Rat isolated right atria obtained 1 wk after sinoaortic denervation
were less sensitive to the chronotropic actions of 
-agonists than
were tissues obtained from animals that underwent sham surgery or no
surgery at all. The potencies, but not the maximal responses for two
high efficacy agonists, norepinephrine and isoproterenol, were reduced
about 3- to 4-fold. Sino-aortic denervation (SAD) caused about a 3-fold
decrease in potency and about a 60% decrease in maximal response for a
low efficacy agonist, prenalterol. The changes in the actions of these
agonists occurred in the absence of any changes in the subtype of
beta receptor mediating the chronotropic response. The
results of analyses of the data for prenalterol showed that SAD caused
a decrease in the operational efficacy of this agonist without any
changes in its KD value for
beta-1 adrenoceptors. SAD had no effect on the responses
of the tissue to blockade of uptake 1 and uptake 2, suggesting no
compensatory changes in the removal processes caused the decreased
potency. The results of radioligand binding assays showed that SAD
caused a decrease in the maximal binding of
125I-cyanopindolol without altering its
KD. Also, the results of competition binding
assays confirmed the lack of effect of SAD on the
KD for prenalterol. The SAD-induced changes
in the actions of agonists acting at right atrial beta-1
receptors were caused by a down-regulation of beta-1
adrenoceptors, which probably occurred in response to SAD-induced
increases in sympathetic tone.
 |
Introduction |
SAD disrupts baroreceptor
mediated regulation of blood pressure and heart rate (Krieger, 1964
;
Krieger et al., 1980). Immediately after SAD there is an
increase in sympathetic activity that is characterized by a labile
hypertension and tachycardia. In the rat, the tachycardia is transient
and usually returns to near control rates within 2 wk after SAD. In the
conscious rat, the labile hypertension has been reported to persist for
months (Vasquez and Krieger, 1980; 1982). However, if the animals are
maintained in a quiet, nonstressful, environment, the hypertensive
phase converts to a normotensive state within 20 days (Irigoyen,
et al., 1995). The mechanisms for the reversal of the
tachycardia and hypertension are not fully understood.
Cardiac tissues contain both beta-1 and beta-2
adrenoceptors (Lands et al., 1967 and Carlson et
al., 1972). In the rat, the chronotropic and inotropic responses
to neuronally released and circulating catecholamines are mediated by
the beta-1 subtype (Juberg et al., 1985).
Generally, in response to an increased sympathetic tone, either as a
result of a disease state or pharmacological intervention, a
desensitization of or down-regulation of beta-1 adrenoceptors, but not beta-2 receptors, occurs in cardiac
tissues (Brodde, 1987). However, not all situations that are known to produce an increased sympathetic tone result in a desensitization or
down-regulation of cardiac beta-1 receptors. For example, a supersensitivity to ISO mediated by beta-2 adrenoceptors was
seen in right atria obtained from rats after certain forms of stress (Callia and DeMoraes, 1984; Bassani and DeMoraes, 1988; Spadari and
DeMoraes, 1988) or in an early stage of experimentally induced sepsis
(Barker et al., 1990). Only a few studies have addressed the
effects of SAD on cardiac beta adrenoceptors.
Chloralose anesthetized rats showed a decreased responsiveness to the
chronotropic actions of ISO at 5 hr after SAD, a time at which
SAD-induced tachycardia was maximal (Vasquez and Krieger, 1982). This
reduced sensitivity to ISO was also seen at 14 to 15 days after SAD, a
time period at which the SAD-induced tachycardia had abated (Vasquez
and Krieger, 1982). The results of further studies using an isolated
heart preparation obtained at 5 hr and 14 days after SAD suggested a
decrease in the potency and maximal chronotropic response to ISO
administered as bolus injections into the perfusion line (Cabral and
Vasquez, 1984). The results of these studies suggested that SAD, as
other models of hypertension, produced an uncoupling between receptor
and G-protein and/or a down-regulation of beta adrenoceptors
mediating chronotropy. In these studies, possible effects of SAD on the
removal processes for catecholamines and/or changes in the subtype of
beta adrenoceptor mediating the chronotropic response were
not evaluated.
Our studies were undertaken to determine the means by which SAD alters
the actions of agents acting at right atrial beta receptors. Functional assays using isolated right atrial preparations were conducted to determine the effects of SAD on the actions of two high
efficacy agonists, ISO and NE, and a low efficacy agonist, PREN. The
advantages of using a low efficacy agonist, such as PREN, is that such
agonists are much more affected by changes in receptor number and/or
coupling than are high efficacy agonists and comparative functional
assays of a low and a high efficacy agonist on the same tissue permit
an estimation of the affinity and efficacy of the low efficacy agent
(Kenakin, 1993; Leff et al., 1990). Additionally, the
effects of SAD were evaluated on the neuronal and nonneuronal uptake
processes; and on the active population of beta receptors
mediating the chronotropic response. Radioligand binding assays were
carried out to determine if agonist affinity and/or the number of right
atrial beta receptor binding sites was altered after SAD.
| |
Methods |
Surgical procedures.
In conducting this research, the
authors adhered to National Institutes of Health guidelines for the use
of animals. Male Wistar rats (300-400 g) were used in all experiments.
Three groups of animals were used: animals with SAD, animals with sham
surgery and naive control animals.
All surgical procedures used aseptic techniques and were carried out
under anesthesia produced by ketamine, 50 mg/kg, i.m., and xylazine, 5 mg/kg, i.m. Bilateral SAD was performed as described by Vasquez and
Krieger (1982). Briefly, after the induction of anesthesia, the
external and internal branches of the carotid arteries were exposed.
The vagas nerve, the sympathetic trunk and surrounding connective
tissue were gently dissected away from the vessels, the superior
laryngeal nerve was resected and a section of the sympathetic trunk
removed. Sham surgery consisted of the same procedures used to expose
and free the arteries, but without denervation.
After SAD or sham surgery, cannulae containing sterile saline were
placed in the left femoral vein and artery for the subsequent administration of drugs and recording blood pressure and heart rate.
The vein and artery were cannulated using sterile PE-50 and PE-10
tubing, respectively. Cannulae were exteriorized at the dorsal neck
region. Following surgery, all animals were treated with benzathine
penicillin, 100,000 U, i.m., to minimize infections.
Twenty-four hr after surgery, the effectiveness of SAD was determined
by the administration of sodium nitroprusside, 4 µg/kg, i.v., to
conscious unrestrained rats. SAD was considered adequate if the animal
responded with no increase in heart rate after a decrease in diastolic
pressure of 30 to 50 mm Hg. Sham surgery was considered successful if
the animals responded with a tachycardia following the depressor
challenge. Only animals meeting the criterion for SAD or sham are
included in our study. Blood pressure was recorded from the femoral
artery using a Statham P23 1D pressure transducer (Grass Instruments,
Quincy, MA) and heart rates were measured with a Grass EKG/tachygraph
and displayed on a Grass model 7D polygraph.
Functional assays using isolated right atria.
A modification
of the procedures described by Kenakin and Beek (1980) was used. Seven
days after surgery, animals were anesthetized with halothane and
euthanized by stunning and exsanguination. The hearts were rapidly
removed and placed in oxygenated KHB. The right atria were removed and
mounted in water jacketed tissue chamber (10 ml volume) containing KHB,
pH 7.3 to 7.5, at 34°C and gassed with 95% O2-5%
CO2. The composition of the KHB was (millimolar): NaCl,
124; KCl, 4.75; MgSO4, 1.30; CaCl2, 2.25; NaHCO3, 25.0; NaH2PO4, 0.6;
dextrose, 10.0; sodium ascorbate, 0.3; disodium EDTA, 0.03 and
17-hydroxyestradiol, 0.005. Ascorbate and EDTA were added to inhibit
the oxidation of catecholamines (Hughes and Smith, 1978).
17-Hydroxyestradiol was added to block the extraneuronal uptake of
catecholamines (Salt, 1972). The neuronal uptake of catecholamines was
inhibited by treatment with 10 µM phenoxybenzamine for 30 min
(O'Donnell and Wanstall, 1985). After treatment with phenoxybenzamine,
the bathing solution was changed by overflow washes every 15 min for 1 hr to remove unreacted phenoxybenzamine and allow a stable basal rate
to develop.
Construction and analyses of concentration-response curves.
Concentration-response curves for the positive chronotropic actions of
ISO, NE and PREN were constructed by the cumulative variation of
agonist concentration at one-half log unit increments (Van Rossum,
1963). In those experiments where PREN was used, concentration-response
curves for both PREN and ISO were generated on the same tissue. The
change in atrial rate was used as the response metameter. Curves were
constructed after the treatment with and removal of phenoxybenzamine
when stable basal rates were established.
In one series of experiments, the effects of SAD on catecholamine
uptake mechanisms were evaluated by generating concentration-response curves in the absence of uptake blockade and then in the presence of
estradiol and after treatment with phenoxybenzamine.
The possible effects of SAD on changes in the subtype of
-adrenoceptor mediating the chronotropic response were evaluated by
constructing concentration-response curves for ISO or NE in the absence
and then in the presence of either ICI118,551, a selective beta-2 antagonist (O'Donnell and Wanstall, 1980) or CGP 20 712A, a selective beta-1 antagonist (Dooley et
al., 1986).
All concentration-response data were evaluated for a fit to a logistics
function in the form:
|
(1)
|
where E is the increase in rate above basal; Emax is
the maximum response that the agonist can produce; c is the logarithm of the EC50, the concentration of agonist that produces
half-maximal response; x is the logarithm of the concentration of
agonist; the exponential term, n, is a curve fitting parameter that
defines the slope of the concentration-response line, and 
is the
response observed in the absence of added agonist. Nonlinear regression analyses to determine the parameters Emax, log
EC50 and n were done using GraphPad Prism (GraphPad
Software, San Diego, CA) with the constraint that = zero. In the
text and tables, the potency parameter, log EC50, is given
as the pEC50, -1·(log EC50).
The dissociation constant, KD, for the partial
agonist, PREN, was estimated by the method of Black et al.
(1985). Initially the data sets for PREN and ISO were analyzed as
described above. The responses for the individual data sets for PREN
were normalized to the maximum response estimated for ISO for each
tissue. The transformed data were then fit to the expression:
![E<IT>=</IT>E<SUB>max</SUB><IT>&tgr;</IT><SUP>n</SUP>[<IT>10</IT><SUP>x</SUP>]<SUP>n</SUP><IT>/</IT>((<IT>10</IT><SUP>K</SUP><IT>+</IT>[<IT>10</IT><SUP>x</SUP>])<SUP>n</SUP><IT>+&tgr;</IT><SUP>n</SUP>[<IT>10</IT><SUP>x</SUP>]<SUP>n</SUP>)](/content/vol280/issue2/fulltext/677/img002.gif)
|
(2)
|
where E is the fractional response relative to that produced by
ISO; Emax is the relative maximal tissue response which was constrained to unity; K is the logarithm of the molar equilibrium dissociation constant; 
is the model definition for efficacy; and n
and x are as defined above. The term is defined as the ratio,
[RT]/KE, where [RT] is the
concentration of active receptors and KE is the
concentration of agonist-receptor complex producing one-half of the
agonist maximal response. The value of n for each data set was
constrained to that value obtained in the fitting to equation 1. This
method for estimating the KD for an agonist is
limited to cases where the agonist maximal response is measurably less
than the tissue maximum response (Leff et al., 1990). Two of
the data sets for PREN obtained from control tissues were not used in
this analysis because they had Emax values that were not different from those for ISO on the same tissues. The dissociation constants are reported in the text and tables as
pKD values, -1· (log
KD).
In the experiments in which competitive antagonists were used to
characterize the active population of beta adrenoceptors, concentration-response curves for agonists were constructed in the
absence of antagonist and in the presence of antagonist after a 2-hr
equilibration period. In all of these studies, only a single concentration of antagonist was used on a single atrium.
Concentration-response data were analyzed as described above.
Concentrations of agonist producing half-maximal response in the
absence, [A], and the presence, [A
], of antagonist were estimated
for use in the Schild equation (Arunlakshana and Schild, 1959):
![log((CR<IT>−1</IT>)<IT>=</IT>n<IT> </IT>log[B]<IT>−</IT>log<IT> </IT>K<SUB>B</SUB>](/content/vol280/issue2/fulltext/677/img003.gif)
|
(3)
|
where CR is [A]/[A]; n is slope; [B] is the concentration
of antagonist and all other terms are as defined above. The apparent molar equilibrium dissociation constant for the interaction of the
antagonist with the receptor, KB, was determined from a
linear regression of log (CR -1) on log [B]. The dissociation
constants are reported in the text and tables as
pKB values, -1· (log
KB). Full Schild analyses were carried out for
ISO and NE on tissues obtained from the naive control group.
In studies using atria from the SAD and SHAM groups, only single
concentrations of ICI118,551 and CGP20712A were used. The concentrations used, 10 nM CGP20712A and 50 nM ICI118,551, were ones
that interact with the receptor for which they have the highest affinity and have only minimal interactions at the other site. A
prediction of the expected behavior of the concentration of antagonists
used was obtained by simulations using the model of Lemoine and Kaumann
(1983) for antagonism at two receptors for a common response mediated
by a single agonist:
![log(CR<IT>−1</IT>)<IT>=</IT>log[B]<IT>−</IT>log{(<IT>&sfgr;<SUB>1</SUB></IT>K<SUB>B<IT>1</IT></SUB><IT>+&sfgr;<SUB>2</SUB></IT>K<SUB>B<IT>2</IT></SUB>)[B]](/content/vol280/issue2/fulltext/677/img004.gif)
|
(4)
|
where CR is as defined above, sigma-1 and
sigma-2 are fractional stimuli of agonist effect mediated
through beta-1 and beta-2 adrenoceptors, and
KB1 and KB2 are the equilibrium dissociation constants for the interaction of the antagonist with beta-1
and fbeta-2 adrenoceptors. In the simulations the values of
sigma for each site were varied between 0 and 1. The
pKB values used were those obtained in this
laboratory and literature values. Those used for CGP 20712 A, were 9.4 (this study) and 5.5 for beta-1 and beta-2
receptors, respectively ((Dooley et al., 1986; Hall et
al., 1990). For ICI118,551 the respective values were 7.0 and 9.4 (Bilski et al., 1983; Barker et al, 1990; this
study).
ICYP binding studies.
Right atria were obtained as described
above. For each experiment, right atria from three rats were pooled.
Membranes were prepared by a modification of the procedure described by
Juberg et al. (1985). The right atria were rapidly removed
and homogenized with a Tissumizer for 30 sec in 10 ml of 20 mM of
NaPO4 (pH 7.6) containing 154 mM NaCl. The homogenate was
centrifuged at 100,000 × g for 1 hr at 4°C to
isolate all particulate bound receptors (Maisel et al.,
1985). The supernatant was discarded and the pellet was resuspended in
1 ml of 0.32 mM sucrose per ten mg wet weight tissue, and stored at
70°C until use. This preparation was used to permit an evaluation
of the effects of SAD on the total number of beta
adrenoceptor binding sites that would not be influenced by changes
produced only by a redistribution between sarcolemal and light
vesicular bound receptors.
Aliquots of the membrane suspension (100 µl containing 35-70 µg
protein) were incubated in triplicate at 37°C for 90 min with varying
concentrations of ICYP (5-300 pM) or a single concentration of ICYP
(100 pM) in the presence of varying concentrations of PREN (0.1-30
µM) in a final volume of 250 µl of a buffer containing: HEPES, 50 mM, pH 7.5; MgCl2, 4 mM; sodium ascorbate, 0.3 mM; and EDTA(Na)2, 0.03 mM. Triplicate samples containing
propranolol (10 µM) were used to define nonspecific binding.
Incubations were terminated by adding 10 ml of 50 mM HEPES buffer at
4°C, followed by rapid filtration through glass fiber filters (GF/B
Whatman, Clifton, NJ) and a wash with 10 ml of the HEPES incubation
buffer. Immediately after filtration, the radioactivity was measured
with a Gamma counter (Beckman Instruments, Fullerton, CA, model 9800). Protein was measured by the method of Lowry et al. (1951)
using bovine serum albumin as the standard.
The apparent number of binding sites and affinity for ICYP were
determined by nonlinear regression analyses using the equation (Kenakin, 1993; Klotz, 1982):
|
(5)
|
where B is the amount of ligand bound expressed as fmol/mg
protein; Bmax is the maximal number of sites expressed as
fmol/mg protein; K is the logarithm of the molar equilibrium
dissociation constant, KD, of the radioligand; x
is the logarithm of the concentration of free ligand; and n is the Hill
coefficient for the binding of the ligand.
The IC50 value for PREN was determined from a fit to
equation 1. The apparent KD for PREN was
calculated from its IC50 value for inhibiting ICYP binding
using the expression (Leff and Dougall, 1993):
![K<SUB>d(PREN)</SUB><IT>=</IT>IC<SUB><IT>50</IT></SUB><IT>/</IT>{(<IT>2+</IT>([ICYP]<IT>/</IT>K<SUB>d(ICYP)</SUB>)<SUP>n</SUP>)<SUP><IT>1/</IT>n</SUP><IT>−1</IT>}](/content/vol280/issue2/fulltext/677/img007.gif)
|
(6)
|
where n is the slope factor for the line describing the
inhibition of ICYP binding by PREN.
Statistical analyses.
The program InStat (GraphPad Software,
San Diego, CA) was used for statistical analyses. Where appropriate,
one-way analyses of variance followed by a Bonferroni multiple
comparisons post hoc test were performed to determine if the
treatments had an effect. In some cases, a paired Student's
t test was used. P < .05 was accepted as
significant.
Drugs.
(-)-NE, (-)-ISO and 17-hydroxyestradiol were
obtained from Sigma Chemical Co. (St. Louis, MO). The following
compounds were kindly provided as gifts: PREN (Dr. Terry Kenakin),
phenoxybenzamine (Dr. Norman Robie), ICI118,551 (ICI, Ltd), and CGP
20712A (Ciba Geigy Pharmaceuticals). 125I-Cyanopindolol,
2000 Ci/mMol, was obtained from Amersham Life Science Inc. (Arlington
Heights, IL)
| |
Results |
Effect of SAD on uptake mechanisms.
The results of the studies
on the combined effect of neuronal and nonneuronal uptake blockade are
given in table 1. The potentiation of NE after the
blockade of neuronal and nonneuronal uptake systems was similar in
atria obtained from all of the treatment groups. The left shifts in the
concentration-response curves for NE on tissues from the naive control,
sham and SAD were not significantly different from each other (one-way
ANOVA, F = 2.6, P > .05).
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TABLE 1
Effect of combined blockade of the neuronal and non-neuronal uptake
systems on the potency of ISO and NE at right atrial beta-receptors mediating chronotropy
|
|
Concentration-response curves for ISO were also significantly (P < .05, paired Students' t-test) shifted to the left after uptake blockade. The shifts were less than those observed for NE. The
leftward shifts for concentration-response curves for ISO on tissues
from the naive control, sham and SAD groups were not significantly
different from each other (one-way analysis of variance, F = 2.0, P > .05). All subsequent studies reported below were obtained
under conditions in which the neuronal and nonneuronal uptake systems
were blocked.
Effects of SAD on the chronotropic actions of ISO, NE and PREN at
right atrial beta adrenoceptors.
The
concentration-response parameters for ISO, NE and PREN on isolated
right atria obtained from control animals and from animals 7 days after
sham surgery or SAD are summarized in table 2. The basal
rates for atria were 235 ± 4 (47), 206 ± 3 (28) and
170 ± 5 (28) for the naive control, SHAM and SAD groups,
respectively. The rates for the SHAM and SAD groups were significantly
different from that for the naive control and that for the SAD group
was significantly different from that for the SHAM group (analysis of
variance, F = 65, P < .05). Despite the differences in basal rates, there were no differences between the Emax values
for the chronotropic actions of ISO and NE on atria from any of the
three treatment groups. The Emax values for the
chronotropic actions of PREN were significantly less than those for ISO
and NE on atria from all treatment groups (analysis of variance, F = 21.0, P < .05).
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TABLE 2
Concentration-response parameters for the chronotropic actions of ISO,
NE and PREN on isolated right atria
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When compared to both control groups, the pEC50 values for
all three agonists were lower on atria obtained from rats 7 days after
SAD (P < .05). For ISO, NE and PREN, SAD was associated with a
2.5-, 4.5- and 2.6-fold reduction in the EC50,
respectively. SAD had no effect on the Emax values for the
full agonists, ISO and NE, but did cause a decrease in the absolute and
relative (as compared to ISO) Emax values for the partial
agonist, PREN. SAD had no effect on the slope parameter for any of the
agonists (P > .05). The concentration-response curves for the
actions of ISO and PREN are shown in figure 1 and those
for the actions of NE are shown in figure 2.
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Fig. 1.
Concentration-response curves for the chronotropic
actions of ISO and PREN on isolated right atria obtained from control
(A), sham (B) and SAD (C) treatment groups. Each point is the mean ± S.E. for six to eight experiments in which curves for ISO and PREN
were generated on the same tissue. The assays were conducted under
conditions of uptake blockade. The y axis is increase in atrial rate in
beats min 1 to provide a direct comparison of the effects
of SAD on the actions of ISO and PREN.
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Fig. 2.
Concentration-response curves for the chronotropic
actions of NE on isolated right atria obtained from control, sham and
SAD treatment groups. Each point is the mean ± S.E. for six
experiments. The assays were conducted under conditions of uptake
blockade. The y axis is fractional response.
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|
Effects of SAD on the apparent affinity and efficacy of PREN at
right atrial beta adrenoceptors.
The effects of SAD on
the KD value and efficacy of PREN are summarized
in table 3. The apparent KD
values for PREN determined by the functional and competition binding
assays were not significantly different. In the competition binding
assays the pIC50 values (calculated
pKD values in parenthesis) and slope parameters
for PREN were 5.83 ± 0.12 (6.39 ± 0.18) and 0.74 ± 0.06, 5.93 ± 0.08 (6.42 ± 0.10) and 0.90 ± 0.14 and
5.77 ± 0.08 (6.38 ± 0.12) and 0.67 ± 0.07, respectively, for the naive, sham and SAD groups. SAD had no
significant effects on the pIC50 values, calculated pKD values or the slope parameters. These data
show that SAD had no effect on the apparent KD
value of PREN for beta adrenoceptors as determined by either
functional or radioligand binding assays. The results of the functional
assays showed that SAD produced a decrease in the efficacy of PREN.
Characterization of the active population of beta
adrenoceptors mediating chronotropy.
In the initial studies,
Schild analyses were done to determine pKB values for the
antagonism of ISO and NE by CGP 20712A and ICI 118,551 on atria from
naive control animals.
The concentration-response curves for the antagonism of ISO by CGP
20712A and ICI 118,551 are shown in figures 3 and
4. Similar results for each antagonist were obtained for
the antagonism of NE (data not shown). The slopes of the initial
regression analyses were not significantly different from unity.
Accordingly, subsequent estimates for the pKB
values were obtained with the slopes constrained to unity (Kenakin,
1993). These data are shown in table 4. The pKB values for the antagonism of ISO or NE by
CGP 20712A were not significantly different (P > .05, unpaired
Student's t test). The pKB values
obtained from a regression of the combined data for the antagonism of
ISO and NE by were 9.36 ± 0.11 and 7.10 ± 0.11 for CGP
20712A and ICI 118,551, respectively. The regressions for the combined
data are shown in figures 5, A and B. These results were
consistent with antagonism at a single population of active receptors.
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Fig. 3.
Antagonism of the chronotropic actions of ISO by
CGP 20712 A. A total of 14 experiments were carried out. The control
data are the mean ± S.E. for 14 replications. For curves
generated in presence of antagonist, each point is the mean ± S.E. for three to four repetitions of each concentration of antagonist.
The assays were conducted under conditions of uptake blockade. The y
axis is fractional response.
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Fig. 4.
Antagonism of the chronotropic actions of ISO by
ICI 118,551. A total of 11 experiments were carried out. The control
data are the mean ± S.E. for 11 replications. For curves
generated in presence of antagonist, each point is the mean ± S.E. for three or mean ± range for two (ICI = 1E-5)
repetitions of each concentration of antagonist. The assays were
conducted under conditions of uptake blockade. The y axis is fractional
response.
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TABLE 4
Estimates of the pKB values for the antagonism of ISO and NE by
CGP 20712A and ICI 118,551 at -receptors in right atria from control
rats
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Fig. 5.
Schild regressions for CGP 20712A (A) and ICI
118,551 (B) antagonism of ISO and NE at beta receptors
in right atria obtained from control rats.
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The next series of experiments were carried out on tissues obtained
after sham or SAD to determine if the treatment altered the active
population of beta adrenoceptors mediating chronotropy. In
these experiments, ISO was used as the agonist and only a single concentration of CGP 20712A or ICI 118,551 was used. ISO was selected as the agonist because it acts equally well at both beta-1
and beta-2 adrenoceptors. The concentrations of the
antagonists used were those that would block the beta
adrenoceptor subtype for which the antagonist is selective and have
little to no measurable effect at the other site, i.e., less
than or about equal to 1 times KB for the least
sensitive sight. For CGP 20712A a concentration of 10 nM was used to
selectively block beta-1 receptors. For ICI 118,551 a
concentration of 50 nM was used to block beta-2 receptors. In tissues obtained 7 days after sham and SAD surgery, the apparent pKB values for CGP 20712A were 9.47 ± 0.06 (n = 6) and 9.55 ± 0.08 (n = 6),
respectively. These data are shown in figure 6. ICI
118,551 at a concentration of 50 nM did not produce a significant change (P > .05, paired Student's t test) in the
pEC50 values for the action of ISO on atria from either
group of animals. The pEC50 values for ISO obtained in the
absence and in the presence of ICI 118,551 were 8.56 ± 0.11 and
8.32 ± 0.12 (n = 5) for atria obtained from rats
7 days after sham surgery. The corresponding values for tissues
obtained from rats 7 days after SAD were 8.33 ± 0.08 and
8.10 ± 0.10 (n = 5).
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Fig. 6.
Antagonism of the chronotropic actions of ISO by
CGP 20712A on tissues obtained from sham (A) and SAD (B) treatment
groups. Each point is the mean ± S.E. for six experiments. The
concentration of CGP 20712A used was one that selectively blocks
beta-1 adrenoceptors and has no measurable effect on
beta-2 adrenoceptors. The assays were conducted under
conditions of uptake blockade. The y axis is fractional response.
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Effects of SAD on ICYP binding.
The isotherms for
ICYP-specific binding to right atrial beta receptors are
shown in figures 7, A and B. The specific binding, as a
percentage of total binding, ranged from 64.5 ± 3.5% (15) for 5 pM ICYP to 53.2 ± 2.8% (15) for 300 pM ICYP. The parameters for
ICYP binding are given in table 5. SAD had no effect on
the affinity of ICYP for beta adrenoceptors binding sites.
Relative to both control groups, SAD caused about a 40% decrease in
the number of binding sites as expressed per mg tissue and about 30% decrease in binding sites as expressed per mg protein. The
Bmax for ICYP per gram tissue in atria from SAD group was
significantly different from both control groups (P < .05). The
Bmax for ICYP per mg protein in tissues from SAD group was
significantly different from the sham group, P < .05, but not
from the naive control group.
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Fig. 7.
Saturation binding of ICYP to beta
adrenoceptors present the total particulate fraction of right atrial
homogenates obtained from control, sham and SAD treatment groups. In
both figures the abscissa is the logarithm of the molar concentration
of ICYP. In A, the ordinate is expressed as fmol/mg protein and in B,
as nmol/g wet weight tissue. Each data point is the mean ± S.E.
for four to five experiments.
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Discussion |
Our results confirm and extend the findings made by Cabral and
Vasquez (1984) where it was shown that the potency of ISO for producing
a positive chronotropic response is reduced after SAD. Our results show
that at 1 wk after SAD, independently of uptake blockade, there were
decreases in the potencies for the chronotropic actions of ISO acting
on isolated right atria. In contrast to the findings made by Cabral and
Vasquez (1984), it was observed that the changes in the potency of ISO
occurred in the absence of changes in its maximal responses. Because
Cabral and Vasquez (1984) had similar observations on hearts taken from
animals at 5 hr and 15 days after SAD, it is not likely that the use of
tissues obtained at different times after SAD is the basis for the
differences in the effects seen on the maximum response to ISO. This
difference may be due to the preparations used to study the effects of
SAD on the chronotropic actions of ISO. Cabral and Vasquez (1984) measured ventricular rates in a perfused isolated heart preparation and
administered ISO, over a relatively narrow dose range, as a bolus
injection into the perfusate. This procedure would provide only a
transient exposure to ISO and a true maximum may not have been observed
because of the limited dose range used. In our study, isolated right
atria suspended in tissue chambers were used and ISO was added
cumulatively to the bath and not removed until the full
concentration-response curve had been developed. Also, an effect of SAD
on conduction that would cause a dissociation between the rates of
atrial and ventricular beating may have contributed to differences in
the results.
Our study also shows that SAD resulted in the decreased potency, but
not maximal response of another high efficacy beta agonist, NE. The potency and the maximal response for the chronotropic actions
of the low efficacy beta agonist, PREN, were reduced 1 wk
after SAD. The KD value of PREN for right atrial
beta receptors was estimated by functional and radioligand
binding assays. The effects of SAD on the actions of PREN were
associated with a decrease in its efficacy for agonism at atrial
beta adrenoceptors. No effects of SAD on the
KD value of PREN for beta
adrenoceptors were observed. The estimates of the
pKD for PREN obtained from the functional and
binding assays were not significantly different. Both assays gave
similar results, about 6.4 from binding assays and about 6.7 from the
functional assays. The pKD values for PREN
obtained in the binding assays are in good agreement with those found
by Brodde et al. (1984) and Hedberg and Mattsson (1981), 6.2 and 6.4, respectively. The estimates obtained from functional assays were also similar to those, 6.4 and 6.6, obtained by Hedberg and Mattsson (1981). However, our estimates of the
pKD value of PREN obtained from the functional
assays are lower than those reported by Kenakin and Beek (1980; 1984)
and Kenakin and Ferris (1983) whose functional assays yielded
pKD values of 7.1 to 7.5. The basis for theses
differences in the estimation of the apparent pKD value of PREN for beta
adrenoceptors are not known.
The results of the studies with selective antagonists showed that the
changes in the potency were not related to changes in the
beta receptor subtype mediating the response. Based on the results of simulations of Schild-plots for the behavior of an antagonist acting at two receptor subtypes (Lemoine and Kaumann, 1983),
it was predicted that if as little as 5 to 10% of the fractional stimuli were mediated by beta-2 receptors in atria, the
concentration-response curve for ISO in the presence of 10 nM CGP20712
A would have been shifted to right about 10-fold as opposed to about a
30-fold shift if only beta-1 receptors were involved. For 50 nM ICI118,551, it was predicted that the curve for ISO would have been
shifted to the right by about 3- to 4-fold if the fractional stimuli by beta-2 receptors was 0.5 and a shift of less than 2-fold, if
it was 0.1 or less. CGP20712 A, at 10 nM which is about thirty times its KB value for beta-1 adrenoceptors
and about 10,000-fold less than its KB value for
beta-2 receptors (Dooley et al., 1986; Hall et al., 1990), shifted the concentration-response curve for
the chronotropic actions of ISO on atria from both the SAD and SHAM treatment groups to the right by a factor of about 30. The estimates of
the pKB for CGP20712 A were 9.47 and 9.55 for
tissues obtained from animals after sham surgery or SAD, respectively.
These estimates obtained from a single concentration of CGP20112 A were
not significantly different from each other or from the
pKB value, 9.36, obtained from a Schild analysis
for its actions on tissues from naive control rats. The estimated
affinity for CGP 20712 A agrees with that reported by others (Dooley
et al., 1986; Hall et al., 1990) for its
interaction with beta-1 receptors.
ICI118,551, at 50 nM which is 30 to 100 times its
KB value for beta-2 adrenoceptors and
slightly less than its KB value for beta-1 receptors (Bilski et al., 1983; Barker
et al, 1990), produced dextral shifts of about 1.5-fold in
the concentration-response curves for the chronotropic actions of
ISO on atria from both the SAD and SHAM treatment groups. In both
cases, the shifts were not significant.
Collectively, these results show that the two antagonists behaved the
same on tissues obtained from the SAD and SHAM treatment groups and in
a manner not different from that seen on atria obtained from naive
control animals. These results indicate that agonism at
beta-1 adrenoceptors produced 90% or more of the fractional stimuli for the chronotropic response in atria obtained from animals that had undergone SAD.
The results of the binding assays showed that SAD had no effect on the
affinity of ICYP for beta receptor binding sites. The Bmax value for ICYP binding expressed either on the basis
of wet weight or protein was decreased in atria obtained from animals after SAD as compared to that for atria obtained after sham surgery. A
statistical difference between the values for the SAD and naive treatment groups was seen only when the comparison was made on binding
sites per gram tissue wet weight. Because the preparation used for the
binding studies contained both sarcolemal membrane bound and
internalized light vesicular bound receptors (Maisel et al.,
1985) possible effects of SAD on the redistribution of sarcolemal bound
and light vesicular bound binding sites were not be determined.
The changes in the agonist actions of ISO, NE and PREN reported here
occurred at a time after SAD in which sympathetic tone is still
elevated as evidenced by increased heart rates, mean arterial pressure
and plasma levels of catecholamines (Alexander et al., 1980;
Vassalo et al., 1991). The effects of chronic exposure to an
agonist to cause either an uncoupling between the receptor and effector
proteins and/or a down-regulation of its receptor are well documented
(Stiles et al., 1984). For example, the decreased sensitivity to ISO seen in tissues from spontaneously hypertensive rats
appears to be due to a down-regulation of cardiac beta
adrenoceptors in conjunction with an increase in the G protein, Gi
,
which can mediate an in inhibition of adenylyl-cyclase (Böhm
et al., 1994).
Classical drug-receptor theory predicts that some decreases in the
population of active receptors, by either an uncoupling or a
down-regulation, can produce decreases in potency of high efficacy
agonists without change in their maximal response. However, the
decreases in the population of active receptors that have no measurable
effect on the maximal responses for high efficacy agonists can cause a
decrease in the potency and in the maximal response of a low efficacy
agonist (Kenakin, 1993). The effects of SAD on the
concentration-response curves for ISO and PREN reported here are
strikingly similar to those reported by Kenakin and Ferris (1983) for
the agonist actions of ISO and PREN on rat left atria after a 4-day
in vivo treatment with ISO. Kenakin and Ferris (1983) found
that the treatment with ISO produced a down-regulation of beta receptors that was associated with a decreased potency
of ISO and a decreased potency and maximal response for PREN. Our results show that the effects of SAD on the actions of the high efficacy agonists, ISO and NE, as well as those on the low efficacy agonist, PREN, can be explained largely by a down-regulation of active
beta-1 receptors that mediate the chronotropic response. Further study is required to determine if changes in G proteins also
contribute to this.
In summary, these results suggest that in the rat SAD produces a
down-regulation of right atrial beta-1 adrenoceptors. This can explain the decreased sensitivity for chronotropic actions of
sympathomimetic amines that have been observed both in vivo and in in vitro and may be, in part, a basis for the
transient nature of the tachycardia. Compensatory changes in other
reflexes, such as the Bezold-Jarish reflex which is exaggerated after
SAD (Chianca and Machado, 1994), may also contribute to the transient nature of the tachycardia that is seen in the whole animal. Also, the
effects of surgery, per se, as well as SAD on basal rates, suggest that
other mechanisms are involved in the adaptation. In view of the
observations which show that chronic beta receptor activation is associated with an up-regulation of cardiac muscarinic receptors (Nomura et al., 1982) and that chronic beta
blockade not only increases responsiveness of atria to
beta agonists, but also to agents acting at atrial
5-HT4 and histamine H1- and
H2-receptors (Saunders et al., 1994; 1996), the
possible effects of SAD on other receptors involved in regulating
cardiac function merit investigation.
The authors thank Drs. Dennis Paul and Renee Bergeron and Ms.
Lerna Minor for technical assistance in conducting the radioligand binding assays. The helpful comments and suggestions made by Drs. A. M. Cabral, D. Vassalo and E. C. Vasquez of the Federal University of
Espirito Santo, Vitória, BR, and Dr. K. Varner of LSUMC are appreciated. Special thanks are extended to Professor E. M. Krieger of
the Heart Institute, São Paulo, BR for initially suggesting this
research.
AR, adrenoceptor;
EDTA, ethylenedaiminetetraacetic acid;
HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid;
ICYP, 125I-cyanopindolol;
ISO, isoproterenol;
KHB, Krebs-Henseleit buffer;
NE, norepinephrine;
PREN, prenalterol;
SAD, sino-aortic denervation.
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Abstract
Introduction
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