cardiac output were responsible for
the indirect adrenergically mediated vasopressor action of ANG II. The
discrepancy between these and our results might be due to species-related
differences, as documented in mammals, and/or to the organizational level
under study (i.e. intact cardiovascular system or in situ heart
versus isolated and denervated working heart) and functional
interactions among its key components. For example, in A. anguilla
the ANG II-mediated cardio-suppressive effect observed at heart level in
vitro could be overridden in vivo by an overall cardiovascular
excitatory stimulation due to the synergism of the adrenergic and RAS
pathways, both of which are activated under stress and emergency conditions
(for the vascular district, see Hazon et
al., 1995
). The intriguing possibility that ANG II exerts
divergent cardiovascular effects at local and systemic levels, i.e. local
cardio-inhibitory protection versus systemic cascades of convergent
excitatory stimuli targeting the heart, would not be surprising in view of the
recently proposed concept of a counter-regulatory hormone in `zero
steady-state error' homeostasis (Koeslag
et al., 1999
). Additional studies are needed to test this
hypothesis.
Transducing receptors and G protein interactions
We used the AT1-selective receptor antagonists losartan and
CV11974 and the AT2-selective antagonist CGP42112 to identify the
receptor subtypes involved in the ANG II inotropic response. In agreement with
functional studies conducted with teleosts
(Olson et al., 1994;
Bernier and Perry, 1997
;
Cobb et al., 1999
), we found
that losartan did not prevent the ANG II-mediated effects. This finding can be
explained by the functional restrictions of the antagonist site of the
receptor (GenBank Accession No. AJ05132;
Tran van Chuoi et al. 1999
)
that in the eel does not contain residues associated with losartan binding
(Russell et al., 2000
). For
this reason we used as AT1 antagonist CV11974, which blocks the
vascular ANG II-mediated effects in vivo and in vitro
(Maillard et al., 2002
) and
also binds cardiac ANG II receptors in an elasmobranch
(Cerra et al., 2001
). CV11974,
but not GCP42112, abolished the negative inotropism elicited by ANG II,
suggesting that it may be mediated by an AT1-like receptor. This
result agrees with those of functional studies conducted with non-cardiac
tissues of other teleosts (see Nishimura,
2001
; Russell et al.,
2000
); however other AT2 antagonists should be tested
before ruling out any role for AT2 receptors in the cardiac effects
of ANG II.
The negative inotropic action of ANG II was prevented by pre-treatment with
PTx, pointing to the involvement of a PTx-sensitive G protein. In mammalian
myocytes, AT1 receptors, acting through a Gi protein,
mediate the inhibitory effect of ANG II on L-type Ca2+
currents and on the adenylate cyclase system
(Ai et al., 1998). Whether the
ANG II-induced negative inotropism in the eel heart involves these mechanisms
or other intracellular cascades (i.e. the phosphoinositide pathway) remains to
be established.
Involvement of the adrenergic and cholinergic systems
There is evidence that some of the cardiovascular effects of ANG II in
teleost are mediated by catecholamines
(Bernier and Perry, 1999;
Oudit and Butler, 1995
) or by
changes in cholinergic tone (Reid,
1992
). However, it is not clear whether the interactions occur at
the adrenergic nervous endings, including the intramyocardial terminals, at
the chromaffin tissue level, or both
(Nishimura et al., 1978
).
Whereas in A. anguilla inhibition of
-adrenergic receptors
only attenuates the ANG II response (Oudit
and Butler, 1995
), in Squalus acanthias the
-adrenergic antagonist phentolamine blocks the ANG II effect
(Opdyke and Holcombe, 1976
).
Moreover, in A. rostrata ß-adrenergic receptor blockade did not
affect the cardiovascular response to ANG II
(Oudit and Butler, 1995
). In
the denervated eel heart preparation, our finding that ANG II-mediated
inotropism was insensitive to
- and ß-adrenergic agonists
(phenylephrine and isoproterenol) and to adrenergic antagonists (phentolamine,
propanolol and sotalol) argues against intra-cardiac adrenergic involvement.
To what extent this adrenergically independent inotropic response to
blood-borne ANG II may apply to the innervated heart remains to be
elucidated.
In the heart, cholinergic stimuli, mediated by the M2 and
M4 muscarinic receptor subtypes (mAChR), preferentially located on
the myocardiocytes and coupled to adenylate cyclase inhibition, elicit
negative chronotropic and inotropic effects (mammals:
Hove-Madsen et al., 1996;
teleosts: Imbrogno et al.,
2001
). Little is known about how the cholinergic system influences
ANG II-mediated inotropy. Antagonism between ANG II and cholinergic effects
has been reported in mammals (Ai et al.,
1998
). In A. rostrata, the ANG II effect was greater
after muscarinic receptor blockade (Oudit
and Butler, 1995
). Our finding that the non-specific muscarinic
antagonist atropine blocked ANG II-mediated negative inotropy suggests that in
A. anguilla the cardiotropic action of ANG II could be partly due to
activation of muscarinic receptors.
Involvement of an EE-NO-cGMP-PKG signal transduction pathway
In the in vitro working eel heart, the EE under basal conditions,
and when activated by chemical (i.e. acetylcholine) or physical stimuli
negatively modulates mechanical performance as a result of the tonic release
of NO, which in turn increases cGMP levels in cardiomyocytes
(Imbrogno et al., 2001).
Interactions between ANG II and endothelial-type NOS have been demonstrated
in vivo and in vitro in the mammalian vascular endothelium
(for references, see Li et al.,
2002), where the AT1 subtype receptor has been
reported. In contrast, except for a study describing AT2 receptors
in the human EE (Wharton et al.,
1998
), there are no reports of ANG II receptors in the EE. The
obligatory role played by the eel EE in transducing the intracavitary ANG II
signal suggests that AT1-like receptors could be located at the EE
level.
ANG II-mediated negative inotropism was significantly enhanced in the
presence of the NOS substrate L-arginine, but abolished by
pre-treatment with NO scavenger (Hb), specific NOS (L-NIO and
L-NMMA) or soluble GC (ODQ) inhibitors, or by exposure to Triton
X-100, which are all consistent with stimulation of EE-NO-cGMP signalling
induced by endoluminal ANG II. The integrity of the EE is a prerequisite for
triggering the ANG II signal transduction pathway, and is further evidence
that EE-NO plays an intracavitary autocrineparacrine role in the
control of fish heart function (Imbrogno
et al., 2001).
An important intramyocardial target of NO signalling is PKG. In addition to
its direct effect on calcium influx in isolated mammalian ventricular
cardiomyocytes (Méry et al.,
1991), PKG, through phosphorylation of troponin I, reduces the
affinity of troponin C for calcium, thereby negatively regulating cardiac
contractility (Hove-Madsen et al.,
1996
). Pre-treatment with the inhibitor KT5328 attenuated the
effect of ANG II, thereby implicating PKG in ANG II-mediated negative
inotropism.
AT1 receptors, G-proteins, G-protein-linked receptors (e.g.
mAChR) and several signalling molecules (e.g. eNOS, protein kinase C,
Ca2+ channels, plasmalemmal Ca2+-ATPase) are
structurally and functionally localized in the endothelial cell caveolae
(Ishizaka et al., 1998). Being
the location of many proteins involved in signal-transduction cascades, the
caveolae could be the domain where ANG II signalling is generated. In the
light of our earlier results showing intracardiac cross talk between
EE-NO-cGMP and cholinergic stimuli
(Imbrogno et al., 2001
), we
postulate that the colocalization of AT1 receptors, muscarinic
cholinergic receptors and eNOS in the restricted space of the EE caveolae (see
Fig. 9) may represent a
temporally and spatially delimited pathway for signal transduction.
|
ANG II and the FrankStarling response
Like most fish hearts, the eel heart is very sensitive to the
FrankStarling response, i.e. the heterometric regulation that
contributes to the increased cardiac output associated with increased filling
pressure, as occurs during periods of exercise or increased venous return
(Farrell and Jones, 1992). ANG
II is a potent effector of venoconstriction in teleosts
(Oudit and Butler, 1995
). In
the in vitro isolated working heart of A. anguilla the basal
release of endogenous NO greatly affects the FrankStarling response by
making the heart more sensitive to preload-induced increases in cardiac output
at a constant afterload and heart rate
(Imbrogno et al., 2001
). It is
therefore notable that, although exerting NO-dependent negative inotropism,
ANG II per se did not affect the FrankStarling mechanism in
the eel heart. This indicates that the NO-cGMP mechanism underlying ANG II
negative inotropism may differ from the mechanism underlying the nitrergic
modulation of the FrankStarling response. The finding that in the eel
heart the local cardio-suppressive modulatory action of ANG II is exerted
without detriment of the intrinsic heterometric modulation may be of great
physiological interest. It is also compatible with the hypothesis that the
local ANG II cardio-inhibitory modulation is part of a homeostatic loop that
protects the heart from excessive haemodynamic loads such as those deriving
from activation of the systemic RAS itself and the adrenergic system.
In conclusion, this study provides the first evidence that endoluminal ANG II exerts a direct cardio-suppressive effect on the mechanical performance of the fish heart via interaction with the endocardial endothelium. This interaction activates G protein-coupled AT1-like receptors, which in turn trigger a NO-cGMP-PKG signal transduction pathway. The cardio-depressive effect of ANG II does not influence the FrankStarling response. These data, together with the involvement of the muscarinic receptors in mediating ANG II inotropic stimulation, suggest that the EE, through its sensory function, is able to adapt cardiac performance to the peripheral demands of the fish. The EE caveolae are prime candidates as the domain at which the tonic-phase ANG II-NO signalling is generated.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ai, T., Horie, M., Obayashi, K. and Sasayama, S. (1998). Accentuated antagonism by angiotensin II on guinea-pig cardiac L-type Ca-currents enhanced by ß-adrenergic stimulation. Eur. J. Physiol. 436,168 -174.[CrossRef][Medline]
Baker, K. M., Singer, H. A. and Aceto, J. F. (1989). Angiotensin II receptor-mediated stimulation of cytosolic-free calcium and inositol phosphates in chick myocytes. J. Pharmacol. Exp. Ther. 251,578 -585.[Abstract]
Baker, K. M. and Aceto, J. A. (1989). Characterization of avian angiotensin-II cardiac receptors: coupling to mechanical activity and phosphoinositide receptors. J. Mol. Cell. Cardiol. 21,375 -382.[Medline]
Baker, K. M., Booz, G. W. and Dostal, D. E. (1992). Cardiac actions of angiotensin II: role of an intracardiac renin-angiotensin system. Annu. Rev. Physiol. 54,227 -241.[CrossRef][Medline]
Bernier, N. J. and Perry, S. F. (1997). Angiotensins stimulate catecholamine release from the chromaffin tissue of the rainbow trout. Am. J. Physiol. 273,R49 -R57.[Medline]
Bernier, N. J. and Perry, S. F. (1999).
Cardiovascular effects of angiotensin-II-mediated adrenaline release in
rainbow trout Oncorhynchus mykiss. J. Exp. Biol.
202, 55-66.
Bernier, N. J., Mckendry, J. E. and Perry, S. F. (1999). Blood pressure regulation during hypotension in two teleost species: differential involvement of the renin-angiotensin and adrenergic systems. J. Exp. Biol. 202,1677 -1690.[Abstract]
Cerra, M. C., Tierney, M. L., Takei, Y., Hazon, N. and Tota, B. (2001). Angiotensin II binding sites in the heart of Scyliorhinus canicula: an autoradiographic study. Gen. Comp. Endocrinol. 121,126 -134.[CrossRef][Medline]
Cobb, C. S., Williamson, R. and Brown, J. A. (1999). Angiotensin II-induced calcium signalling in isolated glomeruli from fish kidney (Oncorhynchus mykiss) and effects of losartan. Gen. Comp. Endocrinol. 113,312 -321.[CrossRef][Medline]
Crackower, M. A., Sarao, R., Oudit, G. Y., Yagil, C., Kozieradzki, I., Scanga, S. E., Oliveira-Dos-Santos, A. J., Da Costa, J., Zhang, L., Pei, Y. et al. (2002). Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417,822 -828.[CrossRef][Medline]
De Gasparo, M., Husain, A., Alexander, W., Catt, K. J., Chiu, A.
T., Drew, M., Goodfriend, T., Harding J. W., Inagami, T. and Timmermans, P. B.
W. M. (1995). Proposed update of angiotensin receptor
nomenclature. Hypertension
25,924
-927.
Drimal, J. and Boska, D. (1973). Effects of angiotensin-II on myocardial mechanics and contractile state of heart muscle. Eur. J. Pharmacol. 21,130 -138.[CrossRef][Medline]
Farrell, A. P. and Jones, D. R. (1992). The heart. In Fish Physiology, volXIIA (ed. W. S. Hoar and D. R. Randall), pp.1 -88. London: Academic Press.
Freer, R. J., Pappano, A. J., Peach, M. J., Bing, K. T., Aclean, M. J., Vogel, S. and Sperelakis, N. (1976). Mechanism for the positive inotropic effect of angiotensin II on isolated cardiac muscle. Circ. Res. 39,178 -183.[Abstract]
Grinstead, W. and Young, J. B. (1992). The myocardial renin-angiotensin system: Existence, importance and clinical implications. Am. Heart J. 123,1039 -1045.[Medline]
Habuchi, Y., Lu, L. L., Morikawa, J. and Yoshimura, M. (1995). Angiotensin II inhibition of L-type Ca2+ current in sinoatrial node cells of rabbits. Am. J. Physiol. 268,H1053 -H1060.[Medline]
Hazon, N., Tierney, M. L., Hamano, K., Ashida, K. and Takei, Y. (1995). Endogenous angiotensins, angiotensin II competitive binding inhibitors and converting enzyme inhibitor in elasmobranch fish. Neth. J. Zool. 45,117 -120.
Hazon, N., Cerra, M. C., Tierney, M. L., Tota, B. and Takei, Y. (1997). Elasmobranch renin angiotensin system and the elasmobranch receptor. In Advances in Comparative Endocrinology (ed. S. Kawashima and S. Kikuyama), pp.1307 -1312. Bologna, Italy: Monduzzi Editore.
Hove-Madsen, L., Mery, P. F., Jurevicius, J., Skeberdis, A. V. and Fishmeister, R. (1996). Regulation of myocardial calcium channels by cyclic AMP metabolism. Basic. Res. Cardiol. 91,1 -8.[Medline]
Imbrogno, S., De Iuri, L., Mazza, R. and Tota, B.
(2001). Nitric oxide modulates cardiac performance in the heart
of Anguilla anguilla. J. Exp. Biol.
204,1719
-1727.
Imbrogno, S., Scorpiniti, D., Cerra, M. C. and Tota, B. (2002). The inotropic influence of angiotensin II on the working heart of the eel (Anguilla anguilla): paracrine aspects and subcellular mechanisms. In Cardiovascular Physiology of Fish. 5th International Congress on the Biology of Fish (Vancouver, Canada, 21-26 July) (ed. K. Gamperl, T. Farrel and D. Mac Kinlay), pp.55 -57. Vancouver, Canada: American Fisheries Society.
Ishizaka, N., Griendling, K. K., Lassegue, B and Wayne, A.
(1998). Angiotensin II type receptor relationship with caveolae
and caveolin after initial agonist stimulation.
Hypertension 32,459
-466.
Kobayashi, H. and Takei, Y. (1996). Biological actions of ANGII. In The Renin-Angiotensin System. Comparative Aspects. Zoophysiology, vol 35 (ed. S. D. Bradshaw, W. Burggren, H. C. Heller, S. Ishii, H. Langer, G. Neuweiler and D. J. Randall), pp 113-171. Berlin, Heildleberg: Springer Verlag.
Koeslag, J. H., Saunders, P. T. and Wessele, J. A. (1999). The chromogranins and the counter-regulatory hormones: do they make homeostatic sense? J. Physiol. 5127,643 -649.
Lipke, D. W. and Olson, K. R. (1988). Distribution of angiotensin-converting enzyme-like activity in vertebrate tissues. Physiol. Zool. 61,420 -428.
Li, H., Wallerath, T. and Förstermann, U. (2002). Physiological mechanisms regulating the expression of endothelial-type NO synthase. Nitric Oxide 7, 132-147.[CrossRef][Medline]
Maillard, M. P., Perregaux, C., Centeno, C., Stangier, J.,
Wienen, W., Brunner, H. R. and Burnier, M. (2002). In
vitro and in vivo characterization of the activity of
telmisartan: an insurmountable angiotensin II receptor antagonist.
J. Pharmacol. Exp. Ther.
302,1089
-1095.
Marsigliante, S., Muscella, A., Vilella, S., Nicolardi, G., Ingrosso, L., Ciardo, V., Zonno, V., Vinson, G. P., Ho, M. M. and Storelli, C. (1996). A monoclonal antibody to mammalian angiotensin II AT1 receptor recognises one of the angiotensin II receptor isoforms expressed by the eel (Anguilla anguilla). J. Mol. Endocrinol. 16,45 -56.[Abstract]
Méry, P. F., Lohmann, S. M., Walter, U. and Fischmeister, R. (1991). Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc. Natl. Acad. Sci. USA 88,1197 -1201.[Abstract]
Meulemans, A. L., Andries, L. J. and Brutsaert, D. L. (1990). Does endocardial endothelium mediate positive inotropic response to angiotensin I and angiotensin II? Circ. Res. 66,1591 -1601.[Abstract]
Nishimura, H. (1985). Evolution of the renin angiotensin system and its role in control of cardiovascular function in fishes. In: Evolutionary Biology of Primitive Fishes (ed. R. E. Foreman, A. Gorbman, J. M. Dodd and R. Olson), pp.275 -293. New York: Plenum.
Nishimura, H. (2001). Angiotensin receptors-evolutionary overview and perspectives. Comp. Biol. Physiol. 128,11 -30.
Nishimura, H., Norton, V. M. and Bumpus, F. M. (1978). Lack of specific inhibition of angiotensin II in eels by angiotensin antagonists. Am. J. Physiol. 235,H95 -H103.[Medline]
Olson, K. R. (1992). Blood and extracellular fluid regulation. In Fish Physiology, vol.XIIB . The Cardiovascular System (ed. W. S. Hoar, D. J. Randall and A. P. Farrell), pp. 135-254. New York: Academic Press.
Olson, K. R., Chavez, A., Conklin, D. J., Cousins, K. L.,
Farrell, A. P., Ferlic, R., Keen, J. E., Kne, T., Kowalski, K. A. and Veldman,
T. (1994). Localization of angiotensin II responses in the
trout cardiovascular system. J. Exp. Biol.
194,117
-138.
Opdyke, D. F. and Holcombe, R. (1976). Response
to angiotensins I and II and to AI converting-enzyme inhibitor in a shark.
Am. J. Physiol. 231,1750
-1753.
Oudit, G. Y. and Butler, D. G. (1995). Angiotensin II and cardiovascular regulation in a freshwater teleost, Anguilla rostrata LeSueur. Am. J. Physiol. 269,R726 -R735.[Medline]
Paton, J. F. R., Deuchars, J., Ahmad, Z., Wong, L. F., Murphy,
D. and Kasparov, S. (2001). Adenoviral vector demonstrates
that angiotensin II-induced depression of the cardiac baroflex is mediated by
endothelial nitric oxide synthase in the nucleus tractus solitarii of the rat.
J. Physiol. 531,445
-458.
Reid, I. A. (1992). Interactions between ANG II, sympathetic nervous system and baroreceptor reflexes in regulation of blood pressure. Am. J. Physiol. 262,E763 -E778.[Medline]
Russell, M. J., Klemmer, A. M. and Olson, K. R. (2000). Angiotensin signaling and receptor types in teleost fish. Comp. Biochem. Physiol. 128, 41-51.
Saavedra, J. M., Viswanathan, M. and Shigematsu, K. (1993). Localization of angiotensin AT1 receptors in the rat heart condution system. Eur. J. Pharmacol. 235,301 -303.[CrossRef][Medline]
Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murray, J., Hasegawa, M., Matsuda, Y. and Inagami, T. (1991). Cloning and expression of a complementary DNA encoding a bovina adrenal angiotensin II type-1 receptor. Nature 351,230 -233.[CrossRef][Medline]
Schorb, W., Booz, G. W., Dostal, D. E., Conrad, K. M., Chang, K. G. and Baker, K. M. (1993). Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ. Res. 72,1245 -1255.[Abstract]
Sekine, T., Kusano, H., Nishimaru, K., Tanaka, Y., Tanaka, H. and Shigenobu, K. (1999). Developmental conversion of inotropism by endotelin I and angiotensin II from positive to negative in mice. Eur. J. Pharmacol. 374,411 -415.[CrossRef][Medline]
Sys, S. U., Pellegrino, D., Mazza, R., Gattuso, A., Andries, L.
J. and Tota, B. (1997). Endocardial endothelium in the
avascular heart of the frog: morphology and role of nitric oxide.
J. Exp. Biol. 200,3109
-3118.
Tierney, M. L., Takei, Y. and Hazon, N. (1997). The presence of angiotensin II receptors in elasmobranchs. Gen. Comp. Endocrinol. 105,9 -17.[CrossRef][Medline]
Timmermans, P. B. M. W. M., Wong, P. C., Chiu, A. T., Herblin, W. F., Benfield, P., Carini, D. J., Lee, R. J., Wexler, R. R., Saye, J. M. and Smith, R. D. (1993). Angiotensin II receptors and angiotensin II receptor antagonists. Pharmac. Rev. 45,205 -251.[Medline]
Tran van Chuoi, M., Dolphin, C. T., Barker, S., Clark, A. J. and Vinson, G. P. (1999). Molecular cloning and characterization of the cDNA encoding the angiotensin II receptor of European eel (Anguilla anguilla). GenBank Database AJ005132.
Wharton, J., Morgan, K., Rutherford, R. A. D., Catravas, J. D., Chester, A., Whitehead, B. F., De Leval, M. R., Yacoub, M. H. and Polak, J. M. (1998). Differential distribution of Angiotensin AT2 receptors in the normal and failing human heart. Pharmacol. 284,323 -336.[CrossRef]
Wu, K. K. (2002). Regulation of endothelial
nitric oxide synthase activity and gene expression. Ann. NY Acad.
Sci. 962,122
-130.