The heart of Ciona intestinalis: eicosanoid-generating capacity and the effects of precursor fatty acids and eicosanoids on heart rate
School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, Wales, UK
* Author for correspondence (e-mail:a.f.rowley{at}swansea.ac.uk )
Accepted 23 March 2002
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Summary |
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Key words: eicosanoid, heart, vascular system, eicosapentaenoic acid, sea squirt, Ciona intestinalis
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Introduction |
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Two main PGs of significance to the mammalian vascular system are
prostacyclin (PGI2) and thromboxane A2
(TxA2). Prostacyclin is synthesized by the vascular endothelium and
causes vasodilatation as well as inhibiting platelet aggregation
(Bunting et al., 1976). These
activities are opposed by TxA2, which is strongly pro-aggregatory
for platelets and causes vasoconstriction
(Hamberg et al., 1975
). The
balance between these two compounds may be of importance in maintenance of the
vascular tone and in haemostasis in general
(Fitzgerald et al., 1987
;
Ullrich et al., 2001
). As well
as these two prostaglandins, other eicosanoids including prostaglandin
E1 (PGE1) have vasoactive effects such as vasodilation
(e.g. Carlson et al.,
1969
).
An increasing body of literature has revealed not only that eicosanoids are
present in every major invertebrate phylum
(Stanley and Howard, 1998) but
also that they play key roles in a great variety of processes in invertebrates
such as reproduction, immunity and ion transport
(Stanley, 2000
). Our
understanding of whether eicosanoids play any role in the vascular system of
invertebrates is limited to a single study by Agnisola et al.
(1994
), who studied the effects
of AA on the systemic heart of the octopus Octopus vulgaris. They
found that perfusion with AA (10-7 to 10-5
moll-1) caused a concentration-dependent increase in heart rate
that had a biphasic effect on inotropism (positive for the lowest
concentration, 10-7 moll-1, but negative for the two
higher concentrations, 10-6 and 10-5 moll-1).
A potent vasoconstrictory effect was also noted, associated with an increase
in coronary resistance.
The tunicate heart makes an excellent subject for studying the potential
effect of eicosanoids for several reasons. The animals are readily obtainable,
and the size and simplicity of organization of the hearts make them easily
excisable. In the sea squirt Ciona intestinalis, the heart is a
simple V-shaped tube enclosed in a fluid-filled, transparent pericardium (see
Fig. 1). The heart forms as an
invagination of the pericardial wall and remains attached to the pericardium
along its length by a raphe (Anderson,
1968). The heart itself is a single layer of myoepithelial cells
with an area of non-muscle cells, termed the undifferentiated line, running
the length of the heart approximately opposite the raphe
(Martynova and Nylund, 1996
).
The heart and circulatory system are valveless, and blood is pumped around the
body by a series of peristaltic waves passing along the heart. There is no
innervation of the heart; pacemaker regions, present at either end of the
heart, alternate in periods of activity, thus changing the direction of
contraction. These heart-rate reversals occur in all tunicate hearts
(Goodbody, 1974
) and are
unique among the chordates (Kriebel,
1968a
,b
).
Heart function, with special reference to reversals, has been extensively
studied in tunicates with respect to mechanical properties and the effects of
drugs and salinity (e.g. Scudder et al.,
1963
; Kriebel,
1968a
; Anderson,
1968
; Goodbody,
1974
; Shumway,
1978
). Blood cells circulate through the body of tunicates through
distinct channels, and in C. intestinalis a complete circulation
takes approximately 1 min (Skramlik,
1929
). Circulation in the test can be extensive in many tunicates,
but in C. intestinalis it is limited to the peduncle region
(Goodbody, 1974
).
|
The evidence concerning the effects of cardioregulatory drugs on the
tunicate heart is contradictory, in that some workers have found that
acetylcholine, which normally depresses cardiac activity in mammals, has no
effect (Scudder et al., 1963),
while others have found that it either increases the heart rate
(Ebara, 1953
) or enhances the
dominance of the abvisceral pacemaker (Waterman,
1942
,
1943
). Similarly, adrenaline
has been found to increase the heart rate in tunicates by some workers
(Scudder et al., 1963
) or to
decrease it by others (Keefner and Akers,
1971
).
The present study examines the nature of the eicosanoids synthesized by the heart and blood cells of C. intestinalis and investigates the potential action of the precursor PUFAs and their eisosanoid derivatives on heart rate.
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Materials and methods |
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Eicosanoid biosynthesis by heart and blood cells
The heart was excised from individual C. intestinalis using a
binocular dissecting microscope. After complete removal of the test, the
underlying mantle was cut through longitudinally to expose the visceral
cavity. The heart was removed by cutting through the hypobranchial, visceral
and test blood vessels, ensuring that the pericardium remained intact and that
any associated tissue was removed. Blood cells were obtained by cutting
through the heart in situ immediately after excision.
Five hearts (approximately 0.2 g wet mass) were obtained using the above
method. Each heart was cut into small pieces and disrupted in 1 ml of marine
saline (0.5 moll-1 NaCl, 12mmoll-1
CaCl2.2H2O, 11 mmoll-1 KCl, 26
mmoll-1 MgCl2.6H2O, 50 mmoll-1
Tris; pH 7.4) using a glass homogenizer before the resulting suspensions were
pooled. Similarly, blood cells were pooled from approximately five individuals
and collected into ice-cold marine anticoagulant (0.45 moll-1 NaCl,
0.1 moll-1 glucose, 30 mmoll-1 trisodium citrate, 26
mmoll-1 citric acid, 10 mmoll-1 EDTA; pH 4.6), washed
twice by centrifugation in marine anticoagulant (1500 g, 10
min, 4°C) and resuspended in 5 ml of cold marine saline. Both suspensions
were incubated with 5 µmoll-1 calcium ionophore A23187 (Sigma
Chemical Co. Ltd, Poole, UK) for 20 min at 16°C and subsequently
centrifuged (1500 g, 10 min, 4°C) to remove debris. An
internal standard, PGB2 (200 ng), was added, and the supernatants
were extracted using Sep-Pak C18 minicolumns as detailed previously
(Knight et al., 1999).
Lipoxygenase products were separated by reverse-phase high-performance
liquid chromatography (RP-HPLC) using an Ultrasphere C18 ODS column
(250 mmx4.6 mm; Beckman Coulter, High Wycombe, UK) and a linear gradient
changing from 100% water:methanol:acetonitrile:acetic acid (45:30:25:0.05 by
volume, apparent pH 5.7) to 100% methanol, over 40 min, with a flow rate of
0.6 ml min-1. Material eluting was detected using Waters 991 or 996
diodearray ultraviolet detectors (Waters Chromatography, Cheshire, UK). Peaks
were identified by co-chromatography with authentic standards and by reference
to previous mass spectrometric analysis
(Knight et al., 1999).
Quantification of products was by reference to the extraction efficiency of
the internal standard (PGB2) using published molar extinction
coefficients.
To quantify the cyclo-oxygenase products, PGE2/3, hearts were
excised, ionophore-incubated and Sep-Pak-extracted as previously described but
without the addition of an internal standard. The eluate was tested with a
commercially available PGE enzyme immunoassay (EIA) kit (Amersham Pharmacia
Biotech UK Ltd, Little Chalfont, UK) as directed in the protocols provided.
The sensitivity and range of the assay was 2.5-320 pg per well, and its
published cross-reactivity with other eicosanoids was 25% with
PGE1, 0.04% with PGF2, <0.1% with
6-keto-PGF1
and <0.001% with AA.
Effect of eicosanoids and precursor fatty acids on heart rate
Hearts were dissected from adult C. intestinalis as described
above and placed in solid watch-glasses containing 2 ml of sea water at
16°C. Any associated tissue was carefully removed prior to observation.
The heart rate was counted for two periods of 60s by direct observation using
a binocular microscope, and the mean value was used to calculate the heart
rate (beats min-1). The heart rate was determined for a period of
30 min at 16°C prior to the addition of test material. Hearts with erratic
or unusual rates were not used further. Arachidonic acid (AA; 0-200
µmoll-1), eicosapentaenoic acid (EPA; 0-200
µmoll-1), 12-(R/S)hydroxyeicosapentaenoic acid
(12-HEPE; 0-5 µmoll-1), 8-(R/S)-hydroxyeicosapentaenoic
acid (8-HEPE; 0-10 µmoll-1) and PGE3 (0-50
µmoll-1) were added to each heart preparation, and heart rate
was monitored for up to 6h. In all cases, in parallel experiments, the effect
of the addition of the appropriate amount of the vehicle (ethanol) was also
tested. All eicosanoids and precursor fatty acids were obtained from Cayman
Chemical Co. (Ann Arbor, USA).
Statistical analyses
Data are presented throughout as mean values ± 1 S.E.M. Significance
of differences between conditions was determined using univariate analysis of
variance (ANOVA) with Dunnett's post-tests. Significance of differences at
each time period was determined with a one-way ANOVA with Dunnett's
post-test.
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Results |
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Eicosanoid profiles of the heart and blood cells
Previous studies have examined the eicosanoid-generating capacity of the
ovary, branchial basket, intestine and tunic of C. intestinalis
(Knight et al., 1999). The
principal lipoxygenase (LO) products of heart were found to be
8-hydroxyeicosapentaenoic acid (8-HEPE) (peak 4) and 12-HEPE (peak 2) (Figs
2,
3). In all cases, smaller
amounts of 8,15-dihydroxyeicosapentaenoic acid (8,15-diHEPE; peak 1) were
found (Figs 2,
3). As well as these identified
products, two other compounds with conjugated dienes (indicative of
monohydroxy fatty acid derivatives) were observed (peaks 3 and 5 in
Fig. 2). No further attempt was
made to identify the compounds under these peaks. C. intestinalis
heart was also found to display PGE-immunoreactivity consistent with a level
of 6.8±0.5 ng g-1 wet mass (mean ± S.E.M.,
N=4).
|
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Blood cells produced a similar range of products except at lower levels. For example, levels of the principal product observed, 8-HEPE, were only 1.45±0.36 ng 10-6 cells (mean ± S.E.M., N=4).
Effects of eicosanoids and their precursor molecules on heart
rate
After a brief period of acclimation, the basal heart rate for C.
intestinalis specimens collected in 1999 was 31.9±0.7 beats
min-1 (N=113), which was significantly higher than that
for those collected in 2000 (20.3±1.3 beats min-1,
N=21) (means ± S.E.M., independent Student's t-test,
P<0.01). Although there was an apparent slight decrease in the
basal heart rate of specimens collected between August and December 1999, this
was not statistically significant (Fig.
4). The difference between the year classes, in addition to the
normal variation among hearts, made it necessary to express each heart rate as
a percentage of the basal reading for that particular heart rather than as
beats min-1.
|
Over a period of 180 min, the heart rate was found to decrease by approximately 34.3% (Fig. 5). After 24 h, some heart preparations were still beating, but erratically and slowly. Addition of the vehicle (10 µl of ethanol) was found to have a significant negative chronotropic effect on heart rate over a period of 180 min. This general retarding effect was shown to be individually significant at two time periods (60 and 120 min; Fig. 5). When the effects of the PUFAs were investigated, it was observed that EPA and AA produced an overall significant stimulatory effect on heart rate at all concentrations used (50-200 µmoll-1; Fig. 6A,B). However, in the case of AA, this was only individually significant at one time point (180 min) and with one concentration (200 µmoll-1) (Dunnett's post-test, P<0.006). With EPA, individual time-related effects were seen at 60 (P<0.004), 120 (P<0.001) and 180 min (P<0.001) with 50 µmoll-1 only (Dunnett's post-tests). Overall, PGE3 at 50 µmoll-1, had a stimulatory effect, but only at one time point (120 min) was it individually significant (Fig. 7). At 10 µmoll-1, PGE3 was without significant effect at all time periods (results not shown). 12-HEPE at 5 µmoll-1 had a statistically significant overall stimulatory effect, but this was not observed at individual time points using a Dunnett's post-test. Unlike PGE3 and 12-HEPE, 8-HEPE was without significant effect at the concentration tested (5 µmoll-1).
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Heart reversals occurred at variable intervals, with successive reversal periods often of different lengths, although the frequency of contractions was fairly consistent for any given animal. There was no clear effect on the frequency of beat reversal following exposure to EPA, AA, 8-HEPE, 12-HEPE or PGE3 (data not shown). Although not quantified, there were no visible effects of any of the compounds tested on stroke volume.
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Discussion |
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Knight et al. (1999) found
that most of the major LO products of C. intestinalis were
EPA-derived, but they also noted smaller amounts of
8,15-dihydroxyeicosatetraenoic acid (an AA-derived product) and a lipoxin-like
material (potentially AA-derived). The finding of only EPA-derived products in
this present investigation probably reflects the smaller quantities of heart
tissue and blood cells used. Hence, if a larger mass of heart tissue or a
greater number of blood cells had been used, the smaller AA-derived products
would probably have been detected. Other urochordates, such as the ascidian
Botryllus schlosseri and the thaliacean Dolioletta
gegenbauri, are known to contain EPA as the main C20 fatty acid component
of cellular phospholipids (Carballeira et
al., 1995
; Pond and Sargent,
1998
). The dominance of n-3 over n-6 fatty acids
in marine phytoplankton is well-documented (e.g.
Ackman et al., 1968
), and the
general abundance of n-3 PUFAs in marine lipids is attributed to this
(Sargent and Whittle,
1981
).
The only cyclo-oxygenase product quantified in the present study was
PGE2/3. The approach used was enzyme immunoassay which, although
highly sensitive (picogram levels), is not totally specific in that other
compounds can cross-react with the antibodies used. Hence, the small amounts
of PGE-immunoreactive material observed in the heart of C.
intestinalis could have resulted from other contaminating eicosanoids,
and care should therefore be taken in any interpretation of these results.
Furthermore, in the present study, no attempt was made to determine whether
the heart or blood cells of C. intestinalis can generate either
TxA2/3 or PGI2/3, both compounds with vascular
activity in mammals (Ullrich et al.,
2001). To date, no researchers have conclusively demonstrated the
presence of these compounds in any invertebrate. For example, Hampson et al.
(1992
) reported the generation
of low levels of thromboxane B (TxB) (the stable breakdown product of
TxA) immunoreactive material by blood cells of the shore crab
Carcinus maenas, but it could be argued that this resulted from
non-specific binding of the antibodies employed leading to false
positives.
Other workers have observed considerable variance in heart rate between
C. intestinalis individuals. Kriebel
(1968a) noted that heart rate
depended upon the size and temperature of the animal. In the present study,
the experimental temperature was constant, but the animals ranged in size and
it was observed, in agreement with Kriebel
(1968a
), that the smaller
individuals displayed higher heart rates than the larger specimens. Indeed,
the significantly lower heart rates observed for animals obtained in 2000
could be explained because they were collected several months later in the
year and they would have reached a greater size before collection. Shumway
(1978
) and Kriebel
(1968a
) recorded mean in
vivo heart rates of 32.9 and 20.7 beats min-1, respectively,
for C. intestinalis at 10°C, and Kriebel
(1968b
) noted that the beat
frequency remained the same following excision. Interestingly, the mean heart
rates of 31.9 and 20.3 beats min-1 observed in vitro in
the present study, from animals collected in 1999 and 2000 respectively, are
similar to the two in vivo rates reported
(Kriebel, 1968a
;
Shumway, 1978
). The
differences in reported rates probably represent different experimental
conditions compounded by natural variation, especially that caused by the size
differences of the animals tested.
The frequency of heart reversal in tunicates has been widely reported (e.g.
Anderson, 1968;
Kriebel, 1968a
;
Shumway, 1978
;
Jones, 1985
). In the present
study, it was found that these occurred infrequently and with no clear
pattern. Jones (1985
) found
that heart reversals in Ascidiella aspersa were also erratic at the
population level but consistent within individuals. Shumway
(1978
) reported regular beat
reversals in C. intestinalis in vivo approximately 2 min apart.
It is clear that both AA and EPA had a significant positive chronotropic
effect on the heart of C. intestinalis, particularly after a 60 min
incubation. What is not clear, however, is whether this is a direct effect on
membranes in the heart tissues caused by the incorporation of exogenous fatty
acids or one involving the generation of eicosanoids. Both cyclooxygenase
(PGE3) and LO (12-HEPE) products also have stimulatory effect on
sea squirt hearts, suggesting that at least some of the effect of fatty acids
may be via such a route. In the only other study on the effects of
fatty acids on invertebrate hearts, Agnisola et al.
(1994) showed that AA was
readily converted by Octopus vulgaris heart into several different
eicosanoids, but the potential role of these compounds in the observed effects
of the parent fatty acid on the heart was not directly investigated. Instead,
they examined the effect of inhibitors of the biosynthesis of eicosanoids on
the heart preparations. They found that indomethacin (a cyclo-oxygenase
inhibitor) and nordihydroguaiaretic acid (NDGA; a non-selective LO inhibitor)
had some effect. In particular, it was found that indomethacin potentiated the
stimulation in coronary resistance induced by AA while NDGA slightly reduced
this effect. A possible explanation of these observations was that AA induced
the generation of vasoactive eicosanoids (either vasoconstrictive or
vasodilatory) and that the presence of indomethacin shunted product generation
from cyclooxygenase-derived towards vasoconstrictive LO products, with
resulting effects. Unfortunately, they failed to carry out key experiments
using exogenous eicosanoids to determine whether any had activity so as to
test this hypothesis. Furthermore, the inhibitors employed are not totally
specific and could have had effects independent of LO or cyclooxygenase
inhibition.
Several other mechanisms may exist that could explain the effects of EPA
and AA on sea squirt hearts independent of eicosanoid generation. For example,
in bovine coronary artery, EPA has been shown to induce nitric oxide (NO)
production in the endothelial cells, resulting in vasodilation
(Omura et al., 2001). While NO
has been shown to be a potent vasodilator in mammals
(Ignarro, 1990
), to our
knowledge there are no reports of such activity in any invertebrate. These
possibilities deserve further investigation.
Finally, mention should be made about the potential in vivo
effects of PUFAs and eicosanoids in the regulation of the vascular system of
tunicates. It is clear that free PUFAs are highly unlikely to be present
in vivo at concentrations as high as the maximum (200 µmol
l-1) employed in the present study. However, some of the
eicosanoids tested in this study were biologically active in the range 5-10
µmol l-1, and such concentrations may be present within the
heart on the basis of the amounts of these compounds observed following
ionophore challenge (see Fig.
3). The observed stimulatory effects of PUFAs and some
eicosanoids, although significant, are relatively small, and it is difficult
to envisage how such changes would affect a sessile organism such as C.
intestinalis in which there may not be a requirement for the vascular
system to adjust cardiac output rapidly in response to sudden environmental
change. While the circulatory system of C. intestinalis has been
shown to be closed (Skramlik,
1929), the vessels are not lined by an endothelium and, hence,
cannot be said to be true blood vessels
(Goodbody, 1974
). Any change
in blood pressure or blood flow would therefore have to be caused by the
action of the heart itself rather than by vasoconstriction or vasodilation
elsewhere in the circulatory system.
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Acknowledgments |
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