Screening Methods in Pharmacology

Contributors A L L E N BARNETT S A M P BATTISTA CLYDE M BURNETT E S A M Z D A J A N I E GlLLIARD JEROME M GLASSMAN PETER HEBBORN P R HEDWALL R KADATZ MARIAN M A Y JACK N MOSS K M U L L E N CHARLES J PAGET R STEGER ROBERT L STONE V C S W A M Y ROBERT I TABER ROBERT A TURNER H J WILKENS Screening Methods in Pharmacology Edited by ROBERT A TURNER Turner Associates Greenwich, Connecticut PETER HEBBORN Department of Biochemical Pharmacology School of Pharmacy State University of New York at Buffalo B.

K MULLEN CHARLES J PAGET

R STEGER ROBERT L STONE

V C SWAMY ROBERT I TABER ROBERT A TURNER

H J WILKENS

Screening Methods in Pharmacology

Edited by ROBERT A TURNER

Turner Associates Greenwich, Connecticut

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List of Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin

Corpora-tion, Bloomfield, New Jersey

Incor-porated, Cambridge, Massachusetts

New York

Research Laboratories, Spring House, Pennsylvania

Depart-ment of CIBA, Ltd., Basel, Switzerland

Pharma-cology, Denver Chemical Manufacturing Company, Stamford, Connecticut

School of Pharmacy, State University of New York at Buffalo, Buffalo, New York

P R. HEDWALL (249), Biological Laboratories of the Pharmaceutical Department of CIBA, Ltd., Basel, Switzerland

Thomae GmbH, Biberach an der Riss, Germany

IX

MARIAN MAY (85, 101), Center for Theoretical Biology, State University

of New York at Buffalo, Buffalo, New York

Research Laboratories, Spring House, Pennsylvania

Depart-ment of CIBA, Ltd., Basel, Switzerland

Indiana

Pharmacy, State University of New York at Buffalo, Buffalo, New York

Indiana

V C SWAMY (1), Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo, Buffalo, New York

Cor-poration, Bloomfield, New Jersey

H J. WILKENS (61), Department of Biochemical Pharmacology, School

of Pharmacy, State University of New York at Buffalo, Buffalo, New York

Preface

The second volume of "Screening Methods in Pharmacology" has the same basic purpose as Volume I, namely, to present sufficient practical information about techniques so that it would be possible for the reader, even with little experience, to establish a screening program for a partic-ular pharmacological activity The contributors to this volume have pre-sented typical results obtained for selected reference compounds, which are intended to show the responses with a known substance and to guide the reader during the initial use of a test method so that he may select suitable doses of the reference drugs and may know the intensity of the response expected for a certain dose level

Because the progress in developing methods has been so rapid since the appearance of the previous volume, it became impossible for one person

to review the pharmacological literature Thus, unlike Volume I, Volume

II is a multiauthored, coedited work

ROBERT A TURNER PETER HEBBORN

xi

Introduction

A Brief Review of the Biochemistry

of the Nervous System

The Organization of Screening

Antiemetic Agents Bronchodilatant Agents Curariform Agents Anabolic, Androgenic, and Antian-drogenic Agents

Potentiators and Antagonists of Tryptamine

Vasopressive Peptides Diuretic and Natriuretic Agents Anticholinesterase Agents Anticholesterol Agents Uricosuric Agents Antishock Agents Hemostatic Agents Local and Spinal Anesthetics Abortifacient Agents

Thymoleptic Agents Dermal Irritants Teratogenic Agents Appendix

References Author Index-Subject Index

Introduction

Numerous methods often exist for screening a series of compounds for a given pharmacological activity Many, but not all, available methods are described in this volume They have been selected because they are the most reliable, the simplest, and, in the opinion of the respective authors, the preferred of the available methods The sensitivity of the assay procedure and the possibility of ranking the compounds that have proved clinical effectiveness are important factors in the selection of a screening method

Those who have been involved with screening drugs for logical activity for even a short time have realized that only a few in a group of substances have activity An alternative situation exists if one has a group of compounds, all of which have varying degrees of activity

pharmaco-In both cases, the screening process is an attempt to identify, by one or more tests, those few substances which are gems among a group of pebbles

Generally it is better to use a screening method which may give a few false positives rather than one which will yield some false negatives

If a substance has no true activity and is shown by a test to have activity,

a false positive results Sooner or later, as testing with the substance is continued, its inactivity will be revealed Some time may be wasted in studying the compound, but in the end the investigator is not misled

On the other hand, a false negative may result in the removal of a substance from further study, so that its activity will remain forever undetected

The developer of a new drug is always seeking a relation between

xv

chemical structure and biological activity, which, if found, is rare and retrospective, rather than deductive Sometimes structural changes

in a molecule that appear minor cause unpredictable and extensive changes in the pharmacological activity, including loss of all activity and introduction of new side effects Often the first member of a homolo-gous series of compounds is the most active pharmacologically Because the biological consequences of small changes in chemical structure are not understood, the structural changes cannot be programmed logically New drugs of a unique character will probably be derived in the future from novel structures rather than from modifications of old structures, study of enzyme systems involved in the disease state, unexpected clin-ical observations, and an understanding of the metabolism of known, active drugs

Experience and scientific intuition play their important roles Screening efficiently for certain pharmacological activities is necessary for pro-gress Since activity is unpredictable, the number of activities covered

by the screening program should be considerable If several tests have indicated that a compound has some activity, it is usually advantageous

to study it further rather than to start with a new compound ab initio

Contemporary investigators of new drugs tend to screen with a broad program

No procedure for screening can be perfect Therefore, anyone forming screening in pharmacology should always be vigilant for border-line results and for results indicating an inactive substance when one strongly suspects that activity is present If one has good theoretical grounds for anticipating activity of a substance, one should continue to study it, even if one screening procedure indicates that activity of a certain kind is absent One should not rigidly accept the results of screening procedures, if, by doing so, one would relegate to the shelf a substance which might be valuable clinically

per-It is possible for a drug to be metabolized or eliminated very rapidly

by laboratory animals and yet to have a prolonged half-life in man Phenylbutazone is an example of a drug having antirheumatic activity in man, but whose activity as an antiinflammatory agent in rodents is de-monstrable only at doses approaching a lethal level Moreover, in some disease states, available, clinically effective drugs are only palliative and not curative It is reasonable to conclude that pharmacological screening tests in which such clinically active drugs have a positive effect can be used to select new drugs which are also palliative and not curative One should, therefore, be continually searching for new screening methods based on animal models of human disease processes

Elucidation of the etiology of clinical disease states still requires

ex-tensive effort When an abnormality in cellular function can be identified

as the consequence of a biochemical lesion, then the primary screening method for new drugs will involve a biochemical assay procedure In the meantime, the pharmacological screening methods of the types described

in this volume will be needed for the discovery of new drugs

Finally, there are no screening methods that do not require the exercise

of judgment and discretion on the part of the researcher

ROBERT A TURNER PETER HEBBORN

A Isolated Organ Systems 4

B Intact Animal Systems 15

References 18

I General Considerations

A ADRENERGIC RECEPTORS

If receptors may be defined as tissue components with which a drug

interacts to produce its characteristic physiological effects, then the

adrenergic receptors specifically refer to those components of the effector

cells through which the sympathomimetic amines exert their actions

The adrenergic receptors have been further classified into a- and

ß-re-ceptors on the basis of their relative responsiveness to sympathomimetic

amines ( Ahlquist, 1948 ) Although the catecholamines act on both kinds

of receptor, some compounds stimulate or block adrenergic responses

specifically at either a- or ß-receptors; those agents, therefore, can be

1

divided into a- and ß-adrenergic stimulants and a- and ß-adrenergic

blocking agents

Blockade at the α-adrenergic receptors can be recognized by

compari-son of a test substance with the actions of two established sympatholytic

agents, now more precisely termed α-adrenergic blocking agents, namely,

phentolamine and phenoxybenzamine The former compound causes a

parallel and rightward shift of the agonist ( catecholamine )

dose-re-sponse curve, and the inhibition of redose-re-sponse to a dose of an agonist

may be reversed by larger doses of the agonist Phentolamine, thus,

is termed a competitive, reversible antagonist The blocking action of

phenoxybenzamine (POB) and other 2-halogenoethylamines has been

described by a variety of terms: nonequilibrium antagonism (Nickerson,

1957), insurmountable antagonism (Gaddum, 1957), and competitive,

irreversible antagonism (Furchgott, 1955; Kimelberg et al, 1965)

In contrast to phentolamine, phenoxybenzamine does not form a

dis-sociable complex with the receptor Its binding to the receptor probably

involves covalent bond formation and the blockade is prolonged

Experi-mentally, an effective adrenergic blockade produced by

phenoxybenza-mine cannot be overcome even by large doses of the agonist

Conse-quently, in experiments performed in vitro, increasing the concentration

of phenoxybenzamine results in a progressive depression of response

to the agonist until complete abolition of the response is achieved

The use of pA# values (Schild, 1947) is a convenient method for

evaluating competitive antagonism pA* is defined as the negative

log-arithm of the molar concentration of the antagonist which will reduce

the effect of a multiple dose of an agonist to that of a single dose

If the interaction of the drugs at the receptor is bimolecular, then

where x is the ratio of equiactive doses of agonist in the presence and

in the absence of antagonist; n and K 2 are constants

Thus, when log ( x — 1 ) is plotted against pA*, a straight line results

with a slope equal to (—n), which intersects the pA* axis at a point

corresponding to pA2 ( Fig 1 ) When n = 1, pA2 — pA10 = 0.95, and

this difference in pA2 and pA10 values can be used as a test for

competi-tive antagonism, although it is preferable to use a plot of log (x — 1)

over a wide range of antagonist concentrations

Antagonist activity may be evaluated, also, in terms of the apparent

dissociation constant K B of the receptor-antagonist complex (Furchgott,

1967) The theoretical basis for this procedure is the equation

corre-sponds to the point of intersection of the regression line with the abscissa Where the dose ratio equals 0.95, a perpendicular dropped from the regression line to the abscissa gives the pAio value of 6.42 ( From Birmingham and Szolcsanyi, 1965 )

where B is the molar concentration of the antagonist and x is the dose

ratio of agonist in the presence and in the absence of the antagonist

Under true equilibrium conditions —log K B = pA2, as defined by Schild (1947)

An empirical term, pA/„ may be used as a quantitative index of the activity of a compound which reduces the attainable maximum of the dose-response curve for the agonist ρΑΛ is defined as the negative log-arithm of the molar concentration of an antagonist which reduces the maximum response to an agonist to a value which is 50% of the maximum

obtained previously in the absence of antagonist This term does not make any assumptions concerning the mode of action of test compounds and can be used to quantify all antagonists which reduce the maximum

To obtain this value, a series of curves are plotted using doses of the antagonist that cause a flattening of the slope and a progressive decline

of the maximum The pA/> value is obtained by interpolation or close extrapolation from two curves whose maxima were reduced to approxi-mately 501

B FACTORS INFLUENCING DRUG ACTION

A number of factors contribute to the physiological effects of ergic drugs The factors include the processes of uptake and enzymic modification, which regulate the concentration of sympathomimetic amines at the receptor sites ( Trendelenburg, 1966, 1968) In addition, spontaneous changes in tissue sensitivity and interaction of drugs at sites other than receptors may cause misinterpretations in evaluating the activity of antagonists (Furchgott, 1968) Finally, given the multi-plicity of sites of action in the adrenergic system, the tests may reveal the action of potential drugs not only at the receptors but also at the ganglia and sympathetic neurons This last feature, understandably, is more likely to occur in intact animal studies than in tissues studied

adren-in vitro

It may not be possible, therefore, to utilize experimental procedures that are ideal in all respects Selection of a particular method necessarily represents a compromise between its convenience and the qualitative

or quantitative significance of the data obtained For example, tion of receptor-blocking properties is possible when examining the cardiovascular activity of the compound However, a detailed assessment

identifica-of the antagonistic properties identifica-of test compounds, involving determination

of quantitative indexes (e.g., pA2 values), invariably requires the use

of in vitro studies and a careful appraisal of possible experimental

variables

II Methods

A ISOLATED ORGAN SYSTEMS

The use of isolated organ systems offers obvious advantages over in

vivo studies Relatively accurate measurement of responses can be made

from several preparations, usually obtained from one animal The various

complicating factors encountered in vivo such as drug distribution,

humoral activity, and reflex activity are largely minimized or avoided Finally, these methods serve to identify and make comparative estimates

of the receptor-blocking properties of test compounds

The stimulation of «-receptors generally induces contraction of smooth muscle which may be recorded via isotonic or isometric systems The availability of automated recording systems (Vickers Corp.) makes routine determination of pA2 values convenient Ideally, the tissues used

in the experiments should contain only «-receptors and should show only minimal changes in sensitivity over the duration of the experi-ment The use of phenylephrine, in place of more commonly used agonists such as norepinephrine, is more appropriate since it combines low affinity for presynaptic sites (Burgen and Iversen, 1965) with strong, preferential action at «-receptors

The experimental procedure consists of plotting a series of sponse curves—one curve in the absence of an antagonist and the others

dose-re-in the presence of varydose-re-ing concentrations of the antagonist (Fig 1) The tissue, is made to contract maximally 2 or 3 times at the beginning

of the experiment Construction of dose-response curves may be carried out by the method described by van Rossum and van den Brink (1963), where successive doses of the agonist are added to the bath after the tissue has acquired steady-state equilibrium to the previous dose After the maximum response has been achieved, the agonist is washed out

of the bath and a complete relaxation of the tissue occurs The antagonist

is then added to the bath and allowed to equilibrate with the tissue for a given period of time The tissue is then exposed to the agonist and a new dose-response curve is obtained, using a range of doses suffi-cient to duplicate the initial dose-response curve Similar dose-response curves are plotted for a wide range of antagonist concentrations If the curves are parallel, they may be interpreted as indicating competitive

antagonism (Fig 2) The dose ratio x is calculated from the parallel shift of the dose-response curves and is utilized in plotting log (x—1)

against pA*

A common source of error in this procedure is the failure of the antagonist to reach equilibrium The time required to reach equilibrium varies with experimental preparations and the concentration and nature

of the antagonist (Furchgott, 1967; Schild, 1947) It is possible to mine the time for equilibrium for an antagonist by challenging the tissue with the agonist at different periods during continuous exposure to the antagonist In common practice, the duration of exposure is chosen arbi-trarily and reported with the experimentally determined pA2 value

deter-I0" 7 I0" 6 IO" 5 I0" 4 IO" 3

Norepinephrine (molar)

FIG 2 The effect of phentolamine on the contractile responses of the rat vas deferens to norepinephrine Responses to norepinephrine were obtained in the presence of various concentrations of phentolamine Competitive antagonism is in- dicated by a parallel shift of the dose-response curves of norepinephrine (From van Rossum, 1965.)

Changes in the sensitivity of the preparation to the agonist may result

in erroneous estimates of ρΑ# values Such a possibility may be counted for by using a control preparation which is treated in a manner similar to the experimental preparation, except that the antagonist is not added Any shifts in the dose-response curves that occur in the control preparation are then used to correct the shift caused by the antagonist in the experimental preparation Finally, low concentrations

ac-of 2-halogenoethylamines and various nonspecific depressants ac-of smooth muscle may cause parallel shifts of dose-response curves, thereby leading

to the false conclusion that they are competitive antagonists However, employment of a test compound in a wide range of concentrations will confirm the identity of its antagonistic properties Increasing the concen-trations of a nonspecific depressant results in gradual loss of parallelism, and a progressive decline in maximal response becomes evident ( Fig 3 )

1 Vas Deferens

The vas deferens fulfills many of the optimal conditions for tive evaluation of adrenergic antagonists The response of this organ

quantita-9 8 7 6 5 -log Epinephrine concentration

FIG 3 Effect of Dibenamine-HCl (DB) on the response of the rabbit aortic strip to epinephrine Increasing concentrations of DB cause a progressive reduction

of the contractile response to epinephrine Responses to epinephrine were tested

at the end of the exposure period after washing DB from the organ bath (From Furchgott, 1955.)

to α-adrenergic agonists consists of a strong rapid contraction followed

by a quick relaxation on washing the agonists out of the tissue Although the vasa deferentia of both rat and the guinea pig (Leach, 1956) are commonly used, the relative preponderance of α-receptors in the vas deferens of the rat (van Rossum, 1965; Vohra and Reiffenstein, 1967) makes the latter more suitable for evaluation of α-adrenergic antagonists The rat is killed by a sharp blow on the head, the vasa deferentia are dissected free from the extraneous tissues and are suspended in organ baths containing Tyrode's solution or a modified form of Krebs' solution (Hukovic, 1961) The system requires aeration by a mixture

of 02 (95%) and C02 (5%). A simple isotonic lever system (1:15; 0.3 gm) provides satisfactory recordings of contractile responses which remain stable for over 3 hr

Cumulative dose-response curves may be obtained conveniently using this preparation The tissue is allowed to equilibrate for 20-30 min before inducing maximal contractions one or two times Following this initial treatment, the second and third dose-response curves for norepinephrine

are usually identical (Patil et al, 1967) This property of the tissue

may be utilized to make accurate estimations of the parallel shift of the curves caused by reversible antagonists (Fig 2) The magnitude

of the shifts caused by different concentrations of the antagonist are then used to estimate the pA2 value by graphical means (Fig 1) The

pA2 values of some common «-receptor antagonists determined on the vas deferens of the rat and guinea pig are listed in Table I

TABLE I COMPARISON OF pA 2 VALUES OF «-ADRENERGIC ANTAGONISTS

Norepineph-rine Epinephrine

Antagonist Phentolamine Piperoxan Aceperone Droperidol Levopromazine Yohimbine Dihydroergot- amine Thymoxamine Piperoxan Yohimbine Piperoxan

Yohimbine

Macusine B Macusine B Tolazoline Tolazoline Chlorpromazine Chlorpromazine

tact time (min)

4.47

5.57 6.07 4.84 4.85 14.2 14.08

Reference van Rossum (1965) van Rossum (1965) van Rossum (1965) van Rossum (1965) van Rossum (1965) van Rossum (1965) Birmingham and Szolcsanyi (1965) Birmingham and Szolcsanyi (1965) Calculated from data of Leach (1956) Calculated from data of Leach (1956) Calculated from data of Leach (1956) Leonard (1965) Leonard (1965) Bickerton (1963) Bickerton (1963)

with its accompanying hypogastric nerves (Hukovic, 1961; Graham et

al., 1968) Stimulation of the postganglionic nerve induces a strong rapid

contraction of the vas deferens The hypogastric nerve-vas deferens preparation is more difficult to use and possesses no inherent advantage over the isolated vas deferens preparations described here for assaying antagonistic activity It is a useful preparation, however, for detecting depressant activity of a test compound on sympathetic nerve function

2 Vascular Smooth Muscle

A commonly used preparation in this category is one utilizing spirally cut strips of the rabbit aorta (Furchgott and Bhadrakom, 1953; Furchgott, 1960) This preparation has been extensively used in the analysis of the action of sympathomimetic amines and their antagonists

at receptor sites (Bevan, 1960; Furchgott, 1954, 1967) It possess many advantages of an isolated organ system For example, three or four tissue preparations are available from each aorta, enabling "paired-control" studies to be made It is sensitive to low concentrations of adrenergic agonists, and the tissue remains stable for long periods of time Con-tractile responses may be recorded using an apparatus that permits simul-taneous recordings from ten arterial preparations (Nash and Luchka, 1965) However, setting the preparation up requires great care, and

an equilibrium period of approximately 2 hr is needed before drugs can be administered In addition, contractile responses to «-adrenergic agonists are slow, and after washing the preparation relaxation is slow Rabbits, preferably weighing 2-3 kg, are killed by a sharp blow on the head, and the thorax is opened to expose the aorta An incision

is made on the descending part of the aorta, and a glass rod (3-4 mm

in diameter) is slowly inserted The aorta is carefully removed, using the glass rod as a guide, and a continuous spiral is cut to obtain lengths

of tissue 2-3 mm wide and 3 cm long The aortic strips are allowed

to equilibrate for 2 hr in organ baths containing oxygenated Krebs' solution at 37°C Recordings of the aortic contractions may be made via isotonic levers (1:10; 3.0 gm) or through a force displacement transducer Furchgott (1967) recommends evaluation of shifts in the lower part of the dose-response curve (25-50% of maximum contraction) when studying the activity of antagonists Log dose-response curves are plotted for the agonist alone and in the presence of the antagonist The shift of the latter dose-response curve from the control gives an

estimate of x, the dose ratio, which is then used to calculate pA2 or

the apparent K B of the antagonist

The procedures described here for rabbit aortic strips have been cessfully employed to study adrenergic activity in various vascular tis-sues For example, Birmingham and Szolcsanyi (1965) used spirally

suc-cut strips of the aorta from rabbits, guinea pigs, and cats and from the carotid arteries of dogs to assess the adrenergic blocking properties

of thymoxamine The experimental conditions for the aortic strips from guinea pigs differ in one respect While the arterial strips from rabbits, cats, and dogs are suspended in Krebs' solution at 37°C, the tissues from the guinea pig are bathed in Krebs' solution maintained at 32°C Helically cut coronary arteries have been studied for their responses

to catecholamines (Zuberbuhler and Bohr, 1965) Isolated veins, also, have been used in the form of spirally cut strips to characterize their

adrenergic receptors (Sutter, 1965; Gulati et al., 1968) The pA2 values for some common α-adrenergic antagonists, obtained on various vascular

TABLE II COMPARISON OF «-ADRENERGIC BLOCKING ACTIVITY ON VASCULAR TISSUE

nne Norepineph-

rine Norepineph-

rine Norepineph-

rine

Antagonist Dihydroergot- amine Phentolamine Yohimbine Macusine B Piperoxan Piperoxan Thymoxamine Thymoxamine Phentolamine

Phentolamine Thymoxamine Thymoxamine Thymoxamine

tact time (min)

8.69 7.20 6.99 6.10

Reference Calculated from Furchgott (1955) Calculated from Furchgott (1955) Calculated from Furchgott (1955) Leonard (1965) Birmingham and Szolcsanyi (1965) Birmingham and Szolcsanyi (1965) Birmingham and Szolcsanyi (1965) Birmingham and Szolcsanyi (1965)

Gulati et al (1968)

Wohl et al (1967)

Birmingham and Szolcsanyi (1965) Birmingham and Szolcsanyi (1965) Birmingham and Szolcsanyi (1965)

preparations, are given in Table II, and are seen to show good agreement within the limits of experimental variation

Interpretation of experimental results obtained from vascular smooth muscle systems must be accompanied by an awareness of differences

in sensitivities of blood vessels to adrenergic agonists Bevan (1961) and Bevan and Osher (1965) have reported on the variability in function

of α-receptors in the thoracic aorta, pulmonary artery, inferior vena cava, and the anterior mesenteric artery of rabbits Helical preparations of these vessels demonstrated that the adrenergic «-receptors in the aorta and pulmonary artery were identical and that they differed in their adrenoceptive responses from those of the anterior mesenteric artery

or the inferior vena cava The experimentally determined pA2 values for thymoxamine on the cat aorta differed from those obtained on the arterial preparations of rabbits, guinea pigs, and dogs; the noticeably greater thickness of the cat aorta is suggested as an explanation for the discrepancy in the pA2 values (Birmingham and Szolcsanyi, 1965) The pharmacological analysis of the responses of the isolated veins of the rabbit also led to the conclusion that they do not form a homogene-ous system (Sutter, 1965)

Circular segments of the rat aorta are used in the experiments of

Wohl et al ( 1967 ) The aortic segments are suspended between stainless

hooks inserted into the lumen so that the contractions of the circular muscle give rise to increases in isometric tension which are measured

by force displacement transducers (Statham, 0.3-1.0 oz) The tissues are allowed to equilibrate under tension (2 g) for 1 hr before measuring responses to drugs Satisfactory dose-response curves to norepinephrine can be obtained by cumulative addition of the catecholamine in volumes

of 0.05 ml or less Responses of the tissue remain stable and reproducible over long periods of time Antagonists are added to the bath after two

or three dose-response curves for norepinephrine show close similarities

An arterial preparation with high sensitivity to norepinephrine has been described by de la Lande and Harvey ( 1965 ) Lop-eared rabbits are anesthetized with urethane (1.76 gm/kg, i.p.); the central artery

of the ear is exposed, and a segment of this artery 5-7 cm in length

is suspended in the organ bath The artery is cannulated at the proximal end, and the lumen is perfused with oxygenated Krebs' bicarbonate solution maintained at 37°C and containing 5-hydroxytryptamine creatinine sulfate (0.4 jug/ml) The outflow from the artery is allowed

to drain by upward displacement; the rate of perfusion is maintained

at approximately 8 ml/min Drugs are injected into the system through the rubber tubing attached to the proximal end of the cannula Changes

in the diameter of the artery caused by norepinephrine result in changes

in perfusion pressure which are measured by mercury manometers or pressure transducers The main advantage of this preparation is the stability of its responses and its high sensitivity to norepinephrine (1-2 ng/ml) Responses to norepinephrine are consistent for 6 hr after com-mencing infusion, and the tissue may be used after storage at 4°C over-night Responses to norepinephrine in the presence of antagonists may

be studied by adding the antagonist to the perfusion fluid

Male guinea pigs weighing 300-600 gm are killed, and their seminal vesicles are removed The contents of the seminal vesicle can produce excessive distention and prevent maximal contractions A small opening into the lumen, therefore, is made at the proximal end, where the ligature

is tied The vesicles prepared in this manner are straight tubular tures varying from 4 to 7 cm in length The tissues are suspended

struc-in organ baths contastruc-instruc-ing oxygenated Locke's solution with 0.1% dextrose and maintained at 39°C Contractions induced by sympatho-mimetic amines may be recorded via an isotonic lever system (1:15, 1.0 gm)

The seminal vesicles of the rat are removed from animals in essentially the same manner as described for the guinea pig An added precaution

to follow is to dissect carefully the coagulation glands from the vicinity

of the seminal vesicles The removal of the coagulation glands must

be made without injury to the seminal vesicles to avoid abnormal sponses or reduced sensitivity The vesicles are allowed to equilibrate for 15-30 min before being stimulated by drugs Recordings of drug-in-duced contraction may be made with an isotonic lever system (1:10; 0.3 gm) or by using force displacement transducers

re-The contractile response of this organ to catecholamines may be ized for studying antagonistic activity of test compounds according to the general procedures described in the beginning of this section The reproducibility and stability of the responses over 3-4 hr prove advan-tageous in determining pA2 values (see Table I) The property of the

util-seminal vesicles of the guinea pig to contract to acetylcholine and mine as well as to adrenergic agonists makes this organ particularly suitable for examination of a wide spectrum of potential antagonistic activity In contrast, the seminal vesicles of the rat are insensitive to histamine, and although they respond in a linear fashion to epinephrine and acetylcholine, the tissue shows a greater sensitivity to the former agonist Another noteworthy feature of the seminal vesicles from the rat is that their adrenergic responses are mediated almost exclusively

hista-through the α-receptors (Clark et al., 1961)

4 Spleen

Isolated strips of a cat's spleen are sensitive to catecholamines when suspended in glucose-deficient Tyrode's solution or McEwen's solution ( 1956 ) and are suitable for analyzing the activity of α-adrenergic antag-

onists (Bickerton et al, 1962; Bickerton, 1963; Bickerton et al, 1966)

The spleen is removed from the cat through a lateral abdominal incision and washed in warm Tyrode's solution containing one-half the usual amounts of sodium bicarbonate and dextrose The sides and the ends

of the spleen are trimmed, and the central rectangular portion is divided

to give two or more strips of splenic smooth muscle, each measuring approximately 4.5 cm long and 1.5 cm wide The tissues are allowed

to equilibrate for 30 min in oxygenated, glucose-deficient Tyrode's tion maintained at 39°C The contractions of splenic strips may be re-corded by an isotonic lever system (1:15; 5.0 gm) The response of this preparation to given doses of catecholamines increased in magni-tude over the first four or five trials and then remained relatively uniform

solu-over several hours (Bickerton et al., 1962)

The response of the splenic smooth muscle to catecholamines is slow

An initially rapid contraction is followed by a slower phase that reaches

a sustained peak in 3-5 minutes Significant changes in sensitivity edly occur when epinephrine and norepinephrine are given in cumulative doses (Bickerton, 1963) The largest changes in sensitivity over two consecutive cumulative dose-response curves were observed at lower dose levels of the catecholamine, i.e., below one-half maximal responses This can be a potential source of error in estimating the activity of

report-a reversible report-antreport-agonist becreport-ause the shift of the curves due to chreport-anges

in sensitivity may be construed as an effect of the antagonist This culty may be overcome by using paired strips from each spleen, one which receives the antagonist and one which serves as a control The experiments of Bickerton (1963) have utilized such procedures in deter-mining pA2 values of tolazoline, given in Table I

in an organ bath containing oxygenated (95% 02 + 5% C02) Tyrode's

or Krebs' solution at 37°C A ligature is tied around the central end

of the mesenteric artery, and the mesentery is threaded through trodes connected to a stimulator Stimulation ( 15 Hz ) of the periarterial adrenergic nerves, preferably, is restricted to periods of less than 30 sec at 3-min intervals to avoid fatigue of the preparation The pendular movements of the intestine may be recorded on a kymograph with an isotonic lever system (1:10; 2.0 gm) or with force displacement trans-ducers Apparently, no differences are seen between isotonic and iso-metric recording (Bowman and Hall, 1970)

elec-Stimulation of the sympathetic nerve or the presence of exogenous catecholamines inhibit the intestinal contractions This inhibitory action

is mediated by a- and /^-receptors and is blocked by both types of

adrenoreceptor antagonists Separation of α-adrenergic inhibitory effects from those mediated by ^-receptors is based on the experimental ob-servations that stimulation of a-adrenoreceptors produced rapid onset

of inhibition, whereas the onset of action at the ß-receptor sites was slow (van Rossum and Mugic, 1965; Bowman and Hall, 1970) Estima-tion of α-adrenergic antagonists may best be carried out by using phenylephrine, an α-adrenoreceptor agonist whose inhibitory effects are blocked by phentolamine while remaining unaffected by ß-receptor an-tagonists propranolol and MJ-1999 (Bowman and Hall, 1970) Initially, responses to phenylephrine are obtained and followed by washout of the agonist from the system The antagonist is then added to the bath, and its blocking activity is determined by stimulating the tissue with the doses of phenylephrine used initially Due to the variations in the sensitivity of the tissue, only a rough estimate of pA2 is possible This preparation, however, is a simple and convenient one for rapid qualita-tive characterization of compounds at adrenoreceptive sites

6 Uterus

The uterus from nonpregnant rabbits responds to α-receptor tion by contracting vigorously, and these contractile responses may be

stimula-used to estimate the activity of α-receptor antagonists ( Broom and Clark, 1923) Rabbits weighing at least 2 kg are killed and the abdomens are opened The intestine is pulled aside to expose the two horns of the uterus, which are dissected free from their mesenteric attachments Each uterine horn is cut into lengths of 2-3 cm, and each portion is divided longitudinally to obtain a pair of matched strips An advantage

of this isolated uterus preparation is that a number ( 6-8 ) of such control preparations can be obtained from each animal The tissues are bathed in oxygenated Ringer's solution If necessary, magnesium chloride (0.1 gm/liter) is added to inhibit spontaneous motility An isotonic lever system (1:5; 1.0 gm) provides satisfactory recordings of uterine contractions

paired-B INTACT ANIMAL SYSTEMS

I Arterial Blood Pressure Responses

experi-mental method is that it serves to reveal the activity of the test pound at various sites in the sympathetic neuroeffector system (Smith, 1961) Dogs or cats may be anesthetized with barbiturates, although a-chloralose is preferred for cats because of the stable anesthesia it induces and its generally weaker inhibitory effects on autonomie func-tions The trachea is cannulated routinely, and the vagosympathetic trunks are cut bilaterally The blood pressure is recorded from the femoral or carotid arteries, and the drugs are administered through the femoral or jugular veins

com-Epinephrine is a potent stimulant of a- and ß-reeeptors The

stimula-tion of «-receptors causes vasoconstricstimula-tion with consequent pressor sponse, while vasodilatation and depressor response result from the ac-tivation of vascular ^-receptor system An adrenergic antagonist such

as phentolamine or phenoxybenzamine converts the normal pressor sponse of epinephrine to a depressor response by blocking «-receptors and thereby allowing ^-effects of epinephrine to prevail An experimental sequence designed to demonstrate α-adrenergic antagonism thus should (1) produce a reversal of the pressor response of epinephrine to a de-pressor response and (2) diminish, but not reverse, the pressor response

re-of epinephrine in an animal pretreated with a /^-receptor antagonist, e.g., propranolol ( 1-3 mg/kg, i.V.)

A broader and more sophisticated screening procedure for ing adrenergic blockade is the one described by Levy and Ahlquist

characteriz-(1961) Their method is based on the ability of a test compound to modify some selected responses to the following adrenergic agonists: phenylephrine ( «-agonist ), isoproterenol ( ß-agonist ), and epinephrine

and ethylnorepinephrine (a- and ß-agonists) The responses that are

employed as criteria for classifying the activity of the antagonist include the «-responses of the retractor penis (contraction) and the pressor response; the ^-responses of positive cardiac chronotrophy and the de-pressor response, and the changes in intestinal motility that are mediated

by a- and /^-receptors The characterization of a compound as an

«-or β-antagonist is made by observing its modifying effects on the

a-or ß-responses to these agonists The features of adrenergic blockade that are demonstrated by these procedures are summarized in Table III

TABLE I I I

T H E EFFECTS OF <*-ADRENERGIC BLOCKADE UPON THE RESPONSES

TO SYMPATHOMIMETIC AMINES IN DOGS°

Intestinal inhibition Retractor penis contraction Pressor

Intestinal inhibition Retractor penis contraction Depressor

Intestinal inhibition Tachycardia Depressor

a-Receptor blockade Reversed

No effect Block Reduced Block Block Potentiated

No effect

No effect Potentiated

The validity of such criteria for screening α-adrenergic blockade was established by using dibozane, an α-adrenergic antagonist (Levy and Ahlquist, 1961) The responses were recorded simultaneously from anesthetized atropine-treated dogs Dibozane (1 mg/kg) met the criteria for adrenergic blockade because of the following effects: reversal of epinephrine pressor response; inhibition of the intestinal effects of phenylephrine, but not those of epinephrine or isoproterenol; prevention

of the contractile response of retractor penis to epinephrine and ephrine; and, finally, potentiation of the depressor response to isopro-terenol and ethylnorepinephrine

convenient method for assaying pressor substances (Shipley and Tilden, 1947; Brown and Gillespie, 1957) and has been utilized for testing com-

pounds with α-adrenergic blocking activity (Graham, 1962) Rats ing 200-300 gm are given atropine (3 mg/ kg, i.p.) 10-15 min prior to being anesthetized with ether Cannulation of the trachea and bilateral vagotomy are performed routinely The brain is pithed by keeping its head taut and in line with the vertebrae and inserting the pithing rod through the eye socket and down the vertebral canal The animal is connected immediately to the respirator, and the respiration rate is main-tained at 50-60 inspirations /min The femoral vein is cannulated for injection of drugs, and the cannulated carotid artery is used for measur-ing pressor responses The rat is injected with heparin and hexa-methonium (5 mg/kg) and allowed to equilibrate for 30 min

weigh-The prepared rat has a steady blood pressure between 30 and 50

mm H g and responds consistently to intravenous injection of phrine or epinephrine for 4-6 hr The activity of the α-adrenergic antag-onists may be evaluated in terms of ED50 values, i.e., the quantity

norepine-of the compound required to reduce by 50% the pressor response to

a standard dose of epinephrine or norepinephrine Care must be taken

in adjusting the respirator to avoid hyperventilation or distention of the lungs Solutions injected are restricted to less than 0.2 ml

2 Nictitating Membrane

The contractile responses of the nictitating membrane of the cat to exogenous norepinephrine or epinephrine provide a means for demon-strating sympathetic blocking activity The distribution of nerves and blood vessels in the region of the nictitating membrane and the virtues

of this preparation for use in analysis of adrenergic mechanisms have been described by Trendelenburg (1963) The adrenergic receptors of the nictitating membranes are predominently of the a-type, although the presence of /^-function has been indicated

The cat is anesthetized and its head is rigidly clamped on the ating table The trachea is cannulated, and the vagus nerve and cervical sympathetic chain are cut bilaterally An isotonic lever system (1:10; 7.0 gm), attached to the nictitating membrane of one eye, may be used

oper-to record contractile responses The blood pressure may be measured simultaneously from the femoral artery, and the drug injections are made into the femoral or the jugular veins

The response of the nictitating membrane to graded doses of phrine is linear, and the response is antagonized by phentolamine and phenoxybenzamine Drugs may be injected directly into the external carotid artery via a cannulated lingual artery (Morrison and Paton, 1953; Trendelenburg, 1959) This method allows a relatively high con-

norepine-centration of the drug to reach the nictitating membrane and also avoids pronounced changes of blood pressure due to systemic action The in-fluence of anesthetic agents on the activity of test compounds may be avoided by inducing anesthesia in cats with ether and subsequently making spinal preparations as described by Burn (1952)

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2

ß-Adrenergk Blocking Agents

Robert A Turner

I Introduction 21

A Brief Historical Review 21

B Cathecholamines and Their Actions on a- and ß-Receptors 23

C Nomenclature 24

D Stimulation and Blockade 24

II Methods of Screening for ß-Adrenergic Blocking Agents 26

A Isoprenaline-Induced Tachycardia 26

B Pressor and Depressor Responses 29

C Hemodynamic Measurements 30

D Responses of the Rat's Uterus 30

E Relaxation of Trachéal Muscle 31

F Responses with the Dog's Submaxillary Gland 33

III Classification of New ß-Adrenergic Blocking Agents 35

IV Metabolic Effects on /3-Adrenergic Blocking Agents 37

A BRIEF HISTORICAL REVIEW

The adrenergic nervous system plays an important part in the control

of many organ systems functioning below the level of consciousness

21

The chemical transmitters, epinephrine and norepinephrine, are released from terminal nerve endings in the sympathetic nervous system After they reach the receptor sites of the innervated organ, the response to the nervous transmission takes place

Langley (1905) provided the basis of understanding adrenergic ceptors by suggesting that effector cells contain both excitatory and inhibitory receptor substances Thus the response to epinephrine was supposed to depend on which type of receptor substance predominated

re-in the organ Many years later Ahlquist (1948) observed that the relative potencies of sympathomimetic amines, in various effector organs, divided into two distinct groups He proposed that adrenergic receptor sites

can be of two types, which he termed alpha and beta Ahlquist

devel-oped his theory from his observations of the relative order of activity

of six sympathomimetic amines At one group of receptors the order

of potency for his series of six amines was 1, 2, 3, 4, 5, 6, while at another group of receptors the order was 2, 4, 6, 5, 3, 1 His observations caused him to postulate two kinds of adrenergic receptors The a-adreno-tropic receptor was found to be associated generally with an excitatory function (vasoconstriction, uterine stimulation, stimulation of the nic-titating membrane), whereas the β-adrenotropic receptor was associated with an inhibitory function ( vasodilatation, inhibition of uterine muscle ) There were found to be two principal exceptions to the general clas-sification that «-receptors were related to excitatory responses, and /^-receptors, to inhibitory responses The cardiac receptors responsible for increase in rate ( chronotropic ) and in force (inotropic) were classi-fied as ^-receptors Moreover, the relaxation (inhibition) of intestinal muscle was apparently due to stimulation of both kinds of receptor

in the dog, whereas the relaxation of the stomach in the guinea pig and of the cecum in the fowl was due to stimulation of ß-receptors The two principal exceptions to the general classification of adrenergic receptors delayed the acceptance of Ahlquist's theory for almost 10 years

As noted previously, one exception is that the order of potency of phrine and related amines in inhibiting intestinal smooth muscle shares the pattern characteristic of the excitatory «-receptors Yet neither type

epine-of adrenergic blocking agent abolishes the relaxant action epine-of epinephrine

on that muscle The discrepancy led to the suggestion that a third type

of receptor mediated adrenergic relaxation of the gut The difficulty was resolved when it was found that the muscle possesses the usual

^-receptors for inhibition and, in addition, «-receptors that also relax

the muscle Since epinephrine acts on both a- and /^-receptors, neither

type of blocking agent alone blocks the relaxant action When both types of blocking agents are present, epinephrine has no relaxant effect

on the intestinal muscle ( See Table I )

TABLE I SOME ADRENERGIC RECEPTORS 0

Organ or tissue Action Receptor

Contraction Relaxation Contraction Relaxation Relaxation Contraction Relaxation

° The table is taken in part from Shanks (1966)

The other exception is the excitatory action of sympathomimetic

amines on the heart The order of potency of epinephrine-like

com-pounds and the effects of blocking agents show that the cardiac receptors

are of the ß-type It should be noted that the receptor site is responsible

for combination with the drug and not for the kind of response given

by the tissue later

B CATECHOLAMINES AND THEIR ACTIONS ON «- AND /^-RECEPTORS

Sympathetic effector cells may have «- and ^-receptors, or both In

general, the cells of a sympathetically innervated tissue have a majority

of receptors of one type, although a small number of the second type

may also be present For example, the smooth muscle of the blood

vessels supplying skeletal muscles has a preponderance of ß-receptors,

through stimulation of which epinephrine causes vasodilatation It also

has a smaller number of «-receptors that allow norepinephrine to

constrict the vessels since the amine has little effect on ^-receptors in

smooth muscle

Among the common sympathomimetic amines, the actions of a few

may be noted Epinephrine affects both kinds of receptor

Norepineph-rine is nearly as active at «-receptors, but its action at ß-receptors

varies much in intensity The most potent stimulant of ^-receptors is

isoprenaline, which has very little action on «-receptors

Some compounds blocking the «-receptors, but not ^-receptors, are

Dibenamine, phenoxybenzamine, and phentolamine

Some compounds recognized as blocking agents for ^β-receptors are dichloroisoprenaline, pronethanol, and propanolol

The location of adrenergic receptors is shown in Table I

The ^-adrenergic blocking agents are the subject of this chapter The blocking agents affecting «-receptors are the subject of Chapter 1 Both kinds of agent are to be distinguished from ganglionic blocking agents such as hexamethonium

C NOMENCLATURE

Nickerson (1965) defines an "adrenergic blocking agent" as one that

selectively inhibits adrenergic nervous activity and antagonizes the

re-sponses to sympathomimetic amines at the effector cell He prefers the term to "adrenolytic agent," which implies blockade of responses to epinephrine, and to "sympatholytic agent," which often implies blockade

of adrenergic nervous activity within the cerebrospinal axis or along peripheral neurons Adrenergic blocking agents antagonize the responses

to circulating catecholamines more than the responses to the adrenergic mediator

Compounds like guanethidine and bretylium inhibit the release of norepinephrine, and Nickerson (1965) terms them "antiadrenergic" agents As would be expected from their interference with the release

of norepinephrine, the responses mediated by a- and ^-receptors are suppressed equally by antiadrenergic agents

D STIMULATION AND BLOCKADE

The first agent to exhibit blockade of ^-receptors specifically was chloroisoprenaline, discovered by Powell and Slater (1958) The com-pound is structuraly interesting, for isoprenaline (isoproterenol) is the most potent of the known activators of /^-receptors, and the replacement

di-of the phenolic hydroxyl groups with chloro groups forms the most specific ^-adrenergic blocking agent known at this time

From accepted pharmacological theory, it should be possible to study any tissue having a /^-receptor and to produce evidence that a new substance is a ^-adrenergic blocking agent by allowing the substance

to contact the receptor and observing the responses at the receptor fore and after stimulation In other words, the effect of the agonist, e.g., isoprenaline, is observed before and after introduction of the antag-onist, e.g., dichloroisoprenaline (See Table II.)

be-STRUCTURES OF SOME OF THE ORGANIC COMPOUNDS M E N T I O N E D IN THIS CHAPTER

Abbreviations:

O P h - i CH3—, Me—

PhCHOHCHMeNHCMe3

Butoxamine (JV-f-butylmethoxamine)

(PhCH2)2NCH2CH2Cl

Dibenamine

HO—(, V-CHOHCHjNHMe

HO Epinephrine

HO Ethylnorepinephrine

ethanolamine)

H 0 \v V7 C H O H C H 2 N H 2

HO Norepinephrine PhCHOHCHMeNHCHMe2

N-Isopropylmethoxamine

OCH2 CHOHCHj NHCHMe2

Me

Kö-592 [ l-(3-Methylphenoxy)-3-

isopropy lamino-2-propanol ]

L V-CHOHCH 2 NHMe

HO Phenylephrine

PhOCH2CHMeNCH2CH2Cl

CH2Ph Phenoxybenzamine CH3S02NH—<\ Λ-CHOHCHMeNHMe

Not all ^-receptors are suitable for routine study Among those that are, some are much more suitable than others

II Methods of Screening for β-Adrenergic Blocking Agents

A ISOPRENALINE-INDUCED TACHYCARDIA

The most selective and commonly employed test of ß-adrenergic ing action is the antagonism to isoprenaline-induced tachycardia In the method of Levy and Ahlquist (1961), dogs were anesthetized with morphine sulfate (10 mg/kg) and atropine sulfate (1 mg/kg), given subcutaneously, and followed after 30 min by an intravenous injection

block-of pentobarbital sodium (30 mg/kg) The heart rate was recorded tinuously with an electronic linear tachometer, triggered by the ECG, but a less elaborate recording system would be adequate Into a jugular vein was injected 2 /xg/kg of a 0.001 M solution of isoprenaline The heart rate changed from about 160 to 250 beats/min If dichloroiso-prenaline was given first (2 mg/kg), the tachycardia was only slight The anesthetized, atropinized dog is useful for several other measure-

con-ments, such as respiration and intestinal motility Moreover,

measur-ing the tachycardia alone, the unanesthetized dog may be used In the

method of Lish et al (1965), isoprenaline (2 /xg/kg) was given

sub-cutaneously The time of maximal increase in heart rate was 10 min later, the increase ranging from 67 to 167 beats/min In the same animal, isoprenaline provoked approximately the same increase at different times Within 30 min the rates returned to the value before injection The test was repeated at 30-min intervals after oral administration of several ß-adrenergic blocking agents Results are shown in Table III

For comparison of the blocking potency, the unanesthetized dog has also been used Ninety minutes after injection of the blocking agent the heart rate was measured After suitable periods of rest different doses of the agent were tried, until the ED50, the dose needed for antag-onizing 50% of the action of isoprenaline, could be estimated

Other simple methods for measuring the chronotropic response to catecholamines have been described The heart rate in anesthetized cats

was measured by Black et al (1965) Cats weighing 2-3.5 kg were

chosen and were anesthetized by an intravenous dose of chloralose (80 mg/kg) After trachéal cannulation, the femoral arterial pressure was measured by means of a mercury manometer, and the heart rate, by

TABLE I I I PREVENTION OF ISOPRENALINE-INDUCED TACHYCARDIA

IN THE UNANESTHETIZED D O G Oral dose Time after Mean change of heart Drug (mg/kg) drug (min) rate (beats/min)

means of a cardiotachometer Records were traced to a kymograph Through a femoral vein, isoprenaline was introduced (0.2 jug/kg) at intervals of 8 min The heart rate increased from about 190 to 250 beats/min The rate fell back to 190 beats/min after about 5 min Repetition of the injections of isoprenaline after commencing an intra-venous infusion of propranolol (2.5 /xg/kg/min) resulted in increases

in heart rate from about 160 to 180 beats /.min Thus an established /?-adrenergic blocking agent caused the chronotropic response to a ß-adrenergic stimulant to be greatly diminished

The contraction rate of isolated muscular strips from the right atriun

of the guinea pig has been measured in vitro (Black et al, 1965)

Ani-mals in the weight range 620-1000 gm were stunned The heart of each animal was revealed, and the anterior vena cava was ligated Another ligature was made on the wall of the right atrium, close to the atrio-ventricular groove A narrow atrial strip, taken between the ligatures, and including the sinoatrial node, was excised The strip was attached

to a strain gauge transducer and was placed in a bath, 10 ml in volume, containing McEwan's solution (NaCl, 7.60; KC1, 0.42; CaCL, 0.24; NaHCOs, 2.10; N a H2P 04H20 , 0.142; glucose, 2.00; sucrose, 4.50 gm/liter) at 37.5°C, through which was bubbled a mixture of oxygen (95%) and carbon dioxide (5%) Muscular contractions were recorded

on a kymograph After a steady rate of contraction had been reached, dose-response curves could be plotted by adding the stimulant drug

in geometrically increasing doses to the bath without changing the bath fluid Successive doses were added after the maximal response to the preceding dose After the end of the dose-response titration, the bath

fluid was changed repeatedly until the contractile rate was constant The titration was repeated with various doses of antagonist present in the bath The pA2 value was then computed (Schild, 1947)

The strips usually had a steady rate of 200 beats/min The graph

constructed by Black et al (1965) was a plot of the increase in heart

rate, as the percent of the maximal (ordinate), versus the logarithm

of the dose of antagonist in the bath Thus the method allows tative evaluations among a group of blocking agents A similar procedure has been described by Koch-Weser ( 1964 )

quanti-Quantitative differences among a group of ß-adrenergic blocking agents were determined by measuring their inotropic effects on electri-cally driven atria (Levy and Richards, 1966) The isolated left atrium

of a rabbit was suspended in a tissue bath containing 50 ml of Ringer solution, and was stimulated at a rate of 120 beats/min with the aid of a square-wave stimulus, having a pulse duration of 5 msec and a strength of 3 times the threshold voltage The atrium was equil-ibrated for 100 min under those conditions before drugs were added

Krebs-to the bath Changes in contractile force were expressed in terms of percent of the postequilibration force The maximal positive inotropic effect of a 9.4 X 10-8 M concentration of isoprenaline was measured

TABLE IV ANTAGONISM OF SOME /3-ADRENERGIC BLOCKING AGENTS TO THE POSITIVE INOTROPIC ACTION OF ISOPRENALINE ON THE ISOLATED, ELECTRICALLY DRIVEN L E F T ATRIUM OF THE RABBIT

Agent dZ-Kö-592

potency 7.98 6.73 6.34 2.08 1.85 1.00 0.76 0.62 0.14 0.12 0.06

caus-ing 50% inhibition of the maximal response to

isoprenaline

Concentration Relative

By using a plot of 2 or 3 points, in which the percent inhibition of the maximal effect of isoprenaline (ordinate) was plotted against the concentration of the blocking agent (abscissa), it was possible to find the concentration causing 50% inhibition The blocking agent was added

to the bath 3-5 min before the isoprenaline was added Results are shown in Table IV

B PRESSOR AND DEPRESSOR RESPONSES

Levy and Ahlquist (1961) have proposed that "ethylnorepinephrine reversal," i.e., conversion of the depressor response to pressor, may serve

as a simple test in screening compounds for ß-adrenergie blockade They suggested that the properties of producing the ethynorepinephrine re-versal and of diminishing but not reversing the depressor response to isoprenaline should constitute a test for β-adrenergic blockade Accord-ing to the findings of Levy and Ahlquist (1961), ethylnorepinephrine

(50 /Ag/kg) activates both a- and ^-receptors In the anesthetized dog

the effects on ^-receptors predominate since ethylnorepinephrine vokes vasodilatation, tachycardia, and a lower blood pressure Blockade

pro-of ß-receptors leaves the effect on the «-receptors, causing phrine to become a pressor, and vasoconstrictive, agent However, ethyl-norepinephrine reversal is not a sufficient condition for ^-blockade, for phenylephrine and methoxamine also accomplish the reversal A true ß-adrenergic blocking agent, such as dichloroisoprenaline, can be dis-tinguished because it will diminish or annul the depressor effect of iso-prenaline but will not convert isoprenaline into a pressor agent That

ethylnorepine-is true because dichloroethylnorepine-isoprenaline blocks the cardiac effects of ethylnorepine-prenaline, mediated through ^-receptors The authors stated that ethyl-norepinephrine reversal alone is conclusive evidence of /^-blockade if the agent is not a vasoconstrictor

iso-In the method of screening used, dogs were anesthetized, as described previously, with morphine and pentobarbital After anesthetization, atropine sulfate (0.5 mg/kg, i.v.) was given Blood pressure was recorded from a carotid artery with a Statham transducer Recordings were made with an optical oscillograph or a cathode-ray tube camera system The latter system permitted the recording of the electronically integrated mean pressure, but a simpler system such as a kymograph would probably be satisfactory for screening

Results with dichloroisoprenaline and isoprophenamine may be cited Intravenous administration of isoprenaline (2 /xg/kg) and ethylnorepi-nephrine (50 /xg/kg) at different times caused the dog's arterial pressure

to fall from about 95 to 45 mm Hg After administration of isoprenaline (1 or 3 mg/kg, i v.) isoprenaline had little effect on the blood pressure, which fell about 20 mm Hg, or less Following the ad-ministration of ethylnorepinephrine, the blood pressure increased about

dichloro-30 mm Hg The results with isoprophenamine were similar; the two activators caused the blood pressure to fall from about 100 to 45 mm

Hg After administration of isoprophenamine (1 mg/kg) the response

to isoprenaline was a fall from about 85 to 70 mm Hg, while that to ethylnorepinephrine was a transient rise from 80 to 110 mm Hg There are methods for testing for ^-blockade with anesthetized cats and dogs, with the use of isolated papillary muscle or with the Langendorff heart, in all of which ß-adrenergic activators cause an in-crease in the force of cardiac contraction The response is blocked by

ß-adrenergic blocking agents (Koch-Weser, 1964; Black et al., 1965)

As the methods are less convenient than others, they are cited only through reference However, if the equipment is on hand, and the animal

is prepared for other measurements on the cardiovascular system, then

it may be convenient to measure the inotropic responses simultaneously with other responses

C HEMODYNAMIC MEASUREMENTS

Blood flow in the femoral artery of the dog is increased transiently

by an injection of isoprenaline distal to the location of a Shipley-Wilson rotameter ß-Adrenergic blocking agents antagonize the increase in flow For example, the flow increase was measured after an injection of iso-prenaline in a dog Dichloroisoprenaline (1 mg/kg) was given by the same route, and then repetition of the isoprenaline injection produced only 40% of the increase in flow noted earlier (Powell and Slater, 1958)

A similar result has been reported by Levy and Ahlquist ( 1961 )

D RESPONSES OF THE RAT'S UTERUS

From a careful study of the rat's uterus, it has been shown that the adrenergic receptors are only of the ß-type (Levy and Tozzi, 1963),

so that the organ is a suitable object for demonstrating ß-adrenergic activation and blockade

Rats weighing 300 gm or more were used Uterine strips were suspended in baths 10 ml in volume, at 37°C, containing Locke's solution (NaCl, 0.9%; KC1, 0.042%; CaCL, 0.024%; glucose, 0.1%; NaHC03, 0.05%) aerated with 95% oxygen and 5% carbon dioxide Spontaneous activity was recorded isotonically by means of a muscle-lever writing on a

kymograph, or isometrically by means of a force displacement ducer, recording through a cathode-ray tube camera system

trans-Control inhibitory responses were obtained with epinephrine (0.02 /*g/ml) and isoprenaline (0.02 /xg/ml) Then a β-adrenergic blocking agent was added to the bath After spontaneous motility had returned

to control values, epinephrine was added and allowed to act for 3 min Without washing, isoprenaline was added Both dichloroisoprenaline and pronethalol (5 /Ag/ml) caused the inhibitory actions of epinephrine and isoprenaline to be abolished, i e, the uterine contractions were of the same character as they were during the control period, before the addi-tion of epinephrine

E RELAXATION OF TRACHÉAL MUSCLE

Another method for evaluating ß-adrenergic blockade involves the use of the trachéal chain The preparation was originally developed

by Castillo and de Beer (1947) Although trachéal muscle has a high natural tone, allowing relaxation to be produced without the prior' use

of a spasmogen, the preparation permits little relaxation even with active drugs, often exhibiting a slow response and a slow recovery Besides, the sensitivity of the muscles from different animals varies greatly Foster (1960) has overcome those difficulties through the use of two animals and by cutting the rings in the trachéal chain

Two guinea pigs weighing 600-800 gm were stunned by a blow on the neck and were exsanguinated The tracheae were removed as sections from the larynx to the carina, and each was cut into 8 rings of equal width, with the aid of scissors Each ring was opened by cutting through the cartilage and counted, the first ring from the larynx being number

1 The odd-numbered rings from one animal and the even-numbered rings from the other animal were tied together Thus two preparations were formed, each with 8 parts Each chain was immersed in Krebs'

solution at 30°C and aerated with 95% oxygen and 5% carbon dioxide

The solution was contained in a cylindrical vessel to whose bottom

it flowed from a reservoir The trachéal chain was attached to a bar

of the inlet tube near the bottom The other end of the trachéal chain was attached to a lever, and the chain was held vertical by adding sufficient weight (approximately 120 mg) to the lever on the side of the fulcrum opposite that holding the chain The tension applied to the vertical chain was about 240 mg The chamber in which the chain was suspended had a water jacket for maintaining temperature Responses were recorded by a pen attached to the lever The spon-taneous elongation of the tissue, caused by permanent support of the