Sex-dependent effects of gonadal steroids and cortisol on cardiac contractility in rainbow trout
Department of Biological Sciences, Idaho State University, Pocatello, ID 83209-8007, USA
* Author for correspondence (e-mail: rodnkenn{at}isu.edu)
Accepted 22 March 2004
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Summary |
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Key words: steroid hormone, cardiac function, cortisol, 17ß-estradiol, androgen, inotropism, rainbow trout, Oncorhynchus mykiss
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Introduction |
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Androgens promote dramatic sex differences in ventricle size and function
during sexual maturation of salmonid fishes
(Franklin and Davie, 1992). As
rainbow trout reach sexual maturity, plasma levels of androgens and estrogens
reach peak concentrations (Scott et al.,
1980a
,b
;
Lou et al., 1986
). Elevated
levels of 11-ketotestosterone (11KT), both during sexual maturation
(Franklin and Davie, 1992
) and
as a result of chronic administration
(Thorarensen et al., 1996
),
induce ventricular hypertrophy (Davie and
Thorarensen, 1997
), presumably through genomic mechanisms.
Further, Slater et al. (1995
)
demonstrated binding of [3H]testosterone to heart cytosolic
fractions from rainbow trout, providing evidence of specific androgen
receptors and the possibility for direct effects of androgens on
cardiomyocytes. Despite the recognized chronic effect of androgens on
ventricle size in salmonid fishes, the acute effects of gonadal steroids on
myocardial contractility or cardiovascular function in fishes has not been
studied.
Another steroid hormone of considerable interest in fishes is cortisol (C).
This glucocorticoid is elevated by stress and reduces immune function, disease
resistance and reproductive success in salmonid fishes
(Carragher et al., 1989;
Campbell et al., 1992
). C
levels are elevated in sexually maturing salmonids
(Sower and Schreck, 1982
).
However, there is a decrease in stress-induced C levels in both sexes during
spawning compared with non-reproductive periods
(Donaldson and Fagerlund,
1968
; Pottinger et al.,
1995
). Similar to the sex steroids, positive inotropic actions of
glucocorticoids have been demonstrated in cardiac tissue of mammals
(Yano et al., 1994
;
Wehling, 1997
;
Falkenstein et al., 2000
) and
amphibians (Hajdu and Szent-Györgyi,
1952
). Thus, we felt that it was also important to determine
whether C serves as a potential modulator of cardiac function in fishes. To
the best of our knowledge, only one previous study has examined the effects of
exogenous C on acute cardiac function in fishes
(Farrell et al., 1988
). These
authors saw no effect of C on isolated cardiac preparations.
Given this background, the objectives of the present study were to (1) examine whether gonadal steroids and/or C can rapidly promote cardiac contractility in male and female rainbow trout and (2) define potential mechanisms for steroid-induced positive inotropism in fishes. We hypothesized that, similar to mammals, sex steroids and C would increase cardiac contractility in fishes. Our results support this hypothesis and provide evidence for rapid inotropic mechanisms involving NO and polyamines.
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Materials and methods |
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Ventricle strip preparation
Fish were netted rapidly and euthanized by a sharp blow to the head. The
ventricle was excised, weighed and immediately placed in ice-cold modified
teleost Ringer solution. This solution contained (in mmol
l1): 111 NaCl; 5 KCl; 5 NaH2PO4; 10
NaHCO3; 1.5 CaCl2; 1.0 MgSO4 and 5 glucose
and was equilibrated with 0.5% CO2:99.5% O2 (pH of 7.6
at 14°C). To address potential effects of chemical anesthesia on the
response of ventricular tissue to steroids, additional fish (N=10)
were anesthetized with a buffered (0.2 g l1
NaHCO3) solution of MS-222 (tricaine methanesulfonate; 0.2 g
l1) or benzocaine (ethyl p-aminobenzoate; 0.2 g
l1) prior to excision of the ventricle. The sex of each fish
was determined by visual or microscopic examination of the gonads, and four
uniform strips (approximate dry mass, 1.5 mg) were cut from each ventricle.
Each strip (approximate dimensions, 4.5 mm long x 0.7 mm wide x
0.5 mm thick) was clamped at its base, tied at the other end with surgical
silk (3-0) and attached to a Kent isometric transducer (model TRN002;
Litchfield, CT, USA). Strips were suspended in 30 ml tissue baths containing
Ringer solution, between platinum wires, and oxygenated throughout the
experiment. The temperature of the muscle baths was maintained at 14°C
with a refrigerated recirculating bath. Strips were stimulated with a voltage
that elicited full contraction (60 V) at 0.5 Hz with 5 ms square wave pulses
(Grass S88 Stimulator; Grass Medical Instruments, Quincy, MA, USA), and the
length of each strip was adjusted to produce maximal twitch force. After a 1 h
equilibration period, we measured twitch force (F), time to peak
force (tp), time to 80% relaxation
(t0.8r), resting tension and ±d F/dT for
30 min using a data acquisition system (BioPac MP100; Goleta, CA, USA) and
software (Acqknowledge v.3.5.5; BioPac). As pointed out by Hartmund and Gesser
(1996), this preparation
cannot be regarded as truly isometric because of its nonhomogenous orientation
of contracting myocytes. Thus, changes in twitch force development and resting
tension were normalized (%) to the measurements taken at the end of the
initial equilibration period. Different fish and separate strips were used for
each experimental condition and appropriate controls. All four strips from a
given heart were prepared at the same time and incubated for the same
duration. The same strip was used only once. Given the large number of
incubation conditions (n=15 for males and n=14 for females),
this ultimately resulted in unequal sample sizes for statistical
comparisons.
Experimental protocols
Chemicals
Cyclodextrin (2-hydroxypropyl-ß-cyclodextrin) was obtained from
Research Biochemicals International (Natick, MA, USA). Testosterone
(4-androsten-17ß-ol-3-one) and 11-ketotestosterone
(4-androsten-17ß-ol-3, 11-dione) were purchased from Steraloids (Newport,
RI, USA). Aldosterone (4-pregnen-18, 20-diol-11ß, 18-epoxy-3,20-dione),
cholesterol (5-cholesten-3ß-ol), 17ß-estradiol
[1,3,5(10)-estratriene-3, 17ß-diol], hydrocortisone
(11ß17, 21-trihydroxypregn-4-ene-3, 20-dione), flutamide
{2-methyl-N-[4-nitro-3-(triflouromethyl)-phenyl]propanamide}, mifepristone
[11ß-(4-dimethylamino)phenyl-17ß-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one],
tamoxifen [(Z)-1-(p-dimethylamino-ethoxyphenyl)-1,2-diphenyl-1-butene], DFMO
[2-(diflouromethyl)-ornithine], GDP [guanosine
5'-O-(2-thiodiphosphate)], L-NAME (N
nitro-l-arginine
methyl ester) and bovine serum albumin (BSA)-conjugated steroids were
purchased from Sigma-Aldrich (St Louis, MO, USA). Unless noted otherwise,
additional chemicals were also purchased from Sigma-Aldrich.
Effects of steroid concentration, sex and contraction frequency
In the first series of experiments, ventricle strips from male and female
rainbow trout were exposed to increasing concentrations (from physiological to
pharmacological) of individual steroids for 40 min. Steroids were solubilized
in either absolute ethanol or 10% (w/v) cyclodextrin. Other experimental
compounds were solubilized in either Ringer solution [epinephrine, GDP,
-diflouromethylornithine (DFMO) and L-NAME] or absolute ethanol
(aldosterone, BSA-conjugated steroids, cholesterol, flutamide, mifepristone
and tamoxifen). Concentrations reported for all steroids and other chemicals
are final concentrations in the tissue baths. Concentrationresponse
curves were constructed for T and 11KT (0.3 nmol l130
µmol l1), C (0.1 nmol l1100
µmol l1) and 17ß-estradiol (E2; 0.01 nmol
l1100 µmol l1). Based on these
studies, optimal concentrations (those that elicited the greatest increase in
contractile force) of T, 11KT, C or E2 were administered to strips, with
control strips receiving an equal volume of ethanol or cyclodextrin. The final
concentrations of ethanol and cyclodextrin were 0.7 mmol l1
and 2.4 µmol l1, respectively. Ventricle strips were
exposed to cholesterol (1 nmol l1100 µmol
l1) or aldosterone (1 nmol l11
µmol l1) to evaluate whether a generic or
non-physiological steroid can alter cardiac performance, respectively. We also
determined whether steroid-induced changes in ventricle performance are
frequency dependent by electrically stimulating strips over a physiological
range of contraction frequencies (0.21.0 Hz), at optimal steroid
concentrations.
Mechanisms responsible for steroid actions
To explore whether steroid actions were additive or involved a common
mechanism of action, we treated strips with an optimal concentration of one
steroid and then another after maximal effects of the first steroid were
demonstrated. We also determined whether steroids' effects are mediated
through their respective receptors by pre-treating ventricle strips with
flutamide (0.1 mmol l1), mifepristone (0.1 mmol
l1) or tamoxifen (0.25 mmol l1) to inhibit
androgen, C and E2 receptors, respectively. We chose to pre-treat trout
ventricle strips with these compounds 10 min prior to administration of
steroids for several reasons. First, this time interval corresponds closely to
the extracellular equilibration time of mannitol in eel ventricle strips
(Rodnick et al., 1997) and the
preincubation times in mammalian hearts (flutamide;
Remmers et al., 1997
) and
isolated tissues (DFMO; Koenig et
al., 1983
). We also wanted to avoid the cytotoxic effects
of these drugs, which can occur after extended exposure
(Wang et al., 2002
).
To identify the possibility of membrane receptors for sex steroids and C, we
used BSA-coupled conjugates of T (0.3 µmol l1), C (0.1
µmol l1) and E2 (1 nmol l1). BSA is a
large protein (Mr=68 000) and, when conjugated to
steroids, renders the molecule membrane impermeable
(Stevis et al.,
1999
). BSA-conjugated steroids are commonly used to identify
steroid actions that do not involve classical nuclear receptors
(Falkenstein et al.,
2000
).
Specific intracellular pathways for possible steroid action on the heart were investigated using a single dose of inhibitory compounds prior to steroid administration. Inhibitory compounds included guanosine diphosphate (GDP; 500 µmol l1; an inhibitor of G-protein activation), (DFMO; 10 mmol l1; an inhibitor of ornithine decarboxylase and polyamine synthesis) and L-NAME (1 mmol l1; an inhibitor of NO synthase). In addition, the responsiveness of trout cardiac tissue to epinephrine (1 µmol l1) was tested, both in the presence and absence of steroids to address hormone additive effects and the effectiveness of GDP. Ventricle strips were always pre-treated with inhibitory and stimulatory agents 10 min prior to administration of steroids.
Data analysis
For each ventricle strip, force and other variables were calculated from
data acquired at 0, 1, 2, 3, 5, 10, 20 and 30 min after a 1 h equilibration
period. Data are expressed as means ±
S.E.M. of the percent change of basal
inotropism. Cardiac performance between control (strips exposed only to
Ringer, steroid vehicle or inhibitor) and steroid-treated strips for both
sexes was assessed by a two-way analysis of variance (ANOVA) with Bonferroni
post-hoc corrections using SPSS software. A one-way ANOVA was used to
examine the effects of contraction frequency on steroid-induced inotropism in
males and females. Statistical significance was set at P<0.05.
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Results |
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Although increases in contractile force (F) were observed, no significant changes were observed in tp, t0.8r or ±d F/dT after exposure to sex steroids or C (Table 1). Thus, although a sex-specific, positive inotropic effect was produced by steroid hormones on trout cardiac tissue, the timing of contraction and relaxation was conserved.
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Contraction frequency and steroid-induced positive inotropism
We observed a negative forcefrequency relationship over the range of
contraction frequencies examined. Compared with 0.2 Hz, relative force
production by the same strip was reduced by approximately 11, 31 and 46% at
0.5, 0.8 and 1.0 Hz, respectively (N=4). For males and females, the
maximum inotropic effects of sex steroids and C were observed at 0.5 Hz (males
ANOVA, F3,28=5.19, P=0.017; females
F2,20=6.32, P=0.024), with reduced, yet
significant, hormone effects occurring at 0.8 Hz (males ANOVA
F3,28=4.92, P=0.045; females
F2,20=5.03, P=0.039)
(Fig. 5). The inotropic effects
of steroids were not observed at 0.2 Hz and 1.0 Hz.
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Signaling pathways and possible mechanisms responsible for steroid actions
Flutamide, mifepristone and tamoxifen had no independent effects on
ventricle strip performance (ANOVA, F6,34=1.96,
P=0.382). In males, pretreatment of ventricle strips with flutamide
blocked the increased inotropism induced by T and 11KT (ANOVA,
F6,34=6.96, P=0.018). Pretreatment of female
strips with tamoxifen inhibited the increase of contractile force induced by
E2 (ANOVA, F6,30=4.74, P=0.028). Pretreatment
with mifepristone completely blocked effects of C in both males and females
(ANOVA, F6,37=5.32, P=0.020). By contrast,
flutamide did not inhibit the positive inotropism in C-treated strips, and
mifepristone did not affect inotropism in T- or 11KT-treated strips
(Fig. 6). This suggests that
each steroid is acting through separate receptor types in males. In females,
however, tamoxifen also inhibited the positive inotropism elicited by C. It is
therefore possible that tamoxifen may block both E2 and C receptors in the
heart of female rainbow trout, or act via a common intracellular
mechanism.
|
Potential pathways by which steroid actions can be mediated include: (1) intracellular or membrane-bound receptors; (2) polyamine synthesis; (3) NO production and (4) G-protein activation. Based on negative results with physiological concentrations of BSA-conjugated T, C or E2 and studies using receptor antagonists, it appears that the improved inotropism induced by `free' steroids involves intracellular binding to specific receptors. Pre-treatment of ventricle strips with DFMO had no independent effects on basal contractile performance, and the addition of steroids to DFMO-treated strips from both males and females showed no change in inotropism (Fig. 7). This suggests that polyamine synthesis plays an important role in promoting steroid-induced stimulation of myocardial contractility in rainbow trout. Pretreatment with L-NAME had no independent effect on ventricle strip performance. Strips receiving T, 11KT, C or E2 after treatment with L-NAME showed no improved inotropism (Fig. 8). This finding provides evidence that steroid-induced positive inotropism may also involve production of NO, either by myocytes or nearby endothelial cells, and subsequent intra- or intercellular signaling.
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Unlike polyamines and NO, it doesn't appear that G proteins are mediators of the steroid-induced positive inotropism in trout cardiac muscle. Although GDP had no independent effects on ventricle strip performance, T, 11KT, C and E2 increased contractile force (to a similar degree as strips receiving steroid only) in strips pretreated with GDP. Evidence for GDP effectiveness and G-protein importance to positive inotropism was provided when ventricle strips from male and female rainbow trout were pretreated with GDP and exposed to epinephrine. As expected, and yet in contrast to the steroids, GDP completely blocked the stimulatory effects of epinephrine on contractility (data not shown). In addition, adrenergic stimulation of inotropism with epinephrine was completely additive to the actions of sex steroids and C.
Cholesterol and aldosterone did not affect contractile performance of ventricle strips from male or female rainbow trout (ANOVA, F6,37=1.65, P=0.347). Control strips receiving just ethanol showed an 8% decrease in contractile force independent of any other treatment (ANOVA, F6,32=3.17, P=0.071), whereas cyclodextrin had no independent effects on contractile performance (ANOVA, F6,30=1.27, P=0.431). It is noteworthy that ventricle strips from male and female fish anesthetized with buffered tricaine or benzocaine showed no response to concentrations of steroids that produced maximal positive inotropism in strips from animals that were euthanized by physical trauma to the head (Table 2; ANOVA, F6,12=1.68, P=0.378).
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Discussion |
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Cortisol, the primary stress hormone in both male and female rainbow trout,
fluctuates throughout a fish's life
(Pottinger et al., 1995) and
mediated a functional response in all trout hearts examined. However, cardiac
muscle from females was more sensitive than males to C. This novel finding
suggests that sex differences exist in receptor binding or intracellular
signaling by C. It is noteworthy that Caldwell et al.
(1991
) demonstrated similar
levels of total C in male and female rainbow trout, and yet mature females
exhibit a lower free C compared with mature males and immature fish. This
difference reflects a higher percent C bound to corticosteroid-binding protein
in female fish. Ultimately, the concentration of unbound steroid hormones may
determine whether steroid-induced inotropism has biological relevance and may
explain sex differences in hormone sensitivity. Although it takes just
310 min for cardiac tissue to respond to steroid hormones in
vitro, the fact that the increase in C during stressful periods takes
much longer (
30 min; Gamperl et
al., 1994
) makes it unlikely that C-induced elevations in
cardiac performance would occur following acute stressors.
The finding of steroid-induced inotropism in the heart of male and female
rainbow trout led us to study potential mechanisms of action via
several complimentary approaches. First, we explored whether the type of
anesthesia was important for realization of the inotropic effects of steroids.
Only one previous study had examined the effects of C on cardiac function in
fishes (Farrell et al., 1988),
and these investigators concluded that C had a minimal effect on performance
of perfused isolated hearts from rainbow trout. However, given our data, and
the fact that Farrell et al.
(1988
) used chemically
anesthetized rainbow trout, we were not surprised that they did not report any
positive effects of C on isolated perfused hearts. In the current study, even
short-term (<5 min) exposure of intact fish to common anesthetics prevented
the subsequent response of isolated ventricle strips to C and sex steroids
(Table 2). Anesthetics,
including benzocaine, can compete with steroids for hydrophobic binding sites
on the nicotinic acetylcholine receptor of Torpedo marmorata
(Arias et al., 1990
) and
decrease the affinity of the canine Na+/K+-ATPase for
ouabain (Kutchai et al.,
2000
). Thus, future studies should recognize the possibility that
even brief chemical anesthesia of fishes can cause confounding effects of
steroid hormone responsiveness in isolated cardiac tissue. We also studied the
specificity of steroids for promoting inotropism by adding a generic precursor
(cholesterol) and non-physiological steroid (aldosterone) to the experimental
protocol. Based on the findings that these hormones were devoid of activity,
it appears that there is selectivity for gonadal steroids and C to promote
myocardial contractility in rainbow trout.
This study is also the first to investigate frequency-dependent effects of
steroids on cardiac performance in fish. Under the defined experimental
conditions, optimal steroid effects on increasing contractile force were
observed at 0.5 Hz, were reduced at 0.8 Hz and absent at 0.2 Hz and 1.0
Hz.Physiological cardiac frequencies of rainbow trout at 1415°C
(Clark and Rodnick, 1999) fall
within the range of frequencies tested. Generally speaking, the majority of
teleost species show a negative forcefrequency response
(Shiels et al., 2002
). In the
current study, myocardial contractile force declined with frequencies above
0.5 Hz, which is consistent with the findings of Hove-Madsen and Gesser
(1989
) in rainbow trout. Given
this frequency dependency of steroid effectiveness in vitro, both
body temperature and activity level may influence steroid action on the
cardiovascular system in vivo.
In mammals, steroid hormones affect cellular function by a variety of mechanisms, including binding to membrane or nuclear receptors and several complex signal-transduction pathways. To elucidate the underlying mechanism that steroids use to modulate myocardial contractile performance in trout, we exposed ventricle strips to (1) BSA-conjugated steroids and (2) chemical inhibitors of steroid receptors and signaling cascades involving synthesis of polyamines, NO and G-protein activation. Although it is possible that the observed steroid effects involved a membrane-bound receptor rather than a traditional nuclear receptor, the fact that free steroids, but not BSA-conjugated steroids, had effects on contractility implies that membrane-bound receptors were not responsible for the observed effects. The fact that the positive inotropic actions of T, 11KT, E2 and C were inhibited completely by pretreatment with receptor blockers also suggests that the signaling mechanism for each hormone involves binding to its intracellular receptor. One important limitation of our tissue preparation is that we cannot identify whether the binding of steroids and chemical signal(s) originated in myocytes or some other cell near the myocyte (e.g. endothelial cell). Future studies involving isolated myocytes or nonmyocyte cells will be necessary to identify the target of steroid binding and signaling in trout cardiac tissue.
Based on the rapid timing of enhanced cardiac contractility following
exposure to steroid hormones (observed within 3 min and maximal after 10 min),
it appears that the mechanism of action involves a nongenomic pathway. The
observed time course of inotropic effects in the rainbow trout ventricle
agrees with previous studies showing rapid effects of C on muscle glycogen
metabolism (Milligan, 2003)
and similar effects in rat cardiac tissue
(Rubín et al., 1999
).
However, we cannot rule out the possibility that a genomic mechanism is
responsible. For example, the synthesis of ornithine decarboxylase, which
appears to play an important role in the myocardial response to steroid
hormones, has a 1030 min turnover time in mammalian cells and increases
rapidly after hormone stimulation
(Bachrach, 1984
). Additional
studies of mRNA transcription and protein expression will be required to
define whether the steroid response in cardiac tissue is genomic or
nongenomic.
Rapid effects of steroids are likely to be mediated through signaling
cascades involving polyamines, NO and G-protein activation. The aliphatic
amines putrescine, spermidine and spermine are ubiquitous cellular compounds
that appear important in cell growth and differentiation
(Marton and Pegg, 1995).
Previous research in mammals has indicated that polyamines contribute to
androgenic stimulation of calcium flux and membrane transport
(Koenig et al., 1989
). Based
on the selective inhibition of ornithine decarboxylase, the rate-regulating
enzyme of polyamine synthesis, our results demonstrate that polyamine
synthesis is required for steroid-enhanced force in ventricle strips from
rainbow trout. At this point, however, we do not know which polyamine is the
active species or the link between polyamine synthesis and other signaling
pathways that ultimately promote cardiac inotropism in fishes.
NO, produced autocrinally by cardiomyocytes or paracrinally by endothelial
cells, has also been implicated as a key molecule in the regulation of
contractile performance. In mammalian cardiac muscle, NO can exert positive
and negative effects on myocardial contractility, depending on the
concentration of NO, the status of the endocardial endothelium and the degree
of cholinergic or adrenergic stimulation
(Mohan et al., 1996;
Balligand and Cannon, 1997
). An
NO-induced reduction in stroke volume and work has been described in the eel
heart (Imbrogno et al., 2001
),
although NO signaling is involved in angiotensin II-mediated inotropism
(Imbrogno et al., 2003
).
Recent research in mammals has suggested that E2 has rapid effects on
endothelial cell function, including stimulation of NO synthase
(Chambliss and Shaul, 2002
).
Furthermore, inhibitors of NO synthase, such as L-NAME, have been shown to
increase the response of myocytes to adrenergic agonists
(Balligand et al., 1993
). Our
studies demonstrate that L-NAME effectively blocks the steroid-induced
positive inotropism in the trout ventricle and provide evidence that steroid
effects on contractile performance require products of NO synthase. Whether we
inhibited endothelial and/or cardiomyocyte NO synthase remains to be
determined. Ultimately, elucidation of the signaling mechanism that NO and
polyamines use will enhance understanding of the role of steroid hormones in
modulating cardiac function in fishes. One possible target for both polyamines
and NO is the enzyme guanylate cyclase, which, when activated, could raise
intracellular cGMP and subsequently stimulate cGMP-dependent protein kinase
activity (Tantini et al.,
2001
).
Although several, rapid, nongenomic effects of steroids appear to be
mediated through G-protein activation
(Cato et al., 2002), this was
not the case for the positive inotropic effects of steroids on the trout
heart. This disassociation was inferred from the observations that (1) GDP had
no independent effects on ventricle strip performance, and all the steroids
tested (T, 11KT, C and E2) increased contractile force in strips pre-treated
with GDP, and (2) the positive inotropism induced with epinephrine was
completely additive to the actions of sex steroids and C. Finally, it will be
of interest in future studies to distinguish the separate pathways or
mechanisms that sex steroids and C use to modulate cardiac inotropism in male
and female rainbow trout. As mentioned previously, we observed additive
effects for androgens (T and 11KT) and C on promoting contractility of
ventricle strips from males. Additive effects, and therefore the involvement
of separate signaling pathways, were not observed between E2 and C in
females.
Ultimately, care must be taken when attempting to extrapolate the results of the current study to in vivo cardiac performance and draw conclusions about the overall importance of steroid hormones. One limitation of this study is the use of a multicellular isometric muscle preparation. Future research should include single-cell experiments on cardiomyocytes and endothelial cells to better define the target of steroid hormones, possible intercellular/paracrine and autocoid signaling, and intracellular pathways/mechanisms of action. In addition, it will be important to conduct experiments on whole animals to validate the physiological relevance of steroid-induced cardiac inotropism in fishes. Our study also strengthens the need to document the sex of fishes when conducting studies of hormone responsiveness and, more specifically, to include both sexes when performing studies on the regulation of myocardial contractility in salmonid fishes.
Summary
This study demonstrates that steroid hormones promote myocardial
contractile performance in vitro in male and female rainbow trout.
The observed effects of gonadal steroids and C depend upon (1) hormone
concentration, (2) contraction frequency, (3) sex of the animal and (4)
involve specific receptor types. The positive inotropism of sex steroids and
cortisol was additive in males, but not females, and these effects were
abolished when fish were anesthetized with either benzocaine or MS-222.
Maximal effects are realized within 10 min and it appears that the improved
inotropism involves (1) intracellular binding of steroids, (2) production of
polyamines and NO but (3) no activation of G proteins.
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Acknowledgments |
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References |
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---|
Arias, H. R., Sankaram, M. B., Marsh, D. and Barrantes, F. J. (1990). Effect of local anaesthetics on steroid-nicotinic acetylcholine receptor interactions in native membranes of Torpedo marmorata electric organ. Biochim. Biophys. Acta 1027,287 -294.[Medline]
Bachrach, U. (1984). Physiological aspects of ornithine decarboxylase. Cell. Biochem. Funct. 2, 6-10.[Medline]
Balligand, J. L. and Cannon, P. J. (1997).
Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences.
Arterioscler. Thromb. Vasc. Biol.
17,1846
-1858.
Balligand, J. L., Kelly, R. A., Marsden, P. A., Smith, T. W. and Michel, T. (1993). Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc. Natl. Acad. Sci. USA 90,347 -351.[Abstract]
Batterham, A. M., George, K. P. and Mullineaux, D. R. (1997). Allometric scaling of left ventricular mass by body dimensions in males and females. Med. Sci. Sports Exerc. 29,181 -186.[Medline]
Beyer, M. E., Yu, G., Hanke, H. and Hoffmeister, H. M.
(2001). Acute gender-specific hemodynamic and inotropic effects
of 17ß-estradiol on rats. Hypertension
38,1003
-1010.
Bordallo, C., Rubin, J. M., Varona, A. B., Cantabrana, B., Hildalgo, A. and Sanchez, M. (2001). Increases in ornithine decarboxylase activity in the positive inotropism induced by androgens in isolated left atrium of the rat. Eur. J. Pharmacol. 422,101 -106.[CrossRef][Medline]
Buitrago, C., Massheimer, V. and de Boland, A. R. (2000). Acute modulation of Ca2+ influx on rat heart by 17beta-estradiol. Cell Signal. 12, 47-52.[CrossRef][Medline]
Caldwell, C. A., Kattesh, H. G. and Strange, R. J. (1991). Distribution of C among its free and protein-bound fractions in rainbow trout (Oncorhynchus mykiss): evidence of control by sexual maturation. Comp. Biochem. Physiol. A 99,593 -595.[CrossRef][Medline]
Campbell, P. M., Fostier, A., Jalabert, B. and Truscott, B. (1980). Identification and quantification of steroids in the serum of rainbow trout during spermiation and oocyte maturation. J. Endocrinol. 85,371 -378.[Abstract]
Campbell, P. M., Pottinger, T. G. and Sumpter, J. P. (1992). Stress reduces the quality of gametes produced by rainbow trout. Biol. Reprod. 47,1140 -1150.[Abstract]
Carragher, J. F., Sumpter, J. P., Pottinger, T. G. and Pickering, A. D. (1989). The deleterious effects of C implantation on reproductive function in two species of trout, Salmo trutta L. and Salmo gairdneri Richardson. Gen. Comp. Endocrinol. 76,310 -321.[Medline]
Cato, A. C., Nesti, A. and Mink, S. (2002). Rapid actions of steroid receptors in cellular signaling pathways. Sci STKE 138,RE9 .
Chambliss, K. L. and Shaul, P. W. (2002). Rapid activation of endothelial NO synthase by estrogen: Evidence for a steroid receptor fast-action complex (SRFC) in caveolae. Steroids 67,413 -419.[CrossRef][Medline]
Clark, R. J. and Rodnick, K. J. (1999). Pressure and volume overloads are associated with ventricular hypertrophy in male rainbow trout. Am. J. Physiol. 277,R938 -R946.[Medline]
Davie, P. S. and Thorarensen, H. (1997). Heart
growth in rainbow trout in response to exogenous testosterone and 17-
methyltestosterone. Comp. Biochem. Physiol. A
117,227
-230.[CrossRef]
Donaldson, E. and Fagerlund, U. H. M. (1968). Changes in C dynamics of sockeye salmon (Oncorhynchus nerka) resulting from sexual maturation. Gen. Comp. Endocrinol. 11,552 -561.[Medline]
Falkenstein, E., Tillman, H. C., Christ, M., Feuring, M. and
Wehling, M. (2000). Multiple actions of steroid hormones
a focus on rapid, nongenomic effects. Pharmacol.
Rev. 52,513
-555.
Farrell, A. P., MacLeod, K. R. and Scott, C. (1988). Cardiac performance of the trout (Salmo gairdneri) heart during acidosis: effects of low bicarbonate, lactate and C. Comp. Biochem. Physiol. A 91,271 -277.[CrossRef]
Franklin, C. E. and Davie, P. S. (1992). Sexual maturity can double heart mass and cardiac power output in male rainbow trout. J. Exp. Biol. 171,139 -148.
Gamperl, A. K., Vijayan, M. M. and Boutilier, R. G. (1994). Epinephrine, norepinephrine, and cortisol concentrations in cannulated seawater-acclimated rainbow trout (Oncorhynchus mykiss) following black-box confinement and epinephrine injection. J. Fish Biol. 45,313 -324.[CrossRef]
Hajdu, S. and Szent-Györgyi, A. (1952). Action of DOC and serum on the frog heart. Am. J. Physiol. 168,159 -170.[Medline]
Hartmund, T. and Gesser, H. (1996). Cardiac force and high-energy phosphates under metabolic inhibition in four ectothermic vertebrates. Am. J. Physiol. 271,R946 -R954.[Medline]
Hove-Madsen, L. and Gesser, H. (1989). Force frequency relation in the myocardium of rainbow trout. Effects of K+ and adrenaline. J. Comp. Physiol. 159, 61-69.
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., Cerra, M. C. and Tota, B. (2003).
Angiotensin II-induced inotropism requires an endocardial endothelium-nitric
oxide mechanism in the in-vitro heart of Anguilla anguilla.J. Exp. Biol. 206,2675
-2684.
Koenig, H., Goldstone, A. and Lu, C. Y. (1983). Polyamines regulate calcium fluxes in a rapid plasma membrane response. Nature 305,530 -534.[Medline]
Koenig, H., Fan, C. C., Goldstone, A. D., Lu, C. Y. and Trout, J. J. (1989). Polyamines mediate androgenic stimulation of calcium fluxes and membrane transport in rat heart myocytes. Circ. Res. 64,415 -426.[Abstract]
Kutchai, H., Geddis, L. M. and Farley, R. A. (2000). Effects of local anaesthetics on the activity of the Na,K-ATPase of canine renal medulla. Pharmacol. Res. 41, 1-7.[CrossRef][Medline]
Lin, A. L., Schultz, J. J., Brenner, R. M. and Shain, S. A. (1990). Sexual dimorphism characterizes baboon myocardial androgen receptors but not myocardial estrogen and progesterone receptors. J. Steroid Biochem. Mol. Biol. 37, 85-95.[CrossRef][Medline]
Lou, S. W., Aida, K., Hanyu, I., Sakai, K., Nomura, M., Tanaka, M. and Tazaki, S. (1986). Endocrine profiles in the males of a twice-annually spawning strain of rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 64,212 -219.[Medline]
Marton, L. J. and Pegg, A. E. (1995). Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol. Toxicol. 35,55 -91.[CrossRef][Medline]
Milligan, C. L. (2003). A regulatory role for
cortisol in muscle glycogen metabolism in rainbow trout Oncorhynchus
mykiss Walbaum. J. Exp. Biol.
206,3167
-3173.
Mohan, P., Brutsaert, D. L., Paulus, W. J. and Sys, S. U.
(1996). Myocardial contractile response to nitric oxide and cGMP.
Circulation 93,1223
-1229.
Pottinger, T. G., Balm, P. H. M. and Pickering, A. D. (1995). Sexual maturity modifies responsiveness of the pituitaryinterrenal axis to stress in male rainbow trout. Gen. Comp. Endocrinol. 98,311 -320.[CrossRef][Medline]
Remmers, D. E., Wang, P., Cioffi, W. G., Bland, K. I. and Chaudry, I. H. (1997). Testosterone receptor blockade after trauma-hemorrhage improves cardiac and hepatic functions in males. Am. J. Physiol. 273,H2919 -H2925.[Medline]
Rodnick, K. J., Bailey, J. R., West, J. L., Rideout, A. and
Driedzic, W. R. (1997). Acute regulation of glucose uptake in
cardiac muscle of the American eel Anguilla rostrata. J. Exp.
Biol. 200,2871
-2880.
Rubín, J. M., Hidalgo, A., Bordallo, C., Cantabrana, B. and Sánchez, M. (1999). Positive inotropism induced by androgens in isolated left atrium of rat: evidence for a cAMP-dependent transcriptional mechanism. Life Sci. 65,1035 -1045.[CrossRef][Medline]
Scott, A. P., Bye, V. J., Baynes, S. M. and Springate, J. R. C. (1980a). Seasonal variations in plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 17,495 -505.
Scott, A. P., Bye, V. J. and Baynes, S. M. (1980b). Seasonal variations in sex steroids of female rainbow trout, Salmo gairdneri Richardson. J. Fish. Biol. 17,587 -592.
Shiels, H. A., Vornanen, M. and Farrell, A. P. (2002). The force-frequency relationship in fish hearts a review. Comp. Biochem. Physiol. A 132,811 -826.
Slater, C. H., Fitzpatrick, M. S. and Schreck, C. B. (1995). Characterization of an androgen receptor in salmonid lymphocytes: possible link to androgen-induced immunosuppression. Gen. Comp. Endocrinol. 100,218 -225.[CrossRef][Medline]
Sower, S. A. and Schreck, C. B. (1982). Steroid and thyroid hormones during sexual maturation of coho salmon (Oncorhynchus kisutch) in seawater or fresh water. Gen. Comp. Endocrinol. 47,42 -53.[Medline]
Stevis, P. E., Deecher, D. C., Suhadolnik, L., Mallis, L. M. and
Frail, D. E. (1999). Differential effects of estradiol
and estradiolBSA conjugates. Endocrinology
140,5455
-5458.
Tantini, B., Flamigni, F., Pignatti, C., Stefanelli, C., Fattori, M., Facchini, A., Giordano, E., Clo, C. and Caldarera, C. M. (2001). Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes. Cardiovasc. Res. 49,408 -416.[CrossRef][Medline]
Thorarensen, H., Young, G. and Davie, P. S. (1996). 11-ketotestosterone stimulates growth of heart and red muscle in rainbow trout. Can. J. Zool. 74,912 -917.
Wang, H. X., Ma, X. C., Deng, Q. L. and Li, D. (2002). Cytotoxicity of flutamide and 2-hydroxyflutamide and their effects on CYP1A2 mRNA in primary rat hepatocytes. Acta Pharmacol. Sin. 23,562 -566.[Medline]
Wehling, M. (1997). Specific, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59,365 -393.[CrossRef][Medline]
Yano, K., Tsuda, Y., Kaji, Y., Kanaya, S., Fujino, T. and Niho, Y. (1994). Effects of hydrocortisone on transmembrane currents in guinea pig ventricular myocytes possible evidence for positive inotropism. Jpn. Circ. J. 58,836 -843.[Medline]