Preventing Rapid Receptor Desensitization at the beta-1-Adrenergic Receptor with Agonist/Antagonist Combinations
Richard Lanzara
E-mail: rlanzara@bio-balance.com
Bio Balance, Inc. 30 West 86th Street, New York, New York 10024, USA
Abstract
Background: Desensitization is a serious side effect of many drugs
and is also a fundamental problem for modeling drug-receptor interactions.
Although there has been very little theoretical or experimental work to
describe the pharmacological effects of agonist/antagonist combinations, this study was designed
to test both a theoretical model and a specific method to prevent rapid
receptor desensitization by using agonist/antagonist combinations. Preventing
desensitization may have relevance for many important drugs, including the
-adrenergic agonist drugs, which desensitize yet are
frequently used in medical practice to promote increased heart rate and
contractility. Because
desensitization is a serious side effect, these drugs are no longer the logical
or preferred treatment for heart failure. Subsequently, the
1-antagonist drugs such as metoprolol (Lopressor)
have replaced the
-adrenergic agonist drugs as a standard treatment for heart
failure. From this perspective, it is important to understand how the 
-agonist drugs interact with the 
-antagonist drugs at the level of the initial receptor
response. Results: The
-agonist drugs (isoproterenol (Iso) or dobutamine (Dob)) were
given as intravenous (IV) solutions to rats with or without the
beta-1-antagonist, metoprolol (Met), which was given either as a fixed amount
or as part of a specific agonist/antagonist ratio. The initial experiments
demonstrated that desensitization occurred for all of the animals receiving
either the Iso or Dob solutions alone. The theoretical model fit these initial
experiments with biophysical parameters, which were then used in calculateing a
specific agonist/antagonist ratio for making the agonist/antagonist combination
solution to prevent desensitization. Both the Iso/Met and Dob/Met
agonist/antagonist combination solutions significantly prevented
desensitization while maintaining near maximal responses in all of the animals tested. This theoretical
model predicted these responses and fit the experimental data with reasonable
biophysical parameters. Conclusion:
This study supports the concept that the earliest events of receptor
desensitization can be modeled and controlled at the level of the initial
receptor response. The theoretical model appears to be the only model capable
of describing this behavior with reasonable biophysical parameters. This
explanation for rapid desensitization suggests that the beneficial effects of
metoprolol for heart failure may result from its action on the initial events
of receptor activation. This method may also be useful for describing and
preventing desensitization in other drugs that desensitize their receptors.
Abbreviations
Iso = isoproterenol, Dob = dobutamine, Met = metoprolol, Iso/Met = the combination of isoproterenol with metoprolol in the specific ratio, Dob/Met = the combination of dobutamine with metoprolol in the specific ratio. dP/dt = maximum time-derivative of left ventricular pressure. LVP = maximum left ventricular pressure. IV = Intravenous. GPCR = G protein-coupled receptors. GRK = G protein-receptor kinase.
Background
Receptor desensitization appears counterintuitive because the addition of more of an activating ligand lessens the elicited response. Although many of the most rapid and important biological events desensitize [1-13], the earliest events of the receptor response haven't been examined with suitable biophysical models that suggest a plausible mechanism for the events that produce rapid desensitization. The rat has previously served as a late-stage or chronic desensitization animal model [14-16]; however, there have been no attempts to both model and test methods to prevent desensitization at the level of the initial receptor-ligand interaction. Therefore, this study was designed both to test a theoretical model and to prevent rapid receptor desensitization using a specific method [17].
The drugs isoproterenol (Iso) and dobutamine (Dob) are two b-adrenergic drugs frequently used for the treatment of patients with a variety of conditions including heart block, decreased cardiac output, and acute heart failure. They are sympathomimetic adrenergic agonists that activate the beta-1-adrenergic receptors, and thereby promote increased heart rate and contractility. The undesirable side effects that accompany these drugs include desensitization, tachycardia and arrhythmias. Since many of the abnormalities in adrenergic signaling observed in late-stage heart failure in both human and animal models are considered a result of adrenergic desensitization, the adrenergic agonists have gradually lost favor as the logical treatment for heart failure [13]. Conversely, metoprolol (Lopressor), a relatively selective beta-1-adrenoreceptor blocker, is now frequently prescribed for heart failure [18] although the scientific rationale behind this remains obscure.
Historically beta-1-receptor blockers, such as metoprolol
(Met), were known to depress cardiac function; however, more recent data have
confirmed that moderate doses of beta-1-receptor blockers produce beneficial
effects in most cases of individuals with heart failure [18,19]. Since the
scientific rationale for these observations is uncertain, it is important to understand
the interactions which are possible between beta-1-receptor blockade and 
-agonist induced desensitization in the heart. From this
perspective, this study tests how a 
-adrenergic agonist combined with a beta-1-receptor
antagonist in a known and specified agonist/antagonist ratio can decrease or
prevent the desensitization due to the agonist [17]. Controlling receptor
desensitization would offer new methods for improving drug therapy and provide
a scientific rationale for why beta-blockers improve the cardiac function of
patients with heart failure.
Desensitization also represents a fundamental problem for the theoretical modeling of drug-receptor interactions. Most theories of receptor activation have difficulty modeling the nonlinear interactions between receptor desensitization and competitive antagonists with meaningful biophysical parameters. These difficulties arise primarily because the competition of an antagonist at the receptor should theoretically block the receptor binding with an agonist and thereby diminish the response. However, since some receptor desensitization is very rapid, there must be at least one alternative explanation for rapid desensitization at the earliest level of receptor activation. Although the role played by the receptor-G protein decoupling schemes involving kinases have been most prominent, they are experimentally difficult to verify for very rapid receptor desensitization and may be secondary phenomena, which occur after the initial phase of receptor desensitization has past. Therefore, revealing the relative importance of these phenomena may assist in placing receptor activation and desensitization into an appropriate temporal perspective.
Receptor activation or desensitization schemes usually involve a diagram of the chemical interactions between the ligand and the receptor. An implicit and often overlooked assumption in these schemes is the nature of the reaction quotients or approximate steady state chemical equilibrium constants. These parameters represent collections of various chemical species, which are often lumped together into a bracket sign representing a concentration. How these reaction quotients combine and are altered by unequal ligand binding to two or more receptor states represents a challenge both to pharmacology and to chemical theory. A better understanding of how drugs work at the level of the receptor may result from an understanding of how these reaction quotients are altered and what chemical species they specifically represent.
An underlying premise in most models is that the chemical equilibrium of the receptor controls the response; however, it is increasingly recognized that it is the perturbation in the equilibrium that determines the receptor response although many theories neglect to calculate the net change in this parameter as the response of the system. Normally the underlying chemical ministate equilibria, which compose the overall equilibrium constant are ignored. These perturbations require a deeper understanding of how the underlying equilibria interact and combine with each ministate within the overall chemical equilibrium. This raises much more complicated questions than we can discuss here; however, we can study these chemical perturbations in more accessible systems that are more tightly controlled and easier to recognize. Ironically, the membrane-embedded, cellular receptor may be a better system to witness these chemical perturbations directly. From a chemical perspective, the changes in competing ministate equilibria may be minimized in a large, membrane-embedded molecule such as a receptor. Since these changes are connected to changes in the initial receptor equilibrium that produce observable biological responses, they may be more accessible because these responses often obey Weber's law.
Weber's law gained wide recognition when it was discovered
that all of our sensory perceptions obey this law; however, the underlying
physiological and biochemical basis for this law hasn't been clearly understood
[20]. Weber's law has been previously linked to an equation for the overall
equilibrium that equates equivalent perturbations produced by either the
transfer of a fraction of weight, 
w, or by an external weighting given by, S1 and S2,
(1)
Where w1 and w2 are the weights on each side of a simple balance and S1 and S2 are the external weights added to each side. Equation (1) represents a simple equation of equilibrium that also obeys Weber's law [20].
Interestingly, Equation (1) can be shown to represent a
two-state, chemical equilibrium of a cellular receptor by substituting Langmuir
binding expressions, S1=R1(S)/(S+K1) and S2=R2(S)/(S+K2),
for the binding of a molecule, S, with each receptor state, R1 and R2. Making these substitutions and substituting 
R for 
w into Equation (1) yields,
(2)
Equation (2) calculates an equivalent perturbation between
two receptor states, R1 and R2, in terms of the competing
reaction quotients, K1 and K2 for S, the binding molecule or
ligand. Where 
R represents the net transfer of receptor states.
Surprisingly, Equation (2) is identical to a two-state model that was previously derived and tested for its ability to model cellular receptor activation and desensitization [17]. With a few minor substitutions, Equation (2) can also be expressed in terms of this previously derived, two-state, pharmacological model [17].
For a receptor in an initial chemical equilibrium between
two-states, the selective affinity of agonist drugs, or ligands, for the high
affinity state, RH, will perturb the initial receptor equilibrium
and thereby produce a net shift in this initial equilibrium as the net receptor
response, 
RH, which is equivalent to 
R in Equation (2).
This is similar to most other two-state models with R and R* states
corresponding to inactive and active receptor states except that this model
relates the response to a fundamental equation for physical equilibrium
(Equation (1)), which can be solved for the net shift in the original
equilibrium, 
RH,
(3)
Where RH and RL represent the amount of unperturbed receptor existing in initial high and low affinity states respectively, and D represents the concentration of the binding drug or ligand. Equation (2), which was derived separately from Equation (3), is exactly analogous to Equation (3). Equation (3) with the factor of (1+ [I]/ Ki) for an antagonist, I, binding equally to each receptor state, multiplied times each of the dissociation constants, KDH and KDL, has been shown to accurately model the dose-response behaviors for agonists with and without antagonists in a wide variety of drug-receptor systems [17].
By taking the derivative of Equation (3) with respect to the dose, D, and setting the derivative to zero for the maximum response with the factor (1+ [I]/ Ki) for a competitive antagonist multiplied times each of the dissociation constants, KDH and KDL, the concentration of the antagonist as a fractional dose of the agonist ([I]=f [D]) can be derived as,
(4)
Where f is the agonist to antagonist ratio that is necessary and sufficient to prevent desensitization at the receptor [17]. This is the specific ratio that was tested for its ability to prevent desensitization.
For calculations of the model versus the experimental response, the ratio given by f was inserted into Equation (3) by altering the inhibition expression (1+ [I]/ Ki) for a competitive antagonist multiplied times each of the dissociation constants for the agonist, KDH and KDL. By this alteration the term (1+ [I]/ Ki) for the competitive antagonist, becomes (1+ f [D]/ Ki) where the antagonist concentration [I] has been replaced by f [D]. Where f is the fractional dose of antagonist relative to the dose of the agonist [D] [17]. Substitution produces the following specific modifications to the agonist dissociation constants: KDH(1+ f [D]/ Ki) and KDL(1+ f [D]/ Ki). These were then inserted into Equation (3) in order to model the experimental responses for the agonist/antagonist combination solutions.
Results
Isoproterenol (Iso) Experiments
The time-derivative of the blood
pressure in the ventricle of the heart (dP/dt) is an accepted measurement of the
contractility of the heart. As the strength of the contractions in the
ventricle of the heart goes up, the rate at which the pressure in the ventricle
rises will increase. Increased
dP/dt therefore implies increased contractility and also serves as a measure of
-receptor stimulation and desensitization.
The initial experiments with Iso alone demonstrated desensitization to the IV agonist solution and provided data for the parametric fit of the model (Table 1). From this fit, the agonist/antagonist ratio was calculated and then used to make the specific agonist/antagonist ratio for the Iso/Met solution, which was subsequently tested in the animals. Plots of the experimental responses for the Iso/Met solution were compared to the responses of the Iso solution alone, and to the model plots.
As shown in Figure 1A, the plot of the responses to increasing levels of infusion of the Iso solution shows increasing dP/dt at the lower dosages, but peaks and rapidly declines at the higher infusion dosages. This shows the presence of desensitization in these animals to the agonist solution alone. The fit of the model to these initial experiments is displayed as a line plot in Figure 1A for comparison with the experimental plot (compare model (dark circles) with experiment (light squares)). This initial experiment demonstrates both the degree of desensitization in these animals and the ability of this model to fit the experimental data.
Figure 1 - Average responses for the rats given Iso, Met or Iso/Met solutions
Figure 1A: Plots of the model
in dark symbols compared with the experimental results in light symbols for a
group of animals compared with a separate group of animals that received a
fixed infusion of metoprolol (Met) IV solution at 1mg kg-1min-1
with the Iso infusions varying. The plots show the responses of the heart
"dP/dt (%)" to increasing infusions of the isoproterenol (Iso)
solution alone, or with Met at a fixed level of infusion (experiment -
"Met (fixed)" (light circles), model - "
RH Met (fixed)" (dark triangles)).
Figure
1B: Plots for both the model and experimental data to the isoproterenol (Iso)
solution (model, "
RH Iso" (dark circles) and experiment, "Iso"
(light squares)), which were also plotted in Figure 1A for reference. The plots
of the Iso/Met IV solution are significantly different from the Iso solution at
the 95% confidence interval for the infusion rates of 10 mg kg-1min-1 and above - paired t-test. The Iso/Met solution is also plotted for
comparison with the model (generated by substitution into Equation (3) with the
modified dissociation constants, KDH(1+ f [D]/ Ki) and KDL(1+
f [D]/ Ki)) (compare model, "
RH Iso/Met" (dark triangles) and experiment,
"Iso/Met" (light circles)).
Figure 1A also shows a plot for a separate group of rats that received a fixed concentration of the metoprolol solution, Met (fixed), at 1.0 mg kg-1min-1, which decreased the dP/dt response to less than 40% of the peak. This experiment was done to determine the apparent Ki for Met, and to demonstrate that the Met solution was acting as an antagonist in these animals. The model successfully fit this data with the factor of (1+ [I]/ Ki) for the antagonist multiplied times each of the dissociation constants, KDH and KDL in Equation (3) for the response.
The Iso/Met IV solution was premixed as a 1:85 mg kg-1min-1 ratio, which was calculated by Equation (4) with the biophysical parameters derived from the initial experiments (Table 1). In addition, the expected response was calculated from Equation (3) for the response with the modifications to the dissociation constants as mentioned above and in the Methods section, and plotted for comparison with the experimental results. These animals were tested with either the IV Iso or Iso/Met solutions. Figure 1B shows the responses to the Iso solution with the standard error bars for a direct comparison with the responses to the Iso/Met solution and the predicted response from the model. The Iso/Met solution increases the dP/dt at low dosages, but at the higher dosages the dP/dt levels off at an elevated and sustained level rather than decreasing sharply, as seen previously for the Iso solution (see Iso (light squares) vs. Iso/Met (light circles) in Figure 1B). Therefore, compared to the Iso solution, the Iso/Met solution displays a more sustained and maximal response into infusion ranges where desensitization would have normally occurred.
Table 1 - Biophysical Parameters from the Theoretical Model
|
Experiments: |
Parameters: in mg kg-1min-1 or (approximate nM) |
|||
|
KDH |
KDL |
Ki for Met |
Ratios* "f " |
|
|
Isoproterenol (Iso) |
1.3 (5.2) |
19 (77) |
300 (440) |
1:85 (1:31) |
|
Dobutamine (Dob) |
1.7 (5.2) |
700 (2100) |
40 (58) |
1:1.6 (1:0.8) |
*
Ratios were calculated by Equation (4), where "f ", represents
the agonist to antagonist ratio that is sufficient to prevent receptor
desensitization [17].
The modified Equation (3), based on the initial parameters,
largely predicted these responses (see model, 
RH Iso/Met (dark triangles), and experiment, Iso/Met (light
circles), in Figure 1B). The amount of Met in the Iso/Met IV infusion is insufficient to inhibit the response as
compared with the fixed amount of Met in the initial experiments (Met (fixed)
in Figure 1A). At the infusion level of 10
g kg-1min-1, the dP/dt is sustained at
a significantly higher level for the Iso/Met infusion than the dP/dt for the
Iso infusion alone at 10
g kg-1min-1 (P<0.05 significant at
the 95% confidence interval - paired Student's t-test, n=3). This demonstrates the ability of the agonist/antagonist
combination solution to sustain the maximum response well into infusion ranges
that previously showed severe desensitization with the agonist solution alone.
In Figure 2A and 2B, responses are plotted individually for three rats that served as their own controls. With the Iso IV solution alone, the rats initially showed a range of peak responses and subsequent desensitization; however, their individual responses with the Iso/Met IV solution were more sustained with much less desensitization. These plots show that the desensitization is prevented for each of the individual rats. This might not have been expected given that these rats desensitized at different levels of infusion and to different magnitudes of depression. Also it might not have been expected given that the biophysical parameters for making the Iso/Met solution were derived from initial experiments obtained from a different set of rats. Comparing the individual responses in Figures 2A and 2B, the responses of all the animals to the Iso/Met solution show a steady rise to peak levels of dP/dt and a continuous and sustained response well past previously measured desensitization levels. Therefore, these results demonstrate that this method correctly calculates a specific agonist/antagonist ratio that largely prevents the experimentally observed desensitization.
Figure 2 - Responses of individual rats to the Iso and Iso/Met solutions
Figure 2A: Plots of the dP/dt
responses in mmHg sec-1 of three animals that served as their own controls.
With the higher IV infusion levels of the isoproterenol (Iso) solution the
desensitization is evident as a shape decline in responses for all of the
animals.
Figure 2B: The same animals as in Figure 2A except that they received the Iso/Met IV solution. The dP/dt responses are significantly different from those for the Iso solution at the infusion levels of 10 mg kg-1min-1 and above (P<0.05 significant at the 95% confidence interval - paired Student's t-test).
Modeling of Spare Receptors
Although the phenomenon of spare receptors has been observed for many years, pharmacological theories have had difficulty accounting for this observation in a meaningful, biophysical model. Therefore, in order to further test the abilities of this theoretical model, the apparent spare receptor reserve was modeled by comparing the response curve given by Equation (3) to the total binding curve, which was calculated as the sum of the Langmuir binding equations for each of the high and low affinity states (Total Binding = RH(D)/(D+ KDH) + RL(D)/(D+ KDL)). Previously, Brown, et al. observed that the plots of beta-adrenoceptor occupancy versus responses for rat left atria and papillary muscles had a rather large receptor reserve: 50% of maximal response was produced with only 1-3% of beta-adrenoceptor occupancy [21]. As shown in Figure 3, the percent of the receptors needed for a fifty percent maximal response appears to be five percent or less when compared to the amount of the total bound, which is in good agreement with Brown, et al. [21].
Figure 3 - Spare receptors for the isoproterenol response
Two plots of the theoretical
model: one is the percent total binding and the other is the percent
response. The total binding was
calculated from the sum of the Langmuir binding equations for the high and low
affinity states, Total Binding = RH(D)/(D+ KDH) + RL(D)/(D+
KDL). The response was calculated from Equation (3). The values for
KDH and KDL were taken from Table 1 for Iso. RH
and RL in Equation (3) were set equal to 10 and 190 respectively,
which represents about 5% of the receptors in the high affinity state, RH.
The problem is that there are at least two binding affinities that affect efficacy and binding. It is the interplay between these that determines both response and binding. From this model, the spare receptor reserve arises from the fraction of receptors that are shifted from the total receptor pool. This results because only a relatively small fraction of the total receptor states are shifted to increase the amount of the higher affinity state. Therefore, the phenomenon known as spare receptors becomes understandable, since the total amount of this shift is, not surprisingly, some relatively small fraction (RH from Equation (3)) of the total number of the total bound pool of receptor molecules. Therefore, this model is consistent with the experimental findings of a large receptor reserve observed for beta-adrenoceptor agonists in the b1-receptor system of rat heart.
Dobutamine Experiments
Similar to the procedure for the
Iso experiments, the KDH, KDL and Ki were
derived from the fit of Equation (3) to the average values of the experimental
data for the Dob infused rats. The model fit the experimental findings within
the range of error for these experiments (compare model - 
RH Dob (dark triangles) with experiment - 
Dob (light triangles) in Figure 4). A separate experiment was
performed to determine the Ki for Met in the animals receiving the
Dob IV solution alone (not shown). All of the parameters, KDH, KDL
and Ki, were inserted into Equation (4) to calculate the specific
ratio for making the Dob/Met agonist/antagonist solution (see Table 1). This
solution was prepared before hand, and subsequently used for the experimental
comparisons with the Dob solution as discussed below. In addition, the
calculated ratio, "f ", for Dob/Met was entered into Equation (3),
and plotted for a direct comparison with the experimental results.
Figure 4 - Plots of the average responses for the dobutamine exposed rats
These plots show the model
compared with the experimental values for either the dobutamine (Dob) solution
or the combination solution of dobutamine plus metoprolol (Dob/Met). At the 200
mg kg-1min-1 infusion level the
response for the Dob/Met infusion (94%4) was significantly higher than for the
Dob infusion alone (48%10) (P<0.005 significant at the 99% confidence
interval - nonpaired Student's t-test,
n=13). The model, generated by substitution into Equation (3) of the modified
dissociation constants, KDH(1+ f [D]/ Ki) and KDL(1+
f [D]/ Ki), was plotted for comparison with the experimental
findings (compare the model in the dark symbols - "
RH Dob" (dark triangles) and "
RH Dob/Met" (dark circles) with the experiments in the
light symbols - "
Dob" (triangles) with "Dob/Met" (squares)).
Similar to the results obtained for the Iso experiments, the
responses of the animals to the Dob IV infusions showed an initial increase in
dP/dt response with the dP/dt reaching an average peak at 20
g kg-1min-1 (range: 4-100
g kg-1min-1) followed by a sharp
decline (
Dob in Figure 4). The decline in dP/dt was on average 40%
below baseline levels. This decline was present to some degree in all of the
responses (range: 56 to -135%), but had a wide range of variability. This
demonstrates that all of these rats were sensitive to Dob induced
desensitization although some were much more sensitive than others.
Table 2 - Summary of the Responses to the Agonist Solutions Compared to the Agonist/Antagonist Solutions
|
1) Experiments with Iso or Iso/Met solutions: |
|||
|
Iso ( |
dP/dt (%)* Iso (n=6) |
Iso/Met ( |
dP/dt (%) Iso/Met (n=3) |
|
5 |
5521 |
5 |
973|| |
|
10 |
-1535İ |
10 |
962|| |
|
20 |
-27 |
20 |
8515 |
|
|
|||
|
2) Experiments with Dob or Dob/Met solutions: |
|||
|
Dob ( |
dP/dt (%) Dob (n=9) |
Dob/Met ( |
dP/dt (%) Dob/Met (n=4) |
|
200 |
4810 |
200 |
944 |
|
400 |
914 |
500 |
868 |
|
800 |
-3828 |
1000 |
737 |
*
Values of dP/dt (%) are meanss.e.
The data for this column include
data from the three paired rats plus three additional rats tested with the Iso
solution alone.
İAt 10
g kg-1min-1 for the Iso IV infusions,
arrhythmias occurred in 1 rat therefore the data was discarded and n is reduced
from 6 to 5.
At 20
g kg-1min-1 for the Iso IV infusions,
arrhythmias occurred in 4 rats therefore the data was discarded and n is
reduced from 6 to 2.
|| P<0.05 Student's t-test
P<0.005 Student's t-test
In a separate experiment, the Dob/Met (1.0/1.6
g kg-1min-1) solution was administered
as a single IV solution. Comparing the Dob/Met to the Dob infused group of
animals, a maximum response was maintained throughout the infusion range,
whereas the Dob group showed a progressive decline in the average response to
values below baseline (see Figure 4 and Table 2). At 200
g kg-1min-1 the dP/dt average response
for the Dob/Met infusion (94%4) was significantly higher than the dP/dt
average response for the Dob IV infusion (48%10) (P<0.005 significant at
the 99% confidence interval - nonpaired Student's t-test, n=13). In addition, this theoretical model
predicted these experimental findings (compare the model, dark symbols - 
RH Dob (dark triangles) and 
RH Dob/Met (dark circles) with the experiments, light symbols
- 
Dob (triangles)
with Dob/Met (squares) in Figure 4) based upon Equation (3) with modification
for a competitive antagonist and the ratio from Equation (4).
Discussion
All of the experiments with the
agonist drugs alone rapidly desensitized the dP/dt responses for each animal at
the higher infusion levels. After infusions of either the Iso or Dob agonist
solutions, peak responses occurred on average at 5 
g kg-1min-1 (range: 1-5) for the Iso
solution or 20
g kg-1min-1 (range: 4-100) for the Dob
solution and subsequently declined. The declines from these peaks were variable
in their onset with step reductions in responses to as low as 15% to 40% below
baseline levels (Table 2). The increased variability within the desensitization
range (35% Iso and 28% Dob see Table 2) also suggests that there exists the
potential for large variations in response when using these drugs clinically.
However, by combining either agonist (Iso or Dob) with metoprolol in the
specified agonist/antagonist ratio, the reductions in responses were
significantly less than the controls (compare Iso (squares) vs. Iso/Met
(circles) in Figure 1B and 
Dob (triangles)
with Dob/Met (squares) in Figure 4).
Although some animals were much more sensitive than others to the
desensitization potential of these drugs, there may be a genetic component to
the onset of desensitization, which was not explored. However, both in the
concentration of onset and the decline below baseline values (Figure 2A and
Table 2), all of the animals receiving either the Iso/Met or the Dob/Met
agonist/antagonist solutions showed significantly less desensitization for each
individual animal. In addition,
maximum responses were largely sustained for each animal receiving the
agonist/antagonist combination solutions. As seen in Table 2, which summarizes
these experimental results with their statistical significance, the Iso/Met or
Dob/Met solutions produced significantly higher responses past the peak than
the responses for either the Iso or Dob solutions alone.
Interestingly, the group of animals that received the Dob solution alone did not show cardiac arrhythmias comparable to the Iso group, but did show a comparable diminution in dP/dt response. This suggests that arrhythmias and desensitization are not necessarily coupled. However, using the Iso/Met agonist/antagonist combination solution, the occurrences of arrhythmias and variations of these responses were both markedly reduced compare to either of the agonist solutions alone.
These experiments demonstrate that specific agonist/antagonist combinations prevent rapid receptor desensitization over a wide range of infusions and also support the hypothesis that desensitization can be reduced or eliminated at the level of the receptor. This may also relate to observations that partial agonists appear to cause less desensitization than full agonists in some receptor systems [5]. In addition, this study further suggests that a full agonist can be made into a "full partial agonist" by adding a specific amount of an antagonist. This concept may have important implications for the modeling of pharmacological drug-receptor interactions since other receptor reaction schemes appear unable to model these results with meaningful biophysical parameters.
Since some of the abnormalities observed in adrenergic
signaling in late-stage heart failure are most likely due to sustained
adrenergic stimulation and concomitant receptor desensitization [13], this
study may provide an additional, scientific rationale for how beta-blocker
therapy improves cardiac function in patients with heart failure [18,19]. This
study suggests that this improvement results from the ability of beta-1-antagonists
to inhibit desensitization in the presence of desensitizing levels of 
-agonist drugs or the naturally occurring 
-agonist hormones such as epinephrine.
From a modeling perspective, this model was able to both describe
and predict the experimental responses for the animals receiving either 
-agonist drugs, or 
-agonist/antagonist combinations (compare the model, dark
symbols, with the experiments, light symbols, in Figures 1B and 4). One reason
why other theories of drug-receptor activation have difficulty modeling these
types of agonist/antagonist interactions is because the additional competition
of a competitive antagonist at the receptor should, theoretically, hinder the
receptor binding with an agonist and diminish the maximal response. Although it
appears surprising that the combination of an agonist with a competitive
antagonist in a specific ratio can maintain the receptor in an active
conformational state, this theoretical model predicts these results and
supports the concept that desensitization can be controlled primarily at the
level of the initial receptor response.
Although there have been extensive studies on the downstream mechanisms associated with receptor desensitization and the decoupling of receptors from their intracellular signaling pathways, the timing and sequence of these events in relation to receptor desensitization still need further clarification. This study raises the prospect that the receptor may remain in an activated state without being desensitized. Therefore, the theory that the phosphorylation of receptors by heterotrimeric guanine nucleotidebinding protein (G protein)coupled receptor kinases (GRKs) is a universal regulatory mechanism that leads to desensitization of G protein signaling (18) may need revision based upon the findings of this study. Whether activated GPCRs are first phosphorylated by GRKs and then bound by molecules of arrestin, which block the binding of the G proteins, or whether there are other states of GPCRs that are phosphorylated to modulate their activi