J. Auton. Pharmac.

(1982), 2, 277-295

REVIEW
IMPORTANT CONCEPTS OF RECEPTOR THEORY

R. R. RUFFOLO, Jr
Department of Cardiovascular Pharmacology, Lilly Research Laboratories, Eli Lilly and Company,
Indianapolis, Indiana 46285, USA

CONTENTS page number

1. Introduction 27 7

2. Receptor Theories 278

2.1 Occupancy Theory -Historical Perspective 278
Clark Treatment 278
Ariens Treatment 278
Stephenson Treatment 279
Furchgott Treatment 280
2.2 Rate Theory 280
2.3 Macromolecular Perturbation Theory 281
2.4 Dynamic Receptor Hypothesis 282
2.5 Mobile Receptor Hypothesis 282
2.6 Summary o f Receptor Theories 282

3. Important Concepts o f Current Occupancy Theory 283

3.1 Full Agonists and the Concept o f Spare Receptors 283
3.2 Partial Agonists 285
3.3 Relationship between intrinsic activity and efficacy 288
3.4 Competitive Antagonists 288
3.5 Threshold Phenomenon 289

4. Summary and Conclusions 290

Appendices
Appendix A 290
Clark Treatment (1 926) 290
Ariens Treatment ( 1 954) 291
Stephenson Treatment ( 1 956) 291
Appendix B
Calculation o f Dissociation Constants 292
Determination o f Dissociation Constants o f Agonists 292
Determination of Dissociation Constants of Partial Agonists 292
Determination o f Dissociation Constants o f Competitive Antagonists 293
Determination of Dissociation Constants of Agonists, Partial Agonists and Competitive
Antagonists by Radioligand Binding Techniques 294

1. Introduction non-mathematically oriented, but otherwise

Current receptor theory attempts to relate drug
effects in a highly quantitative manner to the
interaction of the drug molecule with its receptor.
In most treatments, receptor theory is presented
in seemingly complex mathematical terms in
which fairly simple concepts may be lost to the
interested, reader. The intent of this review is
to demonstrate many of these important concepts
of receptor theory in a manner that will not disturb
those who are less interested in the underlying
mathematical relationships. Examples of these
important concepts will be presented and verbal
definitions will be used wherever possible. In
278 R. R. RUFFOLO Jr
addition, two appendices have been included to
accommodate those readers who are so inclined to
undertake the more conventional mathematical
approach. Appendix A deals with the mathematical
concepts in the origin and subsequent development
of occupation theory. Appendix B presents tne
mathematical derivations for the procedures
used in determining dissociation constants of
agonists, partial agonistsandcompetitiveantagonists
in isolated smooth muscle preparations (in which
physiological responses are measured) and in
radioligand binding studies. Both appendices
contain, for the sake of completeness and clarity,
most of the algebraic manipulations required for
the various derivations, with very few steps being
eliminated. In this way, it is hoped that these
appendices will also be useful to those who are not
normally interested in the traditional mathematical
approach.
2. Receptor Theories
2.1 Occupation Theory -Historical Perspective
The concept of specific receptors was first
presented by Langley (1878) in relation to his
studies on the antagonism by atropine of pilo-
carpine-induced salivary flow in cats. The term
receptive substance was later used by Langley
(1905) to describe the site where substances
such as nicotine and curare act. It was also proposed
(Langley, 1905) that the receptive substance was
the recipient of stimuli from drugs and hormones
acting upon it, and that the function of the
receptive substance was to transfer these stimuli
to the effector organ. The term receptor was later
coined by Ehrlich (1913) who had previously
studied the high degree of specificity of anti-
bodies and noted the obvious relationship to the
action of drugs. (For a more complete description
of the origin and evolution of the term, receptor,
see DeMeyts & Rousseau, 1980).
Clark Treatment. The first quantitative treat-
ment of receptor theory was presented by Clark
(1926), and this serves as the foundation of
occupancy theory. Clark proposed that the inter-
action of drugs with their pharmacological
receptors was described by a mathematical relation-
ship which was similar to that which had been
applied to the interaction of oxygen and carbon
dioxide with haemoglobin. The reaction between
drugs and receptors was postulated to adhere to
Langmuir’s adsorption isotherm (Langmuir, 1918)
which was described by the law of mass action
(see Apendix A). The most important assumption
of this theory was that the magnitude of the
pharmacological effect of a drug was directly
proportional to the number of receptors occupied
by the drug, and that the maximum response was
obtained only when all receptors were occupied.
Additionally, it was assumed that one molecule
of drug interacted with only one receptor, and
that the quantitiy of drug that occupied the
receptor population was negligible relative to the
total amount of drug present in the bathing
medium. The latter assumption was shown to be
true for acetylcholine in the heart and rectus
abdominis muscle of the frog by Clark (1926)
and for adrenaline in the rabbit uterus by Gaddum
(1926).
Clark (1937) recognized that the action of
drugs depended upon two factors: 1) ‘fixation’
(binding) of the drug by the receptor and 2)
the ability of the drug t o produce its action after
‘fixation’. However, the occupancy theory as
proposed by Clark did not address, in a quantitive
manner, the second factor dealing with the ability
of a drug to elicit a response subsequent to binding.
Arie‘ns Treatment. Noting this deficiency in
the occupation theory as proposed by Clark,
Ariens (1954), intrigued by the observation that
not all members of a homologous series of para-
aminobenzoic acid derivatives were active (although
all appeared to bind to the receptor), proposed
that the biological response should be divided
into two separate and independent parameters:
1) affinity, which described the attachment or
binding of the drug to the receptor and was
governed by the law of mass action and 2) in-
trinsic activity which related to the ability of the
drug t o induce an effect after binding. Thus,
within a homologous series of compounds, those
compounds termed agonists would possess an
affinity for the receptor and would also have a
measurable intrinsic activity. Conversely, those
compounds which would bind to the receptor
but had intrinsic activities of zero (i.e. produced
no pharmacological response) were competitive
antagonists. According t o Ariens, a competitive
antagonist possesses the appropriate structural
requirements necessary for occupying the ieceptor
(affinity), but lacked those separate and inde-
pendent structural requirements necessary for
eliciting a response (i.e. no intrinsic activity).
Recognizing that the pharmacological effect of
a drug could be broken down into the independent
parameters of affinity and intrinsic activity, Ariens
(1954) developed a general formulation of drug
action by incorporating the heretofore neglected
concept of intrinsic activity into the Michaelis-
Menten equation, the latter used to describe the
reaction of an enzyme and substrate (see appendix
A). The quantitative mathematical relationships
that resulted from Ariens’ initial treatment of
 IMPORTANT CONCEPTS OF RECEPTOR THEORY
receptor theory suggested that the ED50could be
used as an index of affinity (i.e. an estimate of
the dissociation constant, or more precisely,
that concentration of drug required to occupy
50 percent of the receptor pool), and that the
maximal effect of the drug could be used as a
measure of intrinsic activity. Clearly, drugs with
high affinity could have either high or low intrinsic
activities, and vice versa. The term partial agonist
was introduced by Stephenson (1956) to describe
those drugs that possessed agonist activity but
which produced lesser maximal responses than
other agonists known to act at the same receptor.
Thus, an intrinsic activity of 1 was characteristic
of full agonists, whereas partial agonists were
characterized by having intrinsic activities greater
than zero but less than 1. A competitive antagonist
produced no overt response of its own and there-
fore had an intrinsic activity of zero. The
assumptions implied by Ariens (1954) were that:
1) the response of a full agonist was proportional
to receptor occupancy with the maximum response
being obtained only when all receptors were
occupied, 2) the binding of the drug to the receptor
was described by the law of mass action, and only
one drug molecule would bind to one receptor,
and 3) the quantity of drug bound to the receptor
population was small relative to the total amount
of drug available.
One major assumption made by Clark (1926),
and by Ariens (1954) in his initial treatment of
occupation theory, was that the percent of the
total receptor pool occupied by a drug would be
equal to the percent (of maximum) response
elicited by the drug in that tissue. Thus, when 50
percent of the receptors were occupied by an
agonist, a response equal to 50 percent of the
possible maximum response was obtained. It
followed, therefore, that all receptors needed to be
occupied for a maximal response. Stephenson
(1956), Furchgott (1955) and Nickerson (1956)
demonstrated that this latter assumption was not
true in all cases.
Stephenson Treatment. Stephenson (1956),
in a classic paper, expanded the concepts of
receptor theory previously presented by Clark
(1926) and Ariens (1954) to include the following:
1) the response of a drug was some unknown
positive function of receptor occupancy; thus,
the pharmacological response did not need to be
linearly proportional to the percentage of receptors
occupied, 2) a maximum effect could be produced
by an agonist when occupying only a small
proportion of the receptors and 3) different drugs
may haw varying capacities to initiate a response
and consequentiy, needed to occupy different
proportions of the receptors when producing
279
equal responses. This latter property of drugs
was referred to as efficacy. The term efficacy in
Stephenson’s treatment of occupation theory
replaced Ariens’ intrinsic activity term. Although
both terms refer to the ability of a drug to evoke
a response subsequent to binding, it must be
emphasized that efficacy, as described by Stephen-
son, and intrinsic activity, as initially described by
Ariens, were not synonymous [although intrinsic
activity had came to have the same meaning as
efficacy in later treatments by Ariens (1966)l.
Theoretically, it was possible for two full agonists
with intrinsic activities of 1 to have different
efficacies. The relationship between intrinsic
activity (as originally proposed by Ariens and
still used today) and efficacy will be discussed
later in this review.
Support for Stephenson’s modification of
occupation theory came from the elegant studies
of Furchgott (1955) and Nickerson (1956) who
simultaneously and independently demonstrated
that in some tissues, irreversible receptor
antagonists would initially produce large parallel
rightward shifts in the dose-response curves of
agonists before depressions in the maximum
responses were observed. These studies suggested
that, in some tissues at least, inactivation of a large
number of receptors had to occur before the
typical noncompetitive type of blockade,
characterized by a reduction of the maximum
response, could be observed. This finding could
not have occurred if, as proposed by Clark and
then by Ariens, maximum responses to drugs
were achieved only when 100 percent of the
receptors were occupied by the drug. It was
therefore proposed by Nickerson (1956) that in
some tissues, an excess number of receptors was
present such that a receptor reserve (or spare
receptors) existed. It followed, therefore, that a
maximal response could be obtained with a con-
centration of a strong agonist well below that
concentration required for saturation of the
receptor population.
The concept of spare receptors implies that there
may be more receptors present than the minimum
number required to produce a maximum response
in many tissues, and that 100 percent receptor
occupancy was not always necessary for a maximum
response, as had been previously proposed by
Clark (1926) and Ariens (1954). Thus, in some
tissues, a significant proportion of the receptor
pool could be irreversibly inactivated, and yet a
maximum response to a strong agonist could
still be attained if higher concentrations of the
agonist were administered. These higher con-
centrations of agonist would presumably increase
receptor occupancy (according to the law of mass
action) of the non-alkylated receptors to the point
280 R. A. RUFFOLO Jr
where sufficient occupancy could still be reached
to elicit a maximum response. Only when the total
number of free receptors was reduced below that
level required for a maximum response (i.e. when
receptor number becomes the limiting factor in
the maximum attainable response), would
depression of the maximum be observed. It was
concluded, therefore, that maximal receptor
occupancy need not be the limiting factor in
determining the maximum response of an agonist,
nor must receptor occupancy be linearly pro-
portional to response.
The existence of a receptor reserve and the non-
linear relationship between occupancy and
response indicated that the EDSO(concentration
of drug required for a half-maximal response) of
an agonist was not necessarily an accurate relection
of the dissociation constant (a measure of affinity)
since the EDSOwas not necessarily that concen-
tration of agonist required for occupancy of
half of the total receptor pool. Therefore the
affinity of an agonist could not be determined
from a simple dose-response curve as previously
believed. The relationship between the EDSOand
dissociation constant becomes apparent when one
considers that the dissociation constant is simply
that concentration of drug required for haff-
maximal receptor occupation, and that the EDSO,
on the other hand, is simply that concentration
of drug required for a half-maximal effect. Since
there need not be a linear relationship between
occupancy and response, and since a receptor
reserve may exist, in theory it is possible for a
half-maximum response to occur with significantly
fewer than 50 percent of the receptors occupied.
Differences between EDSOvalues and dissociation
constants of over 100 fold have been observed.
As a specific example, Besse & Furchgott (1976)
have observed that the EDSOof noradrenaline in
rabbit aorta is approximately 22 nM, while the
dissociation constant of noradrenaline for a-
adrenoreceptors in this same tissue is approxi-
mately 339 nM. Their calculations indicated that
only approximately 6 percent of the total a-
receptor pool needed t o be occupied by noradren-
aline for a half-maximal response in rabbit aorta.
What these findings mean is that noradrenaline,
at a concentration of 339 nM, will bind to 50 per-
cent of the a-receptors in rabbit aorta. But in this
tissue, noradrenaline needs to occupy only 6 per-
cent of the receptors to elicit a response equivalent
to one-half of the maximum, and this 6 percent
receptor occupancy of noradrenaline could be
achieved at a concentration of only 22 nM. In this
tissue and others like it where occupancy is not
proportional to response and where a receptor
reserve exists, the EDSO will obviously occur at
some concentration far below that concentration
which the law of mass action indicates is necessary
for 50 percent receptor occupancy (i.e.dissociation
constant). In the specific example provided by
Besse & Furchgott (1976), the EDSOwas simply
that concentration of noradrenaline required to
occupy ti percent of the a-receptor pool in rabbit
aorta. Thus, the reliability of the EDSO as a
measure of affinity is largely dependent upon the
magnitude of the receptor reserve and the degree
to which occupancy and response deviate from
linearity. These concepts will be addressed in
greater detail with specific examples later in this
review,
A major obstacle in the application of the
modified occupation theory was the difficulty
in determining (in isolated tissues) the affinity
of agonists (particularly full agonists) for receptors.
Furchgott (1966) is largely credited with the
development of the specific techniques for deter-
mining dissociation constants of agonists in iso-
lated tissues. These methods require the use of
irreversible receptor antagonists which had
previously been developed by Nickerson (1949).
These contributions are discussed in greater
detail in appendix B.
Furchgott Treatment. As stated above, Furch-
gott’s greatest contribution to receptor theory was
its validation and the development of methods for
determining dissociation constants of agonists.
However, Furchgott (1966) did modify receptor
theory in an important way by proposing that the
term efficacy (e), as described by Stephenson
(1956), was actually the product of intrinsic
efficacy (f) and the concentration of active
receptors in a given tissue (Le. e = ~ [ R T ] ) .
Although this modification appears subtle, it is
nonetheless an important distinction since it
clearly demonstrates that the term efficacy is not
strictly a drug parameter because it depends, in
part, on the characteristics of the tissue in question
(i.e. receptor number, receptor reserve, coupling
between occupancy and response, etc.). Intrinsic
efficacy (E), in fact, is a drug parameter and its
magnitude is independent of the tissue or test
system used. Thus, the efficacy of a drug, by
virtue of its dependency upon the number of
active receptors, will vary from tissue-to-tissue
as the number of active receptors varies, while
intrinsic efficacy is a property of the drug molecule
alone and relates to the ability of the drug molecule
itself to produce a ‘stimulus’ when acting upon a
given receptor.
2.2 Rate Theory
As an alternative to the occupation and modified
occupation theories which assume that the
biological effect is in some manner related to the
 IMPORTANT CONCEPTS OF RECEPTOR THEORY
proportion of receptors occupied, Paton (1961)
proposed the rate theory of drug action. Funda-
mental to this theory is the postulate that the
biological response is proportional to the rate at
which the drug combines with the receptor. Paton
proposed that association and dissociation rate
constants were paramount in determining the
affinity and the stimulant activity of drugs. It
was assumed that each association of drug with
receptor resulted in a ‘quantum’ of excitation.
Clearly, the number of associations, and there-
fore the number of quanta, would determine the
magnitude of response. It followed that the
greater number of productive drug-receptor
associations, the greater the response.
Central to the rate theory was the necessity
for powerful agonists to dissociate rapidly from
the receptor to enable other successful associations
t o occur with the subsequent generation of
additional quanta of excitation. Hence, the dis-
sociation rate constant was considered to be the
determinant factor in whether a compound was
a full agonist, partial agonist or competitive
antagonist. Compounds with high rates of
dissociation would be able to reassociate more
frequently and would be powerful agonists, where-
as competitive antagonists were proposed to have
very low rates of dissociation and would remain
bound to the receptor for longer periods of time,
and would therefore produce relatively few quanta
of excitation in a given period. Partial agonists
were hypothesized to have dissociation rate
constants between those of full agonists and
competitive antagonists.
According to the rate theory, the ratio of the
association and dissociation rate constants would
determine affinity, while the dissociation rate
constant would describe the stimulant properties
of the drug. Clearly the dissociation rate constant
would replace the terms intrinsic activity and
efficacy used in occupation theory.
Paton (1961) claimed that such a theory would
explain the persistent effects of antagonists and
the relatively transient effects of agonists which
were commonly observed in many laboratories.
He also argued that rate theory would explain,
at the molecular level, the reason that some drugs
were agonists while others were antagonists, a
problem that the various forms of occupancy
theory failed to explain. Furthermore, the rate
theory predicted that agonists, which have to
dissociate from the receptor more rapidly than
antagonists, would have lower affinities than
antagonists, an observation that had also been
made by proponents of occupancy theory
(Stephenson, 1956).
The attractiveness of the rate theory was its
simplicity and relative lack of assumptions. How-
281
ever, this theory could not account for a good
many observations which ultimately led to a
debate over its validity. One assumption required
by rate theory was that the rates of onset and
offset of drug action reflected the association and
dissociation rate constants, respectively. Furch-
gott (1955, 1964), Thron & Waud (1968) and
Roberts & Stephenson (1976) demonstrated
clearly that this was not the case in most tissues.
These investigators established that the rates of
onset and offset of drug action were limited by
anatomically unknown, but kinetically distinct,
diffusion barriers. In few instances could onset
and offset rates be shown to be limited by
association with, or dissociation from, the receptor.
In addition, the rate theory would predict that
even competitive antagonists would possess some
residual agonist activity since they must still
initially associate with the receptor and therefore
must generate, at least initially, some quanta of
excitation. Examples are known where classical
antagonists display the ‘vestiges’ of stimulant
action, but this could not be demonstrated for the
majority of competitive antagonists. Finally,
rate theory would predict the phenomenon of
fade in which the initial response is a peak which
gradually fades t o a lower plateau at equilibrium.
The explanation for this phenomenon is that the
initial rate of drug-receptor interaction will be
greater than the rate at steady state. However,
there are numerous instances in which fade cannot
be demonstrated casting further doubt as to the
general applicability of the theory. In view of
these problems, and the fact that occupation
theory, in its current form, was consistent with the
majority of observations, the rate theory was felt
to be of only limited usefulness.
2.3 Macromolecular Perturbation Theory
The occupation and rate theories attempted
to relate pharmacological response to the inter-
actions of drugs with their receptors. Little
attention was paid to the underlying molecular
interactions that occurred at the receptor level
that ultimately lead to the biological stimulus
which produces the pharmacological effect. In fact,
occupation and rate theories were designed around
our ignorance of what happens to the receptor
when acted upon by the drug. As a result of this
limitation in the occupation and rate theories,
Belleau (1964) developed the macromolecular
perturbation theory in an attempt to explain,
at the molecular level, the concepts of intrinsic
activity and efficacy which were not fully explained
by the preceding theories. The model is more
qualitative in nature than either occupancy or
rate theory and serves to conceptualize the mole-
cular events leading to the observed biological
282 R. R. RUFFOLO Jr
response. It is similar in concept to the model
of enzyme flexibility and substrate induced-
fit developed by Koshland (1958) to describe
enzyme-substrate interactions at the molecular
level.
Receptors, like enzymes, were proposed to be
protein in nature. In view of the unique confor-
mational adaptibility of enzymes, Belleau proposed
that receptors, likewise, would also undergo
conformational changes when acted upon by drugs.
Two types of drug-receptor interactions were
proposed. The first was termed a specific con-
formational perturbation (SCP) induced by agonist
molecules, and the second called a nonspecific
conformational perturbation (NSCP) which was
induced by antagonists. The SCP was proposed to
lead to a biological stimulus, and ultimately a
response, whereas the NSCP was proposed to be
ineffective in leading to a biological stimulus.
Additionally, partial agonists were proposed to
induce an ‘equilibrium mixture’ of both the
specific and nonspecific conformational per-
turbations (SCP +- NSCP). According t o the
macromolecular perturbation theory, the type of
conformational perturbation (i.e. specific, non-
specific or an equilibrium mixture), as well as the
rate at which these conformational changes
occured, were used as parameters of drug action
and replaced the terms affinity, intrinsic activity
and efficacy used in occupation theory.
2.4 The Dynamic Receptor Hypothesis
The dynamic receptor hypothesis as proposed
by Bloom & Goldman (1966) was developed
specifically to describe drug actions at adreno-
receptors. However, applications to other receptor
systems were considered possible (Bloom, 1970).
This theory visualized the receptor as an enzyme-
substrate complex, and the function of an agonist
(or partial agonist) was to bind to the enzyme-
substrate complex and subsequently regulate or
modulate the rate of the ongoing reaction. Thus,
the agonist serves to stimulate or inhibit the basal
rate of a reaction system. One might consider
the dynamic receptor hypothesis as being applicable
to effects mediated via 0-adrenoreceptors and
associated with the ATP-Mg+’-adenylate cyclase
complex (including regulatory units and catechol-
amine binding sites). Catecholamines with 0-
adrenoreceptor agonist activity increase the rate
of cyclic-AMP generation by this enzyme-substrate
complex which, under nonstimulated conditions,
functions at some lower basal rate.
Desensitization by agonists could be readily
accounted for in this theory by overstimulation
of the enzyme-substrate complex and the ultimate
temporary depletion of substrate.
2.5. Mobile Receptor Hypothesis
This model, proposed by Cuatrecasas and co-
workers (Cuatrecasas, 1974, Bennett, O’Keefe
& Cuatrecasas, 1975; Jacobs & Cuatrecasas, 1976)
and by De Haen (1976), envisions separate and
independent recognition and catalytic components
of receptors freely diffusing laterally in a fluid
membrane. Binding of a ligand to the recognition
complex causes the freely diffusing recognition
and catalytic components t o associate in an active
form to initiate a biological response. This theory
has greatest application for those hormone
receptors associated with adenylate cyclase in
which the onset of response is relatively slow
compared to the expected high rates of ligand-
receptor interactions. The interaction of one
ligand-recognition site complex with one of more
catalytic units is possible. The possibility also
exists for one catalytic subunit to be acted upon
sequentially by many ligand-recognition site
complexes. Mathematical treatments of the mobile
receptor hypothesis have been presented by others
(Hollenberg, 1978; De Haen, 1976; Jacobs &
Cuatrecasas, 1976).
2.6 Summary of the Receptor Theories
Examples exist in the literature which support
each of the theories discussed above. In particular,
the treatment of Clark, as modified by Ariens to
include affinity and intrinsic activity, is applicable
in some systems lacking a receptor reserve (i.e.
those systems in which a poor relationship exists
between biological stimulus and response) or in
the case of some very weak partial agonists in
which a linear relationship may exist between
receptor occupancy and response. The rate theory
of Paton, although not presently believed to be
generally applicable, does explain the actions of
some drugs in some tissues where diffusion of the
drug to the region of the receptors has been shown
not to be rate limiting. However, the theory with
the broadest application is the occupation theory
as presented by Stephenson (1956) and validated,
modified and extended by Furchgott (1955,1966;
Furchgott & Bursztyn, 1967) and Nickerson
(1956). This theory is applicable in most situations
and is not limited by the existence of spare
receptors or diffusional barriers. Important
concepts of this modified occupation theory will
be discussed in greater detail below.
The macromolecular perturbation theory,
dynamic receptor hypothesis and mobile receptor
hypothesis may each be valid under different
circumstances. These latter theories are not
mutually exclusive of one another, nor do they
necessarily conflict with occupation theory.
 IMPORTANT CONCEPTS OF RECEPTOR THEORY 283

3. Important Concepts of Current Occupancy

Theory 100 -
7A"""" ' """"
OOSE vs. 'RESPONSE
"""" ' ""-
3.1 Full Agonists and the Concept of Spare
Receptors

40
The major difference between the occupation
theory as proposed by Stephenson (1956), and the
original occupation theory as defined by Clark
(1926) and modified by Ariens (1954), is the
concept of spare receptors (or a receptor reserve)
in the former. This has afforded the Stephenson
treatment greater applicability with fewer limi-
= 200
tations, and has led to 'general' overall acceptance.
Stephenson proposed that a linear relationship 10-8 10-7 10% 145
need not exist between receptor occupancy and (CARBAHYlCHolINE] Y
response, and that in many tissues a maximum
response elicited by a strong agonist could be
attained when only a fraction of the total receptor
pool was occupied. Additional evidence for this OCCUPANCY' vs RESPONSE -
concept was supplied by Furchgott (1955) and
Nickerson (1956). Clearly, the Stephenson treat-
ment had fewer limitations than the other
variations of the occupancy theory. The concept
of spare receptors is illustrated in Fig. 1. In Fig. L
1A is presented a log dose-response curve for
carbamylcholine obtained in circular smooth
muscle from rabbit stomach (Furchgott, 1966).
The EDSOof 0.06 pM may be obtained directly
from the plot. The dissociation constant calculated
by Furchgott (1966) for carbamylcholine in this 1 I
tissue is 16 pM, a value more than 260 times 0 0.05 0. I
greater than the EDSO.The fraction of the receptors PERCENT RECEPTOR OCCUPANCY
occupied by the drug may be calculated from the
relatively simple relationship between the equili-
brium dissociation constant (KD) for the drug C' '"""1 ' ' """' ' ' """' vs
log OCCUPANCY ' ' """' ' ' ""'?
RESPUNSE ' ' "

receptor complex and the concentration of drug 100

/--
in the vicinity of the receptors:
fractional
receptor = [RAI
- = [A1 (1) E ~ = 0 . 1 %OCCUPANCY
occupancy [RT] KD [A]
+ 60
0.5 E~=0.004%OCCUPANCY
L
Equation (1) is simply a statement of the law of
mass action (see appendix A for derivation) which
occupation theory states will predict receptor
occupancy. [RA] refers to the concentration of
receptors occupied by the agonist (i.e. concen-
tration of receptor-agonist complex) and [RT] is
the total concentration of receptors. Clearly, 0.WOl 0.001 0.01 0.1 1 10 100
PERCENT RECEPTOR OCCUPANCY
the fraction [ RA] /[RT] is the fractional receptor
occupancy which will equal 1 when [A] is large
(i.e. [A]>>KD) and receptors are saturated. FIG. 1. Effects of carbamylcholine on circular
KD is the dissociation constant of the agonist smooth muscle from rabbit stomach. A. Log
and [A] is the concentration of free agonist in dose-response curve for carbamylcholine. The
EDSO is that dose required for a half-maximal
the region of the receptors. Since an assumption effect and KD is the dissociation constant. B.
of occupancy theory is that the concentration Response from A replotted as a linear function of
of drug bound to the receptors is negligible relative receptor occupancy, the latter calculated from
t o the total concentration of drug present, [A] equation 1. C. Response from A replotted as a
logarithmic function of receptor occupancy.
is generally taken to be the concentration of drug Data from Furchgott (1966).
284 R. R. RUFFOLO Jr
added to the bathing medium. When the same
response as in Fig. 1A is re-plotted as a function
of receptor occupancy (calculated from equation
1) on a linear scale (Fig. lB), it becomes apparent
that the relationship between occupancy and
response is, in fact, nonlinear, consistent with
Stephenson’s treatment of occupation theory.
A plot of receptor occupancy on a logarithmic
scale us. response produces a sigmoid curve (Fig.
1C). It is obvious from this plot that there exists
a large receptor reserve for carbamylcholine in
the rabbit stomach. This is evidenced by the fact
that a half maximal response is obtained at a
receptor occupancy of only 0.004 percent, and a
maximal response can be obtained when only
approximately 0.1 percent of the receptors are
occupied. Thus, for carbamylcholine in rabbit
stomach strips, a 99.9 percent receptor reserve
exists, and this would account for the observation
made by Furchgott (1966) that even when 96.5
percent of the muscarinic receptors are irreversibly
inactivated in this tissue, a near maximal response
may still be obtained with carbamylcholine.
To place these results in their proper perspective,
one must remember that the dissociation constant
is simply that concentration of drug required to
occupy 50 percent o f the receptor population,
whereas the EDSOis that concentration of agonist
required to produce a half-maximal response.
Due to a large reserve (or excess) of muscarinic
receptors in rabbit stomach, only 0.004 percent
of the total receptor pool needs to be occupied
by carbamylcholine to produce a half-maximal
response. Thus, the EDSO is simply that con-
centration required to achieve 0.004 percent
receptor occupancy in rabbit stomach and clearly,
must be smaller in magnitude than the dissociation
constant which is always the concentration re-
quired for 50 percent receptor occupancy. The
magnitude of the difference between the EDSO
and the dissociation constant reflects the extent
of the receptor reserve.
Since the receptor reserve is responsible, in part,
for EDSO values being lower than their respective
equilibrium dissociation constants in many tissues,
by reasoning, if the receptor reserve is progressively
diminished by irreversible receptor inactivation,
then the EDso should increase and approach the
dissociation constant. This has been demonstrated
in Fig. 2. Dose-response curves to histamine were
obtained in guinea pig ileum (Ariens, Van Rossum
& Koopman, 1960) where, under control
conditions, the EDSO is approximately 50 fold
lower than the dissociation constant (the latter
calculated by Furchgott, 1966). This difference
suggests a significant receptor reserve. The curves
to the right of the control curve represent five
successive 10-minute treatments with the ir-
reversible antagonist, phenoxybenzamine (1Ow6M),
which will inactivate increasing proportions of
the receptor pool with each subsequent treatment.
The number adjacent to each curve represents
the percentage of receptors remaining actiue
following treatment with phenoxybenzamine. It
is apparent that as the number of receptors
remaining active is progressively decreased (and
the receptor reserve progressively diminished until
it no longer exists), the dose-response curves
are displaced in a rightward manner and ultimately
show a depressed maxima at the point where the
minimum number of receptors required by
histamine to produce a maximum response
becomes limiting. [Note that before the maximum
is depressed, parallel curves with no depression
of the maxima are obtained further demonstrating
the presence of spare receptors. Such an observation
was made by Nickerson (1956), and was considered
at the time to be the most important evidence for
the existence of spare receptors]. As the receptor
reserve is progressively decreased by irreversible
blockade, there are progressive rightward shifts
in the dose-response curves such that the EDSO
values begin to approach the true dissociation
constant of 1 x lO-’M, as predicted from theory.
An appreciation of the magnitude of the receptor
reserve may be gained by considering the maximal
response. When 75 percent of the receptor pool
is inactivated with phenoxybenzamine (i.e. 25 per-
[HISTAMINE] M
FIG. 2. Log dose-response curves for histamine
in guinea-pig ileum following 5 successive 10-
minute treatments with the irreversible antagonist
phenoxybenzamine. The numbers to the right of
each curve represent the percentage of receptors
still active (Le. receptors not alkylated) after each
phenoxybenzamine treatment. As rece tors are
progressively inactivated, the ED59 of Ristamine
progressively approaches the dissociation constant.
Data from Ariens et al., (1960) and Furchgott
(1966).
 IMPORTANT CONCEPTS OF RECEPTOR THEORY
cent of the receptors remaining in the active state),
the maximum contractile response to histamine
is still largely unaffected. Even when 92 percent
of the receptor pool is inactivated (Le. 8 percent
of the receptors still active), only a slight depression
of the maximum histamine response is observed in
this tissue (Fig. 2). From these data, one may con-
clude that spare receptors do, in fact, exist in this
tissue when histamine is the agonist, and that the
magnitude of the receptor reserve largely
determines the position of the dose-response curve
along the log dose axis and thereby determines
the EDSO. While the dissociation constant is
truely a constant, the EDSOis not since its value
is critically dependent upon, among other factors,
the magnitude of the receptor reserve which will
differ among drugs and tissues. In addition, the
maximum response of an agonist is depressed
only when the number of receptors becomes
limiting such that a sufficient biological stimulus
required for a maximum response cannot be
attained by saturation of the remaining receptors.
The number of receptors that need to be eliminated
in order to observe depression of the maximal
response is also dependent on the magnitude of
the receptor reserve, which in turn is dependent
on the tissue used and the intrinsic efficacy of
the agonist for that receptor.
The previous example serves to illustrate the
fact that as the receptor reserve is progressively
decreased, the EDSOapproaches the true dissoci-
ation constant and thereby becomes a better
estimate of the dissociation constant. The same
phenomenon may be illustrated in another way
by comparing EDSO values and dissociation
constants of a series of compounds with different
efficacies. The magnitude of the receptor reserve
for any given drug is critically dependent on the
efficacy of that compound. In any given tissue,
if the efficacy of a compound is high and the
compound is a full agonist, the receptor reserve
will by large. If, in the same tissue, the efficacy of
another compound is low such that the compound
is a weak partial agonist, the receptor reserve will
be nonexistent. Thus, by comparing the EDSO
values and dissociation constants of agonists
with different efficacies, we may also see how the
magnitude of the receptor reserve affects the
reliability of the EDSOas an estimate of the dis-
sociation constant. From Fig. 3, it is apparent that
the EDSOvalues of full agonists with high efficacies
(and presumably large receptor reserves) are poor
estimates of the dissociation constant (i.e. KD/
ED&>l) and may differ by more than 100 fold in
rat aorta (i.e. KD/EDsC-~OO).As the relative efficacy
progressively decreases and the compounds become
partial agonists with no receptor reserve, the EDSO
becomes a better estimate of the dissociation
285
constant (i.e. KD/EDsodecreasesand approaches 1).
It may be seen from Fig. 3 that for the very
weakest partial agonists in rat aorta (i.e. relative
efficacy < 0.03 in this instance), the EDSOvalues
are actually quite reliable estimates of the dis-
sociation constants (i.e. KD/EDso equals 1). From
this example, it appears appropriate to conclude
that the existence and magnitude of the receptor
reserve largely affects the difference observed
between the EDSOof an agonist and its dissociation
constant. When the receptor reserve has been
eliminated by irreversible receptor inactivation,
or when it does not exist as in the case for weak
partial agonists, the EDSOand dissociation constant
may be quite close.
3.2 Partial Agonists
It is clear from the discussion above that as
one decreases the size of the receptor reserve, the
EDso of a full agonist approaches the true dis-
sociation constant, and the maximal response
becomes progressively depressed. One may observe
a similar phenomenon when considering partial
agonists which produce maximal responses less
than those of full agonists and which, by their
nature, are associated with no receptor reserve.
That is to say that the maximum response to a
partial agonist is obtained only when all receptors
100
D ELI ABILITY OF E050 AS AN INDEX OF AFFINITY
8
i RAT AORTA
0.1
0.001 0111 a1 La
RELATIVE EFFICACY [PE=1.00)
FIG. 3. The ratio of the dissociation constant to
the EDSOas a function of relative efficac of a
series of compounds in rat aorta. As the erficacy
decreases, so does the receptor reserve, and the
EDSO becomes a more reliable estimate of the
dissociation constant. Data from Ruffolo e t al.,
(1979a,b,c, 1980a,b).
286 R . R. RUFFOLO Jr

are occupied even though a strong full agonist

may show a large receptor reserve in the same
tissue. Partial agonists are agonists with efficacies
lower than those of strong full agonists, but higher 80
than those of competitive antagonists. Since
partial agonists have lower efficacies than full PHENYLEPHRINE
agonists, they need to occupy greater percentages
of the receptor pool to produce the same response
as a full agonist (Stephenson, 1956). Depending
upon the efficacy of the partial agonist for the
receptor system in the particular tissue studied, 20
the EDSO may be a realistic estimate of the
dissociation constant, and the relationship between
receptor occupancy and response may approach
linearity.
To conceptualize the partial agonist and its
relationship to a full agonist, it is first beneficial
to introduce the term relative efficacy (Furch-
gott, 1966). Stephenson (1956) pointed out that

li
Y

the efficacy (ability to activate the receptor I 801

n
subsequent to binding) may vary within a homo- Y I
logous series of agonists. For full agonists, efficacy
will be high, indicating that the compounds are
very effective in 'triggering' the receptor once
bound. Conversely, competitive antagonists will
have very low or no efficacy, although they may
possess high affinity for the receptor. Partial
agonists have efficacies between those of full Y
Y)

agonists and competitive antagonists. Efficacy is

a dimensionless term with no theoretical limit E 0 20 40 60 80 100
e PERCENT RECEPTOR OCCUPANCY
(Stephenson, 1956; Furchgott, 1966). Thus, the Y

absolute efficacy cannot be determined. However,

within a series of agonists, it is possible to assign
relative efficacies (er) such that the er of a reference

agonist (usually a full agonist) is 1.This technique
has been developed by Furchgott & Bursztyn
(1967). The procedure involves plotting the
responses of a series of agonists not as a function
of their doses, but rather as a function of the
logarithm of receptor occupancy. Receptor
occupancy is calculated from mass law (equation 1)
using the dissociation constant of the agonist
and the concentration of agonist in the vicinity
of the receptors (generally taken as the con-
centration of agonist added to the bathing medium;
see previous discussion). An example in rat aorta
(Ruffolo e t al., 1979c) is presented in Fig. 4. In
Fig. 4A are shown log dose-response curves to
phenylephrine, oxymetazoline and naphazoline.
According to Stephenson (1956), different drugs
may have varying capacities to initiate a response
and consequently need to occupy different
proportions of the receptor population when pro-
ducing equal responses. This is evident from Fig.
4B where the responses in Fig. 4A are replotted
as a function of receptor occupancy foreachagonist.
Thus, phenylephnne, oxymetazoline and napha-
zoline each produce a response equivalent to 50
8ol
60
20
1 10 100
PERCENT RECEPTOR OCCUPANCY
FIG. 4. Effects of phenylephrine, oxymetazoline
and naphazoline in rat aorta. A. Log dose-response
curves. B. Response of each agonist from A
replotted as a linear function of receptor occu ancy
the latter calculated from equation 1. InJcated
for each compound is the percentage of the
receptor pool occupied by each agonist to pro-
duce a response equivalent to one-half of the
maximum response for phenylephrine. C. Response
from A replotted as a logarithmic function of
receptor occupancy. Data from Ruffolo et al.,
(1979~).
 IMPORTANT CONCEPTS OF RECEPTOR THEORY
percent of the phenylephrine maximum when
occupying, respectively, 7, 45 and 100 percent
of the receptor population (Fig. 4B). According
to Furchgott & Bursztyn (1967), the ratio of the
proportion of receptors occupied by a full agonist
(phenylephrine in this instance) to that occupied
by a partial agonist (oxymetazoline or napha-
zoline) to produce the same response is the relative
efficacy (er) of the partial agonist (relative to the
full agonist). For the compounds in Fig. 4, relative
efficacies of 1.0 (i.e. 7/7), 0.16 (i.e. 7/45) and
0.07 (Le. 7/100) have been calculated for pheny-
lephrine, oxymetazoline and naphazoline,
respectively. What these numbers indicate is that
oxymetazoline and naphazoline have lower effi-
cacies than phenylephrine and are therefore less
able to elicit a response subsequent to binding.
As a result, oxymetazoline and naphazoline must
occupy greater proportions of the receptor pool
relative to phenylephrine when producing the
same response. Since oxymetazoline and napha-
zoline are less active in ‘triggering’ or ‘activating’
the receptor than is phenylephrine, they must
occupy 6.4-fold (i.e. l/er) and 14.3-fold (I/er)
more receptors, respectively, than phenyiephrine
to produce any given response as is indicated in
Fig. 4C where the occupancy-response relationship
from Fig. 4B is presented on a logarithmic
occupancy scale. Furthermore, inspection of Fig.
4C indicates that the reason oxymetazoline and
naphazoline produce lower maximum responses
relative to phenylephrine is because of their lower
relative efficacies and the need of these two
compounds to occupy a greater number of
receptors to produce any given response. The need
for relatively greater receptor occupancies by
partial agonists creates the situation in which
receptor number becomes limiting in terms of
obtaining a maximum response. In the specific
example in Fig. 4, there are a sufficient number
o f receptors present in rat aorta for phenylephrine
to produce a maximum response, but not for the
partial agonists, oxymetazoline and naphazoline
which, by virtue of their lower efficacies, need to
occupy a greater number of receptors than
phenylephrine to produce any given response.
Thus, the maximal response of a partial agonist
is limited to that response obtainable at saturation
(i.e. 100 percent occupancy) which is, of course,
the limit of receptor occupancy. As a result,
there can be no receptor reserve for a partial
agonist .
The relationship between response and receptor
occupancy (plotted on a linear occupancy scale)
follows a characteristic pattern as one proceeds
from a compound with high relative efficacy to
a compound with low relative efficacy. In Fig. 5
are plotted the responses for the compounds in
287
Fig. 4 as a linear function of their respective
receptor occupancies. The responses of each
compound are expressed as a percentage of each
compounds own maximum response in this
instance. It is clear from Fig. 5 that the response
of the compound with the highest efficacy (i.e.
phenylephrine) is a hyperbolic function of receptor
occupancy, consistent with Stephenson’s treatment
of occupancy theory. However, as efficacy
decreases (i.e. oxymetazoline), the relationship
between occupancy and response begins to flatten
out and, in fact, becomes linear for compounds
with very low efficacies (i.e. naphazoline). Thus,
for very weak partial agonists which have-no
receptor reserve and which approach linear
relationships between occupancy and response,
the occupancy theory as presented by Clark
(1926) and modified by Ariens (1954) will apply
and the EDso will be a reliable estimate of the
dissociation constant. For the weak partial agonist,
naphazoline, in rat aorta, a response equivalent
to one-half of its own maximum is obtained at a
calculated receptor occupancy of approximately
5 1 per cent. Thus, for naphazoline, both the EDSO
and dissociation constant represent concentrations
required for 50 percent occupancy, thereby
explaining the good agreement between the EDSO
and dissociation constant in this case. This only
occurs because naphazoline represents the relatively
unique situation when no receptor reserve exists
and where the response is also linearly related to
occupancy (Fig. 4B and Fig. 5). Clearly, such is
-
4
r
K
r‘
=
z
0
0
Y
*
-
W
u
.)
z
0
a
v)
a
W
0 20 40 60 80 100
jlRd 1
% RECEPTOR OCCUPANCY [RAlx 100
FIG. 5. Responses of phenylephrine, oxymetazoline
and naphazoline in rat aorta expressed as a per-
centage of each compounds o w n maximum and
plotted as a linear function of receptor occupancy,
the latter calculated from equation 1. Data from
Ruffolo et al., ( 1 9 7 9 ~ ) .
288 R. R. RUFFOLO Jr
not the case for the strong full agonist, phenyle-
phrine, which will elicit a response equivalent to
one-half of its own maximum with only 6 percent
of the receptor pool being occupied. It must be
emphasized, therefore, that the suitability of the
Clark and Ariens versions of occupancy theory to
weak partial agonists represents a relatively minor
application.
3.3 Relationship Between Intrinsic Activity and
Efficacy
At this point it seems appropriate to consider
the relationship between intrinsic activity as
originally described by Ariens (1954) and relative
efficacy. Both terms are still extremely useful
to pharmacologists and are used quite commonly.
While both terms relate to the ability of an agonist
or partial agonist to activate the receptor
subsequent to binding, both terms do not have the
same meaning. Intrinsic activity, as originally
described, is determined directly from the dose-
response curve and is simply the ratio of the maxi-
mum response of a test agonist to the maximum
response of a reference agonist, the latter usually
being a full agonist. In the case presented in
Fig. 4, the maximum response of naphazoline
is 50 percent of that obtained with phenylephrine.
Thus, naphazoline has an intrinsic activity of 0.5.
Relative efficacy,as described previously, cannot
be determined from the dose-response curve but
first requires transformation of the log dose-
response relationship to log occupancy-response,
as performed in Fig. 4C, using the appropriate
dissociation constant and equation 1to calculate
receptor occupancy. As discussed above, the
efficacy of naphazoline is 0.07 relative to pheny-
lephrine, a markedly different estimate of stimulant
activity than the intrinsic activity of 0.5 would
indicate. Thus, while the intrinsic activity, as
originally described by Ariens, would suggest that
the ability of naphazoline to activate the receptor
is only one-half that of phenylephrine, cal-
culation of relative efficacies indicates that the
ability of naphazoline to activate the receptor
is actually 14-fold less than phenylephrine. It is
interesting to note that although naphazoline is
14-fold less effective than phenylephrine in eliciting
a response on a-adrenoreceptors after binding to
the receptor, the affinity (ability to bind to the
receptor) of naphazoline is nearly 60 fold greater
than phenylephrine indicating that affinity and
efficacy are, in fact, independent of one another
as proposed by Stephenson (1956), and implied
even earlier by Ariens (1954).
It is also possible for two componds with
intrinsic activities of 1, and hence both termed
full agonists, to differ in efficacy. Such an example
has been provided by Besse & Furchgott (1976)
for noradrenaline and a-ethylnoradrenaline in
rabbit aorta. From the dose-response curves of
each compound, Besse & Furchgott (1976)
observed that both compounds produced the
same maximal response, and would therefore
have intrinsic activities of 1. However, the abilities
of these agonists to activate the a-adrenoreceptor
of rabbit aorta were found not to be the same
when the responses of each of these agonists
were re-plotted as a function of receptor
occupancy, the latter calculated from equation 1
using the respective dissociation constants and
agonist concentrations. In spite of the fact that
both compounds produced the same maximal
responses, the efficacy of a-ethylnoradrenaline
was only 0.41 relative to noradrenaline. Thus,
the ability of a-ethylnoradrenaline to activate the
a-receptor in rabbit aorta was only 4 1 percent of
that for noradrenaline, or to put it another way,
noradrenaline and a-ethylnoradrenaline would pro-
duce identical responses in rabbit aorta only
when a-ethylnoradrenaline occupied 2.4-fold more
receptors than noradrenaline, in spite of the fact
that both compounds were able to elicit identical
maximal responses and therefore had the same
intrinsic activities of 1.
3.4 Competitive Antagonists
A competitive antagonist is a compound that
will occupy a given receptor but will produce no
response subsequent to occupation. In terms of
current occupation theory, a competitive
antagonist is said to possess high affinity, but little
or no intrinsic efficacy. Occupation of the receptor
by the antagonist is also described by the law of
mass action, as was the case for full agonists and
partial agonists. However, subsequent to binding,
either no biological stimulus is produced or an
insufficient subthreshold stimulus is produced,
and no response is observed. It must be empha-
sized that competitive antagonists d o not
necessarily have efficacies of zero. Many com-
petitive antagonists are actually very weak partial
agonists with extremely low efficacies. An example
of such a compound is tolazoline which is con-
sidered to be a competitive a-adrenoreceptor
blocking agent. In those tissues where the response
is very favourably coupled to the biological
stimulus, a weak but real response to tolazoline
may be observed (Ruffolo, Rosing & Waddell,
1 9 7 9 ~ ) .In most tissues, however, the coupling
between stimulus and response is not sufficient
for the weak intrinsic efficacy of tolazoline to
be translated into a measurable response, and in
these tissues, tolazoline behaves as a true com-
petitive antagonist.
The function of the competitive antagonist
is simply to occupy, but not stimulate, the receptor
 IMPORTANT CONCEPTS OF RECEPTOR THEORY
and thereby prevent occupancy of the receptor
by an agonist which will stimulate the receptor
once bound. Thus, both the agonist and com-
petitive antagonist will compete with each other
for occupancy of the receptor. The degree of
binding by the agonist and competitive antagonist
under equilibrium conditions is determined by
their respective dissociation constants and con-
centrations, and is governed by the law of mass
action (appendix B). If, under equilibrium
conditions, occupancy by the antagonist prevents
sufficient occupancy by the agonist from occurring,
only a small response will be observed, and quite
possibly no response at all. However, if the con-
centration of agonist is increased such that
sufficient occupancy by the agonist does occur
even in the presence of the competitive antagonist,
a larger response will be observed. The interactions
between agonists and competitive antagonists
have been developed by Gaddum (1926) and
Schild (1949) and will be presented in quantitative
mathematical terms, along with the procedures
used in the evaluation of competitive antagonists,
in appendix B.
3.5 Threshold Phenomenon
The first quantitative treatment of the threshold
phenomenon was made by Ariens e t al., (1960).
Stephenson (1956) had previously proposed that
the biological effect of an agonist was some
unknown function of the stimulus provided by
the agonist. However, as a result of a certain
inertia in the biological object (Ariens et al.,
1960), an effect may only be observed after the
stimulus reaches some threshold value sufficient
1 BIOLOGICAL STIMULUS
0% 100%
I 1
I THRESHOLD I
BIOLOGICAL RESPONSE [ RECEPTOR RESERVE
ET EM
PERCENT RECEPTOR OCCUPANCY
FIG. 6. Schematic representation of the relationshi
occupancy, biological stimulus and biological response.
the biological object, and ET is the threshold effect.
289
to intiate a response. Thus, in certain systems, a
significant fraction of the receptor pool may have
to be occupied before an effect is observed.
The concept of the threshold as it relates to
receptor occupancy, biological stimulus and
response is presented schematically in Fig. 6.
Because of this ‘inertia’ in the biological object
referred to by Ariens et al., (1960), low levels of
receptor occupancy by the agonist will produce a
biological stimulus which is insufficient to initiate
a response. As dose is increased, so is receptor
occupancy (in accord with masslaw), and it follows
that the biological stimulus will also increase.
When the stimulus reaches the minimum value
required to perturb the ‘inert biological object’,
the response becomes apparent. This minimum
stimulus required for response, delivered to the
tissue by the interaction of the agonist with the
receptor, is termed the threshold stimulus. As
receptor occupancy, and therefore stimulus,
increases further beyond the threshold stimulus,
response increases as a function of dose, and the
typical dose-response curve results. Response will
continue to increase as occupancy and stimulus
increase up to the point where the biological
object is not able to respond further due to the
physical limitations of the biological object,
at which point a maximum effect (EM) is reached.
Since the maximum effect can be a physical
limitiation of the tissue, it may be occur at some
degree of receptor occupancy less than saturation.
When this occurs, there is said to be a receptor
reserve or spare receptors (as discussed previously).
Further occupancy of these excess receptors by
the agonist will produce yet further increases in
I
1
1
between threshold, receptor reserve, receptor
%, is the maximum effect that can be obtained in
290 R . R. RUFFOLO Jr
the biological stimulus supplied to the tissue, but
no additional response will occur since the tissue
has previously reached the maximum to which it
is capable of responding.
The existence of a threshold may be established
by studying the combined effects of two or more
drugs. Thus, when a subthreshold dose of drug A
is administered and allows a subthreshold dose of
drug B to produce an effect, then it is clear that
the two subthreshold stimuli are summing to
produce a stimulus greater than the threshold,
enabling a response to occur. The existence of a
threshold may also be observed by plotting the
dose-response relationship of an agonist on a linear
dose scale. If a threshold exists, the curve will
intersect the linear dose scale at some value greater
than zero. Finally, if the lowest response detected
occurs at a fractional receptor occupancy greater
than the fractional response, the indication is that
a threshold exists for the biological stimulus. As
an example, tolazoline, a very weak partial agonist,
will evoke a measurable response in rat aorta
(Ruffolo et al., 1979c) only after receptor
occupancy reaches 13 percent, implying that a
threshold exists for this drug in this tissue. Thus,
only after tolazoline occupied 13 percent of the
total receptor pool was the biological stimulus
delivered to the tissue sufficient to ‘move’the tissue.
Expanding upon this observation, it is possible to
state that if the occupancy-response relationship
of an agonist, when plotted on a linear occupancy
scale, is concave at the origin, the likelihood is
high that a significant threshold exists. A more
complete description of the threshold phenomenon
with accompanying theoretical curves has been
supplied by Ariens (1966) & Van Rossum and
Ariens (1962).
4. Summary and Conclusions
While many theories have evolved to explain the
interaction of drugs with their receptors, the
occupation theory, as proposed by Clark and
subsequently modified by Ariens and then extended
by Stephenson, has gained widest acceptance
because of its broad applicability. There may be
specific instances, however, when one or more
of the other theories is more appropriate. The
major concepts of present occupation theory may
be presented by specific examples without the
laborious traditional mathematical treatment.
However, for those interested readers, appendix
A that follows presents the development of current
occupation theory using the more traditional
mathematical approach. In appendix B, the deri-
vations of the various procedures used to determine
dissociation constants of agonists, partial agonists
and competitive antagonists in isolated tissue
preparations or radioligand binding studies are
presented. In appendices A and B, most of the
algebraic manipulations involved in the derivations
and rearrangements of the various equations have
been included for the sake of clarity. It is hoped
that even those individuals less interested in the
mathematical approach to receptor theory will
also benefit from these appendices.
Appendices
Appendix A. Historical Development of Occupation
Theory: Mathematical Approach.
Clark ( 1 926). Clark proposed that the magnitude
of the response was linearly proportional to the
fractional receptor occupancy of receptor R by
agonist A such that:
where EA is the observed response to agonist A
and EM is the maximal response attainable in the
tissue with a full agonist acting on the same
receptor system. [RA] is the concentration of
receptor-agonist complex and [RT] is the total
concentration of receptors. Assuming a simple
bimolecular interaction between agonist and
receptor, we may write:
R + A -RA (3)
Assuming, further, that occupancy of the receptor
by the agonist proceeds according to the law of
mass action, the dissociation constant (KA) of
the receptor-agonist complex [ RA] in equation
(3) may be written as follows:
Rearranging equation 4 gives the following:
[RI [A1
[RA] = ___ (5)
KA
The total receptor concentration is the sum of
the concentration of receptors occupied by the
agonist and the concentration of free receptors
such that:
[RT] = [RAI + [Rl (6)
Substitution of [RA] (equation 5) and [RT]
(equation 6) into equation (2) gives:
(7)
[RI + [RAI
Substituting equation (5) once again for [RA] in
the denominator of equation (7) gives:
 IMPORTANT CONCEPTS OF RECEPTOR THEORY
Rearranging and simplifying gives the following
relationship between receptor occupancy, agonist
concentration and dissociation constant (mass
law):
(9)
According to equation (2) we may also write:
Clark proposed that this relationship adequately
described the response of an agonist simply as a
function of fractional receptor occupancy. No
consideration was given to compounds within a
homologous series which produce less than
maximal responses, or to competitive antagonists
which occupy the receptor but produce no
response. Thus, the theory would predict that any
compound occupying the receptor would produce
a response proportional t o the fraction of receptors
occupied. A maximum response would require
100 percent receptor occupancy.
Ariens (1954). Ariens sought to account for the
fact that partial agonists and competitive
antagonists, which also occupied the receptor,
produced smaller maximum responses than full
agonists, or no response at all. Ariens proposed
that the drug would possess an affinity for the
receptor (described by the dissociation constant)
and an intrinsic activity. The latter term, given the
symbol a, was related to the ability of a compound
t o activate the receptor subsequent to binding,
and was given limits from 0 for competitive
antagonists to 1 for full agonists. Partial agonists
could be assigned a value between 0 and 1.
Ariens proposed that the response of an agonist
,would be equal to the product of intrinsic activity
and the concentration of receptors occupied by
the drug such that:
Substituting equation (9) for [RA]/[RT] in equation
(11)gives:
With this relationship, the activities of full agonists,
291
partial agonists and competitive antagonists could
be explained. However, it is apparent from
equation (12) that, according to thismode1,response
was still linearly proportional to receptor
occupancy and that a maximum response would
require saturation of the receptors.
Stephenson ( 1 956). Stephenson proposed that
the response of an agonist was some unknown
function of the biological stimulus (S) such that:
EA
- = f(S)
EM
It was further assumed that identical responses to
two agonists would be obtained when the
biological stimuli produced by each agonist is
the same, regardless o f the receptor occupancy
o f each agonist necessary to produce this Same
stimulus. The stimulus was proposed to be equal
to the efficacy of the drug (e) times the fractional
receptor occupancy, the latter being described by
mass law (i.e. equation 9):
It is clearly seen by substituting equation (14) into
equation (13)that:
where f is an unknown function. This theory will
explain the activity of full agonists, partial agonists
and competitive antagonists, and does not require
that response be linearly proportional tooccupancy.
That is, a maximum response may be obtained, in
theory, with less than 100 percent receptor
occupancy, which appeared to be the case in many
tissues. Furchgott (1966) argued that efficacy,
as defined by Stephenson, was not truly a drug
parameter since its value, although undefined and
with no theoretical limit, would vary among
tissues. Furchgott (1966) extended the theory of
Stephenson by proposing that efficacy, e, was the
product of intrinsic efficacy (E) and total receptor
concentration ([RT]) such that:
e = E [ RT] (16)
Intrinsic efficacy is, in fact, a drug parameter
which describes the stimulant activity of an
agonist. Intrinsic efficacy does not vary among
tissues, but rather is a property of the drug mole-
cule itself for any given receptor type, in contrast
to efficacy (e) which varies among tissues because
it is dependent upon receptor number which, in
turn, is not constant from tissue-to-tissue.
Substituting equation (16) into (15) allows the
following relationship to be written between
292 R. R. RUFFOLO Jr
response, intrinsic efficacy and receptor
occupancy:
(17)
Equation (17) represents current receptor theory.
The function, f, is still unknown, but this does
not prevent receptor theory, as it now exists,
from being utilized (as developed in Appendix
B). An extension of these classical concepts of
receptor theory to the case where cooperativity
exists has been the subject of a thorough review
by Colquhoun (1973).
Appendix B. Methods Used to Determine
Dissociation Constants of Agonists, Partial Agonists
and Competitive Antagonists.
Calculation of Dissociation Constants of
Agonists. Determining the dissociation constant of
an agonist in isolated tissues was a problem until
the mid-1960's. Because of the existence of a
receptor reserve in many tissues, and the non-
linearity between occupancy and response, it was
recognized that the EDSOwould be a poor estimate
of the dissociation constant in most instances.
Building upon the theory of Stephenson (1956),
Furchgott (1966) developed a procedure, which is
in widespread use today, for determining the
dissociation constant of a full agonist provided an
irreversible receptor blocking agent is available.
Furchgott reasoned that if receptor number was
reduced by an irreversible blocker to a fraction,
q, of the original receptor population (i.e. [RT]),
then the response of an agonist after partial
irreversible receptor blockade (EA~)relative t o the
maximum response attainable in the tissue before
partial receptor inactivation (EM) would be
described by the following relationship:
where q is the fraction of free receptors (i.e.
fraction of receptors not alkylated) and S' and
[A'] represent the stimulus and concentration of
agonist, respectively, required to produce effect
EA' after a fraction (1-q) of receptors had been
inactivated by the irreversible blocker. This is so
because after receptor inactivation, the initial
[RT] is reduced to q[ RT] , and by analogy, any
calculated receptor occupancy will be reduced
by this same proportion (i.e. q[ RA] /[ RT] , where
q < 1). According t o theory, equieffective
responses before receptor inactivation (EA/EM)
and following receptor inactivation (EA~/EM)
will occur only when the biological stimulus
before receptor inactivation (S) equals the stimulus
after inactivation (S') (Stephenson, 1956). Thus, i f
EA,EA' (19)
EM
then the following must be true:
f(S) = f(S') (20)
Substitution of equations (14) and (18) intoequation
(20) gives:
for equal responses before and after partial
irreversible receptor inactivation. According to
Furchgott, only receptor number is changed by
irreversible receptor blockade while the unknown
function, f, that exists between stimulus and
response is unchanged by this treatment. Thus,
both sides of equation (21) may be simplified as
follows:
This relationship may be algebraically simplified
to the following:
It is clear from equation 23 that a plot of the
reciprocal of the concentrations of agonist before
receptor inactivation (1/[A] ) against the reciprocal
of the equieffective concentrations after receptor
inactivation (l/[A']) will yield a straight line with
a slope of l / q and intercept of [ ( l - q ) / q K ~ ] .
The value of q is obtained from l/slope and the
KA is determined from the equation:
KA = slope - 1 (24)
intercept
Determination of dissociation constant of a
partial agonist. As stated earlier, with a partial
agonist, the EDSOwill approximate the dissociation
constant since no receptor reserve will exist for
these compounds. However, depending upon the
system studied and the efficacy of the partial
agonist, significant differences between the EDSO
and dissociation constant may still exist since a
nonlinear relationship between occupancy and
response can occur for partial agonists even when
no receptor reserve is present (e.g. oxymetazoline
in rat aorta; see Figs. 4 and 5). It is possible to
use the method of Furchgott described above t o
estimate the dissociation constant of a partial
agonist. However, a technically simpler method
has been developed by Waud (1969). Waud
reasoned that a full agonist and a partial agonist
would produce equal fractional responses (relative

to the maximum response obtainable in that tissue
with the full agonist) only when the biological
stimuli produced by the full agonist and partial
agonist were the same. Therefore:
EA = Ep implies SA = ~p (25)
EM EM
where Ep and Sp are, respectively, the effect and
stimulus produced by the partial agonist. There-
fore, as Waud proposed, and as a direct conse-
quence of equation (25), the following relationship
will hold when the full agonist and partial agonist
produced equal effects (and therefore equal
stimuli):
where KA and Kp are the dissociation constants
of the full agonist and partial agonist, respectively,
eA and ep are the efficacies of the full agonist
and partial agonist, respectively, and [A] and
[PI are the concentrations of the full agonist
and partial agonist, respectively. It was further
assumed that eA>>ep such that a large receptor
reserve exists for the full agonist. When this
assumption is satisfied, Waud argued that for the
full agonist, [A] << KA and equation 26 could
be simplified to:
This relationship may be arranged algebraically
and simplified to:
- eAKP
1 =-i-- eA
[A] ePKA[Pl KAeP
Thus, a plot of the reciprocals of the concen-
trations of full agonist ( l / [ A ] ) against the recipro-
cals of the equieffective concentrations of partial
agonist (l/[P]) will give a straight line. The
dissociation constant of the partial agonist may
be calculated as:
Kp=- slope
intercept
In addition, Ruffolo et al., (1979a,b,c) have
shown that if the dissociation constant of the full
agonist (KA) is known (i.e. method of Furchgott),
the relative efficacy of the partial agonist (er)
may be calculated using the intercept obtained
from the preceding analysis of Waud as follows:
eP -
_ 1 (30)
- er intercept(KA)
eA
The relative efficacies obtained by this method
for partial agonists give identical results t o those
values calculated from occupancy-response curves
IMPORTANT CONCEPTS OF RECEPTOR THEORY 293
as proposed by Furchgott (see Ruffolo et al.,
1979a,b,c).
Determination of the dissociation constant o f
a competitive antagonist. The situation for deter-
mining the dissociation constant of a competitive
antagonist is not subject to the same theoretical
limitations as was the case for a full agonist or
partial agonist. Schild (1949) reasoned that the
dose-response curve to an agonist would be shifted
in a parallel manner to the right by an antagonist
regardless of whether the effect of the agonist
was a linear or nonlinear function of agonist
concentration. It was implied by Schild that equal
responses produced by an agonist in the absence
(EA/EM) and in the presence (EA~/EM)of a
competitive antagonist will occur only when the
agonist occupies equal proportions of the receptor
population such that:
EA
- =EA'
-
EM EM
imDlies:
where [RA] /[RT] is the fractional receptor
occupancy before the addition of the competitive
antagonist and [ RA'] /[ RT] is the fractional
receptor occupancy of an agonist in the presence
of a competitive antagonist. Receptor occupancy
of an agonist in the presence of a simple
competitive antagonist is described as follows:
(28)
where [A'] is the concentration of the agonist
in the presence of the competitive antagonist, B.
The dissociation constant of the competitive
antagonist and its concentration are designated as
KB and [ B] , respectively. Substituting into
equation (32) the receptor occupancy by an agonist
(29) in the absence of an antagonist (equation 9) and
in the presence of a competitive antagonist'
equation 33) will give the following:
Algebraic simplification yields:
(35)
Gaddum (1926) has studied, in great detail, ratios
of [A'] /[A], which are simply ratios of the con-
centrations of agonist giving an equal response in
the presence and in the absence of the competitive
antagonist. This ratio has been termed the dose
ratio. Taking the logarithm of both sides of
equation 35 gives the following relationship:
log (dose ratio -1) = log [ B] -log KB (36)
A plot of log (dose ratio -1) against -log [B]
(commonly called the Schild Plot) will give a
straight line with a slope of unity i f blockade is
competitive (Arunlakshana & Schild, 1959).
The intercept along the abscissa (i.e. when dose
ratio = 2) is the negative logarithm of the dissoci-
ation constant of the competitive antagonist.
This intercept is commonly referred to as pA2
and is simply the negative logarithm of the molar
concentration of antagonist that causes a two-fold
rightward shift in the dose-response curve of an
agonist (i.e. causes a dose ratio of 2). The pA2
is characteristic of a particular antagonist and
receptor type, and is independent of the agonist
used. Thus, the pA2 of a competitive antagonist
is useful in classifying receptors, and this has been
the subject of a recent comprehensive review by
Kenakin (1982). The assumptions involved in this
method for determining dissociation constants of
competitive antagonists have been discussed in
detail by Furchgott (1972).
Determination of dissociation constants of
agonists, partial agonists and competitive
antagonists b y radioligand binding techniques.
Determination of dissociation constants of drugs
by radioligand binding methods is easier to compre-
hend than the methods that employ the pharma-
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(Received 2nd September, 1982)