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Vol. 281, Issue 1, 218-225, 1997
Department of Cardiovascular Research, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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Ethinyl estradiol (EE2), administered orally to ovariectomized (ovex) rats, has been shown to prevent loss of bone mineral density and to decrease serum cholesterol levels. Radioligand binding studies with the dihydropyridine (DHP) [3H]PN200-110 were undertaken to characterize calcium (Ca++) channels in cardiac and aortic tissues from ovex rats treated for 35 days with EE2 (0.1 mg/kg day p.o.) or vehicle, and from vehicle-treated sham-operated controls (sham). Cardiac tissues from EE2-treated rats displayed significant increases in the density (Bmax) of high-affinity DHP binding sites (505 ± 46 fmol/mg) compared with vehicle-treated ovex rats (303 ± 35 fmol/mg); DHP Bmax values from EE2-treated cardiac tissues were not significantly different from vehicle-treated shams (385 ± 76 fmol/mg). Cardiac Ca++ efflux channels from sarcoplasmic reticulum were assessed with [3H]ryanodine. [3H]Ryanodine Bmax values were not affected by EE2 treatment. However, [3H]ryanodine Kd values in preparations from EE2-treated rats were significantly decreased (10 ± 2 nM) compared with ovex rats (35 ± 11 nM) and were similar to values in sham rats (8 ± 2 nM). Cardiac beta adrenoceptors were not affected by EE2 treatment, which thus confirmed the selective regulation of DHP receptors by EE2. Aortic preparations from EE2-treated rats exhibited significant increases in DHP receptors (125 ± 37 fmol/mg) compared with both vehicle-treated ovex rats (32 ± 3 fmol/mg) and vehicle-treated shams (24 ± 9 fmol/mg). There were no differences in the binding affinity (Kd) of [3H]PN200-110 for cardiac or aortic sites among the three groups. To ascertain if EE2-mediated increases in Ca++ channel density and ryanodine binding affinity affected in vivo responses to agonists that use extracellular and intracellular Ca++ stores, responses to BAY k 8644 and norepinephrine were examined in pithed rats from the same three groups. No significant differences in hemodynamic responses occurred among the three groups to BAY k 8644 or norepinephrine. Thus, in female ovex rats, prolonged treatment with EE2 resulted in increased density of cardiac and aortic calcium channels which did not translate into increased calcium-mediated inotropic, rate or pressor responses. Conversely, EE2 treatment in ovex rats prevented the decrease in cardiac [3H]ryanodine binding affinity evident in vehicle-treated ovex rats. These data suggest that EE2 treatment in normotensive ovex rats resulted in modulation of both the L-type and sarcoplasmic reticulum Ca++ channels, and these alterations maintained cardiovascular homeostasis.
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Introduction |
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Abstract
Introduction
Methods
Results
Discussion
References
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Estrogen replacement therapy in
postmenopausal women has a cardioprotectant effect associated with
reduced mortality from coronary artery disease, myocardial infarction
and stroke. Whereas some of the protective effects of estrogen are
thought to be mediated through lipid lowering (Nabulsi et
al., 1993
) and inhibition of atherosclerotic progression in
coronary arteries (Adams et al., 1987; Williams et
al., 1990), estrogen has been shown to act on the blood vessel
wall (Wren, 1992; Mendelsohn and Karas, 1994) and, possibly, directly
on the heart (Raddino et al., 1986; Jiang et al.,
1992). Animal studies showed estrogen increased arterial flow and
cardiac output and decreased vascular resistance (Naden and Rosenfeld,
1985; Stice et al., 1987b; Magness and Rosenfeld, 1989),
effects that may be accompanied by increases in heart rate or decreases
in MAP. These cardiac and vascular effects are consistent with the
possibility that estrogen or a metabolite affects Ca++
influx.
The idea that estrogen may have calcium antagonist properties has been
investigated by several groups. Jiang and co-workers (1991) reported
that, in rabbit coronary artery rings devoid of endothelium, micromolar
concentrations of 17
-estradiol diminished contractile responses to
Ca++ and induced relaxation in ring segments precontracted
with BAY. With rat aortic ring segments with and without endothelium,
Cohen and Susemichel (1996) demonstrated that 17-estradiol (1 and 10 µM) produced a dextral shift and depression of maximum contraction to
increasing concentrations of BAY. 17-Estradiol also was shown to
have a negative inotropic effect on isolated cardiac myocytes (Jiang
et al., 1992). Decreases in cell shortening were accompanied by decreases in action potential duration and Ca++ influx.
Consistent with these in vitro observations, ex
vivo Ca++ uptake into uterine arteries of female pigs
was decreased during periods of high circulating estrogen (estrous and
early pregnancy; Stice et al., 1987a, b), coincidental with
increased uterine blood flow (Ford et al., 1983).
Acute administration of 17-estradiol in cultured myometrial cells
isolated from late-pregnant rats inhibited Ca++ channel
currents, which resulted in a negative shift of the steady-state inactivation curve and suggested an estradiol-mediated depression of
contractile activity (Yamamoto, 1995). However, Garfield (1984) reported that the effect of chronic estrogen was increased myometrial activity. Moreover, Batra and Bengtsson (1978), who had previously noted the inhibitory effect of estrogen in vitro on
Ca++ uptake in rat uterus, reported that continuous
administration of estrogen to ovex rats resulted in increased
Ca++ influx into isolated uterine smooth muscle, and this
was accompanied by increased density of uterine calcium channels,
compared with untreated ovex controls (Batra, 1987). Because
Ca++ uptake into myometrium was significantly increased
after 24 h estrogen treatment, but not at 1 h (Batra and
Sjogren, 1983), and was not evident in urethra or urinary bladder
(Batra, 1986), Batra hypothesized that estrogen treatment resulted in
genomic activation of new Ca++ channels in "target
organs."
The fact that high estrogen concentrations in vitro can inhibit cardiac and vascular Ca++ channel influx, together with the observation that in vivo administration of estrogen can act to increase Ca++ channel density in the uterus, prompted us to examine the effects of orally administered estrogen on the binding affinity and density of cardiac and aortic Ca++ channels from ovex rats. In addition, we quantified cardiac ryanodine receptors and beta adrenoceptors to determine whether estrogen-mediated effects were selective to the L-type Ca++ channel. Further, we asked how the observed effects on Ca++ channel density altered hemodynamic responses to agonists that used extracellular or intracellular calcium in their mechanism of action.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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Animal protocol.
Selection and dosing of rats were
essentially as described by Black et al. (1994).
Sprague-Dawley virgin female rats (6 months old, obtained from Charles
River Laboratories, Portage, MI) were divided into three groups:
ovariectomized (ovex, administered vehicle 1 ml/kg/day p.o.);
ovariectomized and administered ethinyl estradiol (EE2, 0.1 mg/kg/day
p.o.) and sham-operated (sham), which also received vehicle (1 ml/kg/day p.o.). The 0.1 mg/kg dose of EE2 was sufficient to increase
serum estradiol in ovex animals to levels approaching those of
sham-operated animals. Also, uterine weights of the EE2-treated rats
were comparable with the sham. Vehicle was 20%
hydroxypropyl-S-cyclodextrin. Animals were dosed for 35 days and
sacrificed by excess CO2 on day 36. Ethinyl estradiol and
hydroxypropyl-S-cyclodextrin were obtained from Sigma
Chemical Co. (St. Louis, MO) and Aldrich Chemical Co. (Milwaukee, WI),
respectively.
Determination of serum cholesterol and 17-estradiol
concentrations.
Measurements of serum cholesterol were performed
according to the protocol of Black et al. (1994) and were
quantified by blood taken by cardiac puncture in anesthetized animals
on day 36. Estrogen levels were determined by a solid-phase
125I-radioimmunoassay designed to measure 17-estradiol
in serum (Diagnostic Products Corporation, Los Angeles, CA).
Membrane preparation and radioligand binding studies.
Hearts
(trimmed of atria and aorta) and aortas were dissected, quickly frozen
and stored at 
70°C if membranes were not prepared immediately.
Microsomal membrane vesicles (3-4 g) were isolated from minced hearts
or aortas from each group as previously described (Jones et
al., 1979); preparations were stored in 0.25 M sucrose/30 mM
histidine at 70°C.
). Assays were terminated by rapid filtration
onto Whatman GF/C filter paper. Assay (and wash) buffer was 50 mM
Tris/HCl (pH 7.3), 1 mM ethylenediaminetetraccetic acid and 12 mM
MgCl2. Nonspecific binding was defined as amount of bound
ligand remaining in the presence of 1 µM nifedipine (Sigma). For
competition binding assays in cardiac and aortic membrane preparations,
increasing concentrations of BAY (Research Biochemicals Inc. Wayland
MA, 0.01 nM-30 µM) were used to displace
[3H]PN200-110 (approximately 0.7 nM). Protein was
measured by the method of Bradford (1976).
Binding studies with [3H]ryanodine (NEN, 60 Ci/mmol), in
cardiac microsomal vesicle preparations were performed in glass tubes (12 × 75 mm, total volume 100 or 200 µl) at 37°C with 25 µg
protein as described by Pessah et al. (1990). Assay buffer
consisted of 20 mM N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid,
pH 7.1, 250 mM KCl, 15 mM NaCl, 1 mM MgCl2 and 100 µM
free Ca++ (Bers, 1982). Cardiac microsomal vesicles were
incubated with approximately 200,000 dpm of [3H]ryanodine
(6 nM). All [3H]ryanodine binding assays were done at
physiologic Mg++ (1 mM) and optimum Ca++ (100 µM) concentrations. Incubations were terminated after 2 h with 2 ml ice-cold wash buffer (20 mM Tris-HCl, pH 7.1, 250 mM KCl, 15 mM
NaCl, 50 µM CaCl2 and 1 mM CHAPS), followed by rapid filtration onto 0.3% PEI-soaked Whatman GF/C filter paper, with a
Brandel harvester (Gaithersburg, MD). The filters were washed once with
4 ml of ice-cold wash buffer. Retained radioactivity was then
determined. Nonspecific binding was defined as the amount of bound
ligand remaining in the presence of 10 µM ryanodine (Calbiochem, La
Jolla, CA).
Binding studies in cardiac microsomal preparations, with increasing
concentrations of the beta receptor ligand
[125I]CYP (NEN, 2200 Ci/mmol, 0.03-10.0 nM), were
performed in 12 × 75 mm glass tubes (total volume, 500 µl) at
23°C for 90 min with 100 µg protein per tube. Assays were
terminated by rapid filtration onto Whatman GF/C filter paper. Assay
buffer was 50 mM Tris/HCl (pH 7.3), 1 mM ethylenediaminetetraccetic
acid, 12 mM MgCl2. Wash buffer was 20 mM Tris/HCl (pH 7.3),
50 mM NaCl, 12 mM MgCl2 and 1 mM CHAPs. Nonspecific binding
was defined as amount of bound ligand remaining in the presence of 1 µM propranolol (Sigma).
Hemodynamic measurements.
Cardiovascular parameters in
response to BAY and NE (Sigma) were determined in pithed rats from each
of the three groups (sham, ovex and EE2). Rats were pithed with a 19 G
stainless steel rod while under Metofane (methoxyflurane) anesthesia
and immediately connected to a Harvard model 683 small animal
ventilator through PE240 polyethylene tubing inserted into the trachea.
Rats were respired with room air (1.0 ml/100 g b.wt., 80-90
strokes/min). The right carotid artery was cannulated with polyethylene
tubing (PE90) which was connected to a Spectramed P23XL pressure
transducer for recording MAP. The output signal from the pressure
measurement was fed into a 9857B cardiotachometer coupler to measure
heart rate. After a midsternal thoracotomy, left ventricular pressure was measured from a signal derived via a cannula (PE90
tubing) directly inserted into the left ventricle and connected to a
P23XL transducer and 9879 coupler (modified from Hayes and Bowling, 1987). Measurements were obtained of LV
dP/dtmax (dP/dt) by
electrical differentiation of the left ventricular pressure signal. All
signals were recorded on a Beckman R611 physiological recorder.
Pressure transducers and polyethylene tubing were filled with
physiological saline containing 10 U/ml heparin. The right femoral vein
was cannulated with polyethylene tubing (PE50) connected to a syringe for intravenous administration of agonist. Each animal received only
one compound. Dose-response curves were obtained by cumulative administration of increasing i.v. bolus doses of agonist after responses had returned to baseline or plateaued.
Calculations and statistics.
Radioligand binding affinity
and receptor density were determined from saturation isotherm data by
the nonlinear regression analysis program Lundon-1 (Lundeen and Gordon,
1986). Ki values for BAY from competition curves
were determined from Lundon-2 (Lundeen and Gordon, 1986). Multiple data
points within a group were presented as mean and standard error of the
mean (S.E.M.). Statistical significance (P < .05) among groups
was evaluated by analysis of variance followed by Dunnett's
t test, using the ovex group as control. ED50
values (doses denoting 50% maximum response) from hemodynamic
measurements were determined by log-logit transformation of the data.
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Results |
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Abstract
Introduction
Methods
Results
Discussion
References
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Physiological effects and serum lipids.
The effects of
ovariectomy and EE2 treatment on uterine wet weight, body weight and
serum concentrations of cholesterol and 17-estradiol are shown in
table 1. Both sham and EE2-treated animals had
significantly lower body weights (P < .01) and significantly greater uterine weights (P < .01) than the ovex controls. Heart weights were not significantly different among the three groups. The
slightly decreased heart weights of the EE2-treated animals were
consistent with lower body weights, and the heart/body ratio was
similar for all animals (data not shown), thus indicating no myocardial
hypertrophic response to EE2. EE2 at 0.1 mg/kg for 35 days lowered
total serum cholesterol by 60% (P < .0001). No significant
difference in cholesterol levels was observed between sham and ovex
rats. As expected, serum 17-estradiol concentrations were decreased
in ovex rats; and EE2 treatment raised serum estrogen levels to those
measured in sham rats.
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Characteristics of cardiac and aortic
[3H]PN200-110 binding.
[3H]PN200-110 bound with specificity and high affinity
to the DHP receptor of the calcium channel in both heart and aorta. Binding in membrane preparations from all three groups was reversible, protein dependent and saturable. Analysis of saturation isotherms from
cardiac tissues (fig. 1) revealed high-affinity DHP
binding sites (Bmax) that were significantly
increased by approximately 65% in EE2-treated rats compared with ovex
rats, but were not significantly different from sham values (fig.1 and
table 2). Binding affinities (Kd)
were best described by a single-site model. Kd
values were not significantly different among the three groups in
cardiac preparations (188-204 pM), which indicated that neither ovariectomy nor subsequent EE2 treatment affected affinity of [3H]PN200-110 binding to cardiac L-type Ca++
channels. In aortic tissues from EE2-treated rats, the density of DHP
binding sites (Bmax) was increased by greater
than 4-fold compared with both ovex and sham controls (fig.
2 and table 2). Aortic [3H]PN200-110
Bmax values of sham animals were not
significantly different from ovex. Binding affinity for
[3H] PN200-110 in aortic tissue was similar in all three
groups.
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-estradiol. Concentrations of 1 nM to
10 µM estradiol had no effect on binding in either cardiac or
vascular preparations (data not shown).
To compare relative binding affinities of BAY for cardiac and aortic
Ca++ channels, competition binding assays with BAY and
[3H]PN200-110 were undertaken (figs. 3 A
and B). Lundon-2 determination of the best fit model indicated one site
of interaction for BAY for both heart and aorta, although a two-site
model was possible in some of the cardiac preparations.
Ki values for BAY in cardiac preparations were
as follows: sham, 2.8 ± 1.5 nM; ovex, 2.1 ± 1.0 nM; EE2,
2.9 ± 1.6 nM. Aortic Ki values for BAY
were slightly, but not significantly, higher and were as follows: ovex,
7.5 ± 4.6 nM; sham, 7.2 ± 1.5 nM; EE2, 7.8 ± 2.3 nM.
Thus, binding affinities of BAY were similar in both cardiac and aortic
tissue, and EE2 treatment did not alter binding affinity of BAY in
either tissue.
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Effect of EE2 treatment on cardiac SR Ca++ efflux channels and beta adrenoceptors. To determine whether the increases in Ca++ channel density affected the cardiac SR efflux channels, we measured [3H]ryanodine binding in microsomal membrane preparations from sham, ovex and EE2-treated rats. Interestingly, the binding density of [3H]ryanodine was not affected by EE2 treatment when compared with ovex rats (table 3). However, [3H]ryanodine binding affinity was increased significantly (P < .05) in EE2-treated rats compared with ovex rats, with Kd values approximately those of shams (table 3).
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Effect of EE2 treatment on in vivo hemodynamic responses to BAY and NE. To gain insight on how increases in cardiac and vascular Ca++ channels might be manifested as in vivo functional changes, we investigated hemodynamic responses to BAY and NE in open-chest, pithed female rats from ovex, EE2-treated and sham groups. Both agents had inotropic and vasoconstrictor effects in the rat, and NE elicited increases in heart rate of greater than 140 beats/min. The two agonists were chosen to compare responses elicited by 1) a compound that relies primarily on extracellular calcium (BAY is a Ca++ channel agonist that greatly increases the mean "open" time of the channel) and 2) a compound that uses intracellular Ca++ stores (NE is an alpha adrenoceptor agonist in the vasculature and a beta adrenoceptor agonist in ventricular muscle).
Despite the increase in cardiac Ca++ channel binding densities, the potency and efficacy of BAY- and NE-elicited inotropic responses in EE2-treated and sham rats were not significantly different from values measured in ovex animals. Comparative increases in cardiac contractility in response to BAY and NE are shown in figure 4; ED50 values and maximum responses (at 100 µg/kg dosage) are listed in table 4. Because of its direct action on cardiac beta adrenoceptors, NE elicited greater inotropic and chronotropic responses than BAY.
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Discussion |
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Abstract
Introduction
Methods
Results
Discussion
References
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The purpose of this study was to determine whether prolonged EE2
treatment in ovex rats produced any measurable changes in the binding
characteristics of cardiac and SR Ca++ channels and cardiac
beta adrenoceptors, and whether any noted differences in
receptor density or affinity translated in vivo into
measurable hemodynamic effects. We now report that EE2 treatment of
ovex rats for 35 days resulted in significant (P < .05) increases in both cardiac and aortic L-type Ca++ channel density with
no change in receptor binding affinity. The dosage of EE2 (0.1 mg/kg/day) was sufficient to effect the expected increases in uterine
weight and decreases in cholesterol in agreement with Black et
al. (1994). Although increases in Ca++ channel density
in uterine smooth muscle after prolonged estrogen treatment have been
reported by Batra (1987), to our knowledge estrogen-mediated effects on
cardiac or vascular Ca++ channel radioligand binding have
not been reported previously.
DHP binding, with [3H]PN200-110, was conducted in
membrane preparations from all three groups of animals in each binding
assay for comparisons to be made under identical conditions.
[3H]PN200-110 binding parameters in cardiac and aortic
tissue from sham rats in this study were in good agreement with those
reported by Lonsberry et al. (1992) in heart and Ikeda
et al. (1994) in aorta, with the exception of greater aortic
Kd values reported here. The difference in the
binding affinity of [3H]PN200-110 to aortic membranes,
compared with that of Ikeda et al., may be related to
differences in sex (female vs. male), age (29 weeks
vs. 13 weeks), strain (Sprague-Dawley vs.
Wistar-Kyoto) or in the protocols for membrane preparation. It is
possible the apparent lower binding affinity represents a lower
affinity site, although a two-site model was not indicated by analysis
of the binding data. Further, Ki values for BAY
in this study were in good agreement with Kwon et al.
(1989) using heart and Wei et al. (1986) using vascular
tissue. Thus, [3H]PN200-110 binding characteristics in
the present study are consistent with data reported in previous work.
Hormonal regulation of Ca++ channels has been documented by
studies investigating the effects of thyroid function on cardiac Ca++ channels (Hawthorn et al., 1988; Seppet
et al., 1993; Wibo et al., 1995). Similar to the
effects of estrogen, hypothyroidism in rats resulted in increased
density of cardiac Ca++ channels (Hawthorn et
al., 1988). However, hypothyroidism also causes significant
decreases in cardiac beta adrenoceptor binding sites
(Bilezikian and Loeb, 1983; Hawthorn et al., 1988). In
contrast, EE2 treatment in the present study resulted only in the
selective increase in cardiac Ca++ channels, because
neither cardiac beta adrenoceptor nor ryanodine receptor
density was affected. In addition, cardiac Ca++ channel
expression likely was not affected by the decreased body weight of the
EE2-treated animals compared with the ovex controls. Iwashima and
co-workers (1994) reported no significant differences were found in the
cardiac-type Ca++ channel alpha-1 subunit mRNA
levels among fed, fasted and refed rats.
Whereas cardiac [3H]ryanodine receptor density was not
altered by EE2 treatment, [3H]ryanodine binding affinity
was significantly increased in these rats to a
Kd similar to that of the sham animals. It is
not readily apparent why ovariectomy resulted in a decrease in the
binding affinity of [3H]ryanodine for the SR
Ca++ efflux channel. However, recent work by Cannell
et al. (1995) and Sham et al. (1995) provided
evidence that cardiac L-type Ca++ and SR efflux channels
have a functional cross-relationship such that Ca++ flux
through either channel alters the gating kinetics of the other. Thus,
the effect on ryanodine binding affinity by EE2 may be related to the
increase in density of cardiac L-type Ca++ channels in
these animals.
Although the changes in Ca++ channel density occurred in
both cardiac and aortic tissue in EE2-treated rats, determination of hemodynamic parameters in response to BAY or NE did not produce evidence of estrogen-mediated effects on cardiac contractility or blood
pressure. Several explanations may be advanced as to why the increased
Ca++ channel densities observed in response to EE2
treatment did not translate into changes in agonist-induced inotropy or
arterial pressure. 1) It is possible that the in vivo
responses measured, dP/dtmax and MAP,
were insensitive to changes that might have occurred based on
alterations of Ca++ channel densities. 2) It is also
possible that the measure of DHP binding with
[3H]PN200-110 reflected the preferential binding to
Ca++ channels in the inactive state (Hess et
al., 1984), because the use of membrane preparations provides
Ca++ channels that are in the inactive state (Chin, 1986).
In vivo, the population of Ca++ channels in the
"open" state may be a factor in determining the extent that
increased channel density affects hemodynamic function. If chronic
estrogen treatment results in the appearance of excess DHP receptor
sites, the density of functional Ca++ channels may be
unchanged. Aiba and Creazzo (1993) have determined the existence of
excess, nonfunctional DHP receptors in embryonic chick heart. 3)
Studies have documented in vitro the Ca++
channel antagonist effects of estrogen (Batra and Bengtsson, 1978;
Jiang et al., 1991; Cohen and Susemichel, 1996). If
Ca++ channel antagonism were also occurring in
vivo, then the effects of estrogen to block Ca++
influx could be opposed by its ability to increase Ca++
channel density, with the net hemodynamic effects being modest. 4) Not
only can estrogen serve in vitro as a Ca++
channel antagonist, recent work by White et al. (1995) has
documented that estrogen relaxes coronary arteries by opening
Ca++- and voltage-activated K+ channels. Again
these vasodilating actions would be opposed by increased
Ca++ influx mediated through increased Ca++
channel density.
During preparation of this manuscript, further insight on the vascular
actions of EE2 was reported by Rahimian et al. (1996). In vivo administration of EE2 to ovex rats (by the same
protocol described in this paper) resulted in enhanced NO-mediated
vasodilating effects in aortic segments and increased aortic responses
to phenylephrine in the presence of a NO synthase inhibitor. These data
support the idea that prolonged EE2 treatment to ovex rats acts to
increase expression of aortic endothelial NO. Interestingly, basal
contractility of aortas was not significantly different among groups,
and responses to phenylephrine in the absence of NO blockade were also
similar (personal communication, Dr. C. van Breeman). Increases in
Ca++ influx through increased numbers of smooth muscle
Ca++ channels (as described in the present study) could
counteract the vasodilation produced by increased NO, with the end
result being maintenance of vascular tone. Estrogen-mediated increases in eNOS without overt changes in basal vascular tone might allow for
overall greater vessel elasticity, which could afford a
cardioprotectant effect. In another study, Hishikawa et al.
(1995) showed that human aortic endothelial cells treated with 17 -estradiol (8-48 h) expressed increased concentrations of eNOS,
adding further support to the idea that estrogen treatment results in
increased vascular nitric oxide. Whether these changes in eNOS were
accompanied by increases in Ca++ channel density has not
been reported.
In summary, oral treatment of ovex rats with EE2 resulted in significant increases in the binding density of cardiac and aortic L-type Ca++ channels, without alterations in binding affinity of [3H]PN200-110. The increase in Ca++ channel density was selective because cardiac beta adrenoceptor and ryanodine receptor densities were not affected. Increases in Ca++ channel density, detected in both aortic and cardiac tissue, were not sufficient to translate into measurable differences in the hemodynamic responses to BAY or NE in EE2-treated rats. These studies emphasize the need to examine carefully the role that biochemically measured changes in cardiovascular Ca++ channel densities may play in cardiovascular homeostasis.
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Footnotes |
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Accepted for publication December 20, 1996.
Received for publication July 2, 1996.
Send reprint requests to: Nancy Bowling, Department of Cardiovascular Research, DC 0520, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285.
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Abbreviations |
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EE2, ethinyl estradiol; ovex, ovariectomized; DHP, dihydropyridine; Ca++, calcium; sham, sham-operated controls; Bmax, maximum binding sites; Kd, dissociation constant; SR, sarcoplasmic reticulum; BAY, BAY k 8644; NE, norepinephrine; PEI, polyethylenimine; Metofane, methoxyflurane; PE, polyethylene; dP/dt or LV dP/dtmax, left ventricular dP/dtmax; MAP, mean arterial pressure; S.E.M., standard error of the mean; Ki , inhibition constant; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; [125I] CYP, cyanopindolol, iodo.
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References |
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Abstract
Introduction
Methods
Results
Discussion
References
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- and -adrenergic receptor systems and adrenergic responsiveness.
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Naunyn-Schmiedeberg's Arch Pharmacol.
337: 539-544, 1988[Medline]. oestradiol on contraction, Ca2+ current and intracellular free Ca2+ in guinea-pig isolated cardiac myocytes.
Br. J. Pharmacol.
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Arch. Int. Pharmacodyn.
281: 57-65, 1986.This article has been cited by other articles:
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