BETA ADRENOCEPTOR RESPONSE IN ASTHMA

A

Thesis submitted by

JUDITH KATHLEEN1GREENACRE

for

The degree of Doctor of Philosophy

in the

University of London

Department of Clinical Pharmacology

Royal Postgraduate Medical School

Hammersmith Hospital

London W12 OHS

1978 :i :

ABSTRACT

An inherent defect of beta adrenergic receptor function has been suggested as the underlying mechanism in bronchial asthma. Many investigators have presented data to support this hypothesis but few have considered the possibility that treatment with beta adrenergic stimulants might adversely affect beta adrenoceptor function. The experiments described in this thesis were designed to examine this possibility.

Because human lung tissue is not readily available, the peripheral blood lymphocyte was studied. Beta adrenoceptor function was evaluated by measuring cyclic 3' 5' adenosine monophosphate (cyclic AMP) formation in -8 4 response to increasing concentrations of isoprenaline (10 to 10 moles. -1 litre ).

Cells from asthmatics on beta adrenergic bronchodilators in large doses had a significantly reduced cyclic AMP response compared to lymphocytes from normals or from asthmatics on non-adrenergic medication (p< 0.001). Cells from normal subjects given salbutamol orally (12-16 mg/day) or by inhalation in excessive doses (3000pg/day) also showed depressed beta adrenergic responsiveness (p< 0.05 and p<0.01). Patients with phaeochromocytomata and high levels of circulating endogenous catecholamines had reduced lymphocyte cyclic AMP formation compared to normal subjects (p<0.01). A dose-related desensitization of beta adrenoceptor function was produced in vitro by incubating lymphocytes 8 -6 1 from normal subjects with isoprenaline (10 to 10 moles. litre ) for 24 hours (p< 0.05).

Drug treatment with beta adrenergic stimulants depresses beta adrenoceptor function and there is little evidence that asthmatics have an inherent defect in this mechanism. ACKNOWLEDGEMENTS

I would like to thank especially Dr. M.E. Conolly, my close associate and mentor in the work reported in this thesis. I am also grateful to Professor C.T. Dollery for invaluable advice on both the work and on its presentation. Mr. P. Scofield provided expert technical assistance and I must also thank the other members of the Department of Clinical Pharmacology at the Hammersmith Hospital. Aviva Petrie very kindly performed the statistical analysis.

My thanks also go to Dr. F.H. Scadding, Professor C. Fletcher and Dr. N. Pride who allowed me to study some of their patients.

I gratefully acknowledge Mrs. T. Turton, Mrs. J. Ellis and Mrs. J. Taylor who assisted in the preparation of the manuscript and typing.

Finally, I would like to express my appreciation to the Medical Research Council whose generous support allowed me to do this work.

Last, but not least, I would like to thank my husband for his patience. : iii :

The list below is of published and "in press" work, some of which forms part of this thesis.

beta-Adrenoceptor Function - Effect of Prolonged Exposure to beta- Adrenoceptor Agonists. Conolly, M.E. and Greenacre, J.K. (1975) No. 1241, 6th International Congress of Pharmacology Abstracts, Helsinki, Finland.

The beta-Adrenoceptor of the Human Lymphocyte and Human Lung Parenchyma. Conolly, M.E. and Greenacre, J.K. (1977) Br. J. Pharmac., 21, 17-23.

The Lymphocyte beta-Adrenoceptor in Normal Subjects and Patients with Bronchial Asthma: The Effect of Different Forms of. Treatment on Receptor Function. Conolly, M.E. and Greenacre, J.K. (1976) J. clin. Invest., 1307-1316.

Desensitization of the beta-Adrenoceptor of Lymphocytes from Normal Subjects and Patients with PhaeoehromocytomakStudies in vivo. Greenacre, J.K. and Conolly, M.E. (in.press) Br. J. clin. Pharmac.

Desensitization of the beta-Adrenoceptor of Lymphocytes froM Normal Subjects and Asthmatic Patients in vitro. Greenacre, J.K., Scofield, P. and Conolly, M.E. (in press) Br. J. clin. Pharmac. : 1 :

TABLE OF CONTENTS

Page

CHAPTER I INTRODUCTION AND AIMS OF THE PRESENT STUDY 10

1.1 Introduction 11 1.2 Autonomic function in asthma 12 1.3 Cyclic adenosine 3' 5' monophosphate: the second messenger 15 1.4 The theory of partial beta adrenergic blockade in asthmatics 16 1.4.1 Szentivanyi's theory 16 1.4.2 Supporting evidence 18 1.5 Possible tolerance to beta adrenergic agents: the increase in asthma deaths 24 1.6 Evidence of tolerance to beta adrenergic stimulants in animals and man 26 1.7 in vitro production of tolerance to beta adrenoceptor agonists 30 1.8 Possible physiological and pathophysiological importance of tolerance 34 1.9 Purpose of the present study 36

CHAPTER II GENERAL MATERIALS AND METHODS 37

2.1 Introduction 38 2.2 Materials, drugs and chemicals 38 2.3 Preparation of chromatography materials 39 2.4 Preparation of binding protein 40 2.5 Cell separation and viability 41 2.6 Incubation 44 2.7 Cyclic AMP purification 45 2.8 Assay of cyclic AMP 45 2.9 Calculation of results 47 2.10 Statistical methods 50

CHAPTER III CHARACTERISTICS OF THE LYMPHOCYTE BETA ADRENOCEPTOR 51

3.1 Introduction 52 3.2 Methods 53 : 2

Page 3.3 Results 54 3.3.1 Lymphocytes: normal dose response curve to isoprenaline 54 3.3.2 Lymphocytes: response to salbutamol 57 3.3.3 Lymphocyte response to isoprenaline in the presence of beta adrenoceptor antagonists 57 3.3.4 Lung experiments: incubation with isoprenaline and salbutamol 62 3.3.5 Lung experiments: incubation with isoprenaline and propranolol or practolol 62 3.4 Discussion 67 3.4.1 Classification of the lymphocyte beta adrenoceptor 67 3.4.2 Variability of lymphocyte cyclic AMP response to isoprenaline 69

CHAPTER IV LYMPHOCYTE BETA ADRENOCEPTOR RESPONSE IN ASTHMATICS 72 4.1 Introduction 73 4.2 Subjects and methods 73 4.3 Results 77 4.3.1 Asthmatics on large doses of beta adrenergic bronchodilators 77 4.3.2 Asthmatics on non-adrenergic medication 77 4.3.3 Asthmatics studied serially, initially on large doses of beta adrenergic bronchodilators and subsequently on other anti-asthmatic drugs 84 4.4 Discussion 92

CHAPTER V NORMAL SUBJECTS "TREATED" WITH BETA ADRENERGIC STIMULANTS 95 5.1 Introduction 96 5.2 Subjects and methods 97 5.3 Results 100 5.3.1 Normal subjects on oral salbutamol 12-16 mg daily 100 5.3.2 Normal subjects taking excessive salbutamol by inhalation 100 5.3.3 Obstetric patients given prolonged infusions of isoxsuprine 100 5.3.4 Patients with phaeochromocytomata 109 5.4 Discussion 115 •3 :

Ema

CHAPTER VI PRODUCTION OF BETA ADRENOCEPTOR DESENSITIZATION IN VITRO 117

6.1 Introduction 118 6.2 Materials and methods for in vitro studies 119 6.3 Results 127 6.3.1 Cyclic AMP response of lymphocytes from normal subjects cultured with or without isoprenaline 127 6.3.2 Cyclic AMP response of lymphocytes from normal subjects and one asthmatic patient cultured with and without PGE 1 131 6.3.3 Cyclic AMP response of lymphocytes from asthmatic subjects cultured with and without isoprenaline or PGE 1 139 6.3.4 Assay of phosphodiesterase activity in lymphocytes cultured with isoprenaline or PGE1 139 6.4 Discussion 139

CHAPTER VII CONCLUSIONS 148

REFERENCES 153

APPENDIX 1 167 : 4 :

TABLE OF TABLES

Page

Table I Normal subjects - lymphocyte response to isoprenaline in picomoles cyclic AMP above

baseline unstimulated level 55 Normal subjects - lymphocyte response to Table II isoprenaline, percent increase in cyclic AMP 58 Table III pA2 values for propranolol and practolol

observed in human lymphocytes 64

Table IV pA2 values for propranolol and practolol

obtained in other laboratories 68

Table V Clinical details of asthmatic patients on high

doses of beta adrenergic bronchodilators 75 Table VI Clinical details of asthmatic patients on

non-adrenergic medication 76

Table VII Asthmatic patients on large doses of beta adrenergic bronchodilators: lymphocyte cyclic AMP response to isoprenaline, percentage increase

over baseline 79

Table VIII Asthmatic patients on large doses of beta adrenergic bronchodilators: lymphocyte response to isoprenaline, picomoles cyclic AMP over baseline 8o

Table IX Asthmatic patients on non-adrenergic drugs: lymphocyte cyclic AMP response to isoprenaline,

percentage increase over baseline 82

Table X Asthmatic patients on non-adrenergic drugs: lymphocyte response to isoprenaline in picomoles

cyclic AMP over baseline 83

Table XI Asthmatics studied before and after changing from large doses of adrenergic bronchodilators to non-adrenergic drugs: percentage increase in lymphocyte cyclic AMP 88

Table XII Asthmatics studied before and after changing from large amounts of adrenergic bronchodilators to non-adrenergic drugs,absolute increase in lymphocyte cyclic AMP 89

Table XIII Clinical details of patients with

phaeochromocytomata 99 :5

Page

Table XIV Normal subjects taking oral salbutamol (12-16 mg/day): percentage increase in lymphocyte

cyclic AMP in response to isoprenaline 102

Table XV Normal subjects taking oral salbutamol: absolute increase in lymphocyte cyclic AMP 103

Table XVI Normal subjects taking large amounts of inhaled salbutamol (30 inhalations/day): percentage increase in lymphocyte cyclic AMP in response

to isoprenaline 105

Table XVII Normal subjects taking large amounts of inhaled salbutamol: absolute increase in lymphocyte cyclic AMP 106

Table XVIII Obstetric patients studied before and after infusions of isoxsuprine: percentage increase in lymphocyte cyclic AMP in response to isoprenaline 108

Table XIX Obstetric patients studied before and after isoxsuprine: absolute increase in lymphocyte cyclic AMP 110

Table XX Patients with phaeochromocytomata: percentage increase in lymphocyte cyclic AMP in response to isoprenaline 112

Table XXI Patients with phaeochromocytomata: absolute increase in lymphocyte cyclic AMP 113

Table XXII Clinical details of asthmatic patients at time of lymphocyte culture study 124

Table XXIII Percent increase over baseline cyclic AMP response to isoprenaline 10-4 moles. litre-1 in lymphocytes cultured with and without isoprenaline for 24 hours 129

Table XXIV Absolute cyclic AMP response to isoprenaline in lymphocytes cultured with and without isoprenaline 132

Table XXV Percent increase in cyclic AMP response to isoprenaline or PGE1 in lymphocytes cultured with and without isoprenaline for 24 hours 134

Table XXVI Absolute cyclic AMP response to isoprenaline or PGE1 in lymphocytes cultured with and without isoprenaline 135 Page

Table XXVII Percent increase in cyclic AMP response to isoprenaline or PGE1 in lymphocytes cultured with and without Pal for 24 hours 137 Table XXVIII Absolute cyclic AMP response to isoprenaline or PGE1 in lymphocytes cultured with and without Pal 138 Table XXIX Percent increase in cyclic AMP response to isoprenaline in lymphocytes from asthmatic subjects cultured with and without isoprenaline for 24 hours 140

Table XXX Absolute cyclic AMP response to isoprenaline in lymphocytes from asthmatic subjects cultured with and without isoprenaline

Table XXXI Absolute cyclic AMP response to isoprenaline or PGE1 of lymphocytes from an asthmatic patient which had been cultured with or without isoprenaline or PGE1 for 24 hours 143 Table XXXII Effect of 24-hour culture with isoprenaline or PGE1 on phosphodiesterase activity in total lymphocyte homogenate 144

Table XXXIII Effect of 24-hour culture with isoprenaline or Pal on phosphodiesterase activity in lymphocyte membrane 145 : 7:

LIST OF ILLUSTRATIONS

Page

Figure 1 The isolation of protein kinase from rabbit skeletal muscle 42

Figure 2 Influence of pH on cyclic AMP binding of protein kinase from rabbit skeletal muscle 43

Figure 3 Elution pattern of Dowex AG 1 x 8 46

Figure 4 Displacement of cyclic AMP from protein kinase by ATP, ADP and cyclic GMP 48

Figure 5 Plot of standard curve of cyclic AMP binding 49

Figure 6 Absolute increase in lymphocyte cyclic AMP in response to isoprenaline 56

Figure 7 Percentage increase in lymphocyte cyclic AMP in response to isoprenaline 59 Figure 8 Percentage increase in lymphocyte cyclic AMP in response to isoprenaline and salbutamol 60

Figure 9 Response of lymphocyte cyclic AMP to isoprenaline in the presence of propranolol or practolol 61

Figure 10 Schild plots showing effect of propranolol and practolol on lymphocyte cyclic AMP response to isoprenaline 63

Figure 11 Cyclic AMP response of human lung parenchyma to isoprenaline and salbutamol 65

Figure 12 Propranolol or practolol inhibition of cyclic AMP response to isoprenaline in human lung parenchyma 66

Figure 13 Cyclic AMP response of lymphocytes from asthmatic patients on beta adrenergic bronchodilators compared to normal subjects 78

Figure 14 Cyclic AMP response of lymphocytes from asthmatics on non-adrenergic drugs compared to normal subjects 81

Figure 15 Cyclic AMP response of lymphocytes from asthmatic patients on beta adrenergic bronchodilators compared to patients on other drugs 85 :8:

Page

Figure 16 Absolute increase in lymphocyte cyclic AMP in response to isoprenaline: asthmatics on beta adrenergic stimulants, other medication and normal subjects 86

Figure 17 Serial study of lymphocyte cyclic AMP response in asthmatic patients initially on large amounts of beta adrenergic bronchodilators and subsequently on other drugs 87

Figure 18 Percentage increase in cyclic AMP in lymphocytes from Patient No. 2, initially on large doses of beta adrenergic stimulants and subsequently on

other therapy 90

Percentage increase in cyclic AMP in lymphocytes Figure 19 from Patient No. 6, as above 91

Figure 20 Effect of oral salbutamol on lymphocyte cyclic AMP response to isoprenaline in normal subjects 101

Figure 21 Effect of large amounts of irhnled salbutamol on lymphocyte cyclic AMP response to isoprenaline in normal subjects 104

Figure 22 Lymphocyte cyclic AMP response in obstetric patients before and after isoxsuprine infusions 107

Figure 23 Lymphocyte cyclic AMP response in patients with phaeochromocytomata compared to normal subjects 111

Figure 24 Lymphocyte cyclic AMP response in a patient with a phaeochromocytoma before and after operation 114

Figure 25 Flow chart of in vitro culture method 121 14 Figure 26 Elution profile of C adenosine and 3H cyclic AMP with Dowex AG 1 x 2 126

Figure 27 Dose-related reduction in cyclic AMP response in normal lymphocytes cultured with isoprenaline 128

Figure 28 Individual results from lymphocytes cultured with varying concentrations of isoprenaline 130

Figure 29 Response of normal lymphocytes to isoprenaline and to prostaglandin E1 after culture with isoprenaline 133 • 9•

Page Figure 30 Response of normal lymphocytes to isoprenaline and to PGE after culture with PGE 1 1 136 Figure 31 Response of lymphocytes from asthmatics and from normal subjects after culture with and

without isoprenaline 142 : 10 :

CHAPTER I

INTRODUCTION AND AIMS OF THE PRESENT STUDY : 11 :

1.1 INTRODUCTION

Asthma or variable airways obstruction (Ciba Foundation Study Group, 1971) is common and has remained a clinical problem despite major therapeutic and scientific advances in the last 25 years. The Second National Survey of Morbidity in General Practice found that approximately half a million patients consulted a doctor at least once for asthma in 1970-71 (Office of Population Censuses and Surveys, 1974). It is estimated that about one million individuals in Britain are affected, many mildly or suffering only occasional attacks and not seeking medical advice during any one year. One survey of school children in Kent showed a prevalence rate of -3.8 percent (Hamman et al., 1975) while a study in a south London practice over 15 years revealed an incidence of 2.5 percent with an annual prevalence of 1.2 percent (Fry, 1965). The cost is high in terms of morbidity of patients, treatment by the National Health Service and time off work - 2.3 million days of certified absence in the year ending 31st May, 1975 (Department of Health and Social Society).

In the past, many doctors considered the disease to be unimportant, causing only moderate morbidity and negligible mortality. In the nineteenth century the American physician Oliver Wendell Holmes even described it as 'the slight ailment that promotes longevity' (Ellul- Micallef, 1976). However, this is untrue. People do die from asthma - almost 1,200 in England and Wales in 1975 - 145 under the age of 35 (Office of Population Censuses and Surveys, 1977).

The death rate has changed little since the introduction of corticosteroids or of selective bronchodilators. In fact mortality rose in the early 1960s at the same time as beta adrenergic bronchodilators in convenient pressurized aerosols became widely available and extensively used. An excess of 3,500 deaths from asthma occurred between 1961 and 1967 and most tragically with a seven-fold increase in patients between the ages of 10 and 14.

The continuing clinical problem has encouraged a considerable amount of research into the pathophysiology of the disease. Immunological : 12:

mechanisms have been extensively explored since the discovery of IgE in 1966. However, many asthmatics have no demonstrable allergy and the underlying aetiology, at least in these subjects, remains a mystery. Mucous production and character are being investigated, but little is known about it at present.

One hallmark of asthma, whatever its cause, is hyper-reactivity of the airways to a variety of stimuli including allergen challenge in atopic patients, infection, chemical or irritant dusts, cold air, exercise and emotion. Bronchial hyper-reactivity can be demonstrated even during periods of clinical remission and an important area of research has explored this phenomenon, focusing on possible dysfunction of the autonomic nervous system.

The introduction to this thesis will discuss the evidence for autonomic dysfunction in asthma and describe, in particular, an influential theory by Szentivanyi who postulated an underlying defect in beta adrenergic receptors. A considerable amount of work in man and in various in vitro systems appeared to support this hypothesis and this data will be presented.

More recently, the link between the rise in asthma deaths and the use of excess beta adrenergic medication suggested to some investigators that tolerance to these drugs might be important. In support of this, studies showing desensitization to beta adrenergic stimulants in animals, man and in vitro will be discussed.

In this thesis the possibility that beta adrenergic function of both asthmatics and normals can be affected by beta adrenoceptor stimulants was investigated. If this hypothesis is correct, much of the data supporting Szentivanyi's theory of a beta adrenergic defect in the asthmatic can be discarded and alternative theories might be pursued more profitably.

1.2 AUTONOMIC FUNCTION IN ASTHMA

The mammalian bronchial tree is supplied by both parasympathetic and sympathetic nerves; the former are more extensive than the latter. Cholinergic afferent nerve terminals are located superficially in the • 13

epithelium which lines the airways from the trachea to the peripheral airways (Nadel, 1973). However, in mammals sympathetic fibres are found only in the larger airways (Mann, 1971). It is possible that in man adrenergic fibres do not supply the bronchi at all; Richardson and Beland (1976) have demonstrated another inhibitory system and sympathetic influences may be entirely the result of circulating catecholamines.

Normal bronchial tone is maintained by the parasympathetic system. Vagotomy, vagal cooling and anticholinergics in animals, or the latter drugs in man, can be shown to reduce airways resistance (Widdicombe and Sterling, 1970).

Parasympathetic predominance in asthmatics was first suggested as early as 1909 (Eppinger and Hess). Numerous reports were published describing - asthmatic attacks precipitated by pilocarpine or other cholinergic agents (cited in Curry, 1947). Subsequently asthmatics were shown to be extremely sensitive to small amounts of cholinergic drugs (Curry, 1947; Curry and Leard, 1948; Tiffeneau, 1958) as well as to histamine (Curry, 1946) which was known to be an important mediator in anaphylactic shock in animals (Schild, 1937).

Atropine-like alkaloids were used with limited success in the nineteenth and early twentieth centuries in the treatment of asthma (Herxheimer, 1959) but a newer related compound appears to be more promising (Storms et al., 1975). Atropine prevents not only broncho-constriction in asthmatics due to methacholine (Itkin and Anand, 1970) and to non-specific irritants such as citric acid, dusts and cold air, but also in some patients, to histamine. The drug protects against antigen challenge in dogs. Gold et al. (1972) and one group (Yu at al., 1972) found this in man although other workers using smaller doses (Itkin and Anand, 1970; Rosenthal et al., 1974) have not confirmed it. The parasympathetic nervous system therefore, may play an important role in the bronchial hyper-reactivity characteristic of asthmatics as well as in modulating normal bronchial tone.

Sympathetic nervous system effects are mediated by noradrenaline and adrenaline. These catecholamines stimulate adrenoceptors on cells to produce characteristic responses. The classification of alpha and beta : 14:

adrenoceptors origingoly proposed by Alquist (1948) is now generally accepted as a result of the use of selective agonists and antagonists. The subdivision of beta adrenoceptors into betel and beta2 (Lands et al., 1967) is more controversial. Differences between the beta adrenoceptors in the heart which control rate and contractile force and those in vascular smooth muscle can be demonstrated using different agonists and antagonists. However, the separation is not as clear cut as that between alpha and beta adrenoceptors since there is some evidence that beta adrenoceptors in other tissues have differing characteristics (Bristow et al., 1970; Lefkowitz, 1975). Nevertheless, the concept has been useful as drugs primarily stimulating beta2 or blocking betal responses have been developed.

Evidence that the adrenergic system is involved in the control of bronchial tone in normal man is tenuous. Adrenaline applied directly to bronchi in vitro relaxes them (Hawkins and Schild, 1951), but it is difficult to show this in intact normal subjects. Simple tests of airways obstruction such as the peak expiratory flow rate (PEER) show no change. Studies in normal subjects given a beta adrenergic blocking drug, propranolol, also show no change in PEER or forced expiratory volume in one second (FEV) (Zaid and Beall, 1966; Marcelle et al., 1968). Some workers, using sensitive tests with the whole body plethysmograph have found an increase in airways resistance after the drug (McNeill and Ingram, 1966; MacDonald et al., 1967; Jones, 1972) but others have not confirmed this (Richardson and Sterling, 1969). Thus, the sympathetic nervous system and circulating catecholamines are relatively unimportant in maintaining bronchial tone in normal subjects.

Bronchial smooth muscle possesses alpha adrenoceptors which elicit bronchoconstriction (Simonson et al., 1972) as well as beta adrenoceptors which mediate bronchial relaxation. Thymoxamine, the alpha adrenergic blocking agent, will not itself produce bronchodilitation in normal subjects (Astin, 1972), but adrenergic stimulation with phenylephrine after pretreatment with propranolol produces a significant increase in airways resistance which is prevented by pretreatment with thymoxamine (Prime et al., 1972). Thus the effect of the alpha receptor can be demonstrated by blocking the opposing beta2 adrenoceptor. :15:

Increased alpha adrenergic tone has been postulated as the cause of bronchial hyper-reactivity in asthmatics (Patel et al., 1974). In a group of these patients the combination of alpha blockade with thymoxamine and beta stimulation with isoprenaline produced significantly more bronchodil&tation measured in the body plethysmograph than either alone (Patel, 1976).

Another alpha blocking agent, indoramin, which, like thymoxamine, has antihistamine properties and also antagonizes serotonin has been shown to produce some bronchodi4tation and to protect against the bronchoconstriction characteristically produced by exercise in many asthmatics (Bianco et al., 1974).

Derangements of the autonomic nervous system may be important in the pathogenesis of asthma. Increased parasympathetic activity causing increased bronchial smooth muscle tone and hyperirritability of the airways could result in bronchoconstriction and wheeze. Increased alpha adrenergic activity could also result in bronchoconstriction. The next section deals briefly with the intracellular compounds that mediate these effects. This is followed by a discussion of another theory of autonomic dysfunction in asthmatics - Szentivanyi's hypothesis of a defect in beta adrenergic fanction in these patients.

1.3 CYCLIC ADENOSINE 3' 5' MONOPHOSPHATE: Tat, SECOND MESSENGER Stimulation of the beta adrenoceptor activates adenyl cyclase and this membrane bound enzyme catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine 3' 5' monophosphate (cyclic AMP). The latter compound is subsequently broken down by phosphodiesterase (P1k0 to 5' adenosine monophosphate (5' AMP). Cyclic AMP acts as an intracellular or second messenger to activate various protein kinases, an important group of enzymes present in many cell types, which preform the hormone induced work of the cell. In the liver cell increased cyclic AMP results in glycogenolysis, in cardiac muscle it produces a positive inotropic effect (Sutherland and Robison, 1966; Sutherland, 1970) in bronchial smooth muscle bronchodilltation (Andersson et al., 1972).

Previously sensitized lung tissue challenged with antigen will release : 16 :

histamine, slow relaxing substance (SRS-A) and other mediators of bronchoconstriction. Increased cyclic AMP in this preparation results in reduced release of these mediator substances. Stimulation of alpha adrenoceptors by adrenaline in the presence of a beta adrenergic antagonist such as propranolol causes a reduction in cyclic AMP by an unknown mechanism and this leads to increased histamine and SRS-A production (Orange et al., 1971).

The alpha and beta adrenoceptors, therefore, have opposing effects and beta adrenergic blockade would be disadvantageous to the asthmatic because of a predominant alpha adrenergic effect. Both more mediator substances and a direct action on bronchial smooth muscle would produce bronchoconstriction.

Stimulation of the cholinergic receptors on cells in lung tissue results is an increase in cyclic guanosine 3' 5' monophosphate (cyclic GMP). This is associated with decreased response to cyclic AMP and with increased release of histamine and SRS-A (Lefkowitz, 1976).

The levels of cyclic AMP and cyclic GMP within the cell are both important. Any drug or manoevre which increases the former, or reduces the latter, should be beneficial to the asthmatic.

THE THEORY OF PARTIAL BETA ADRENERGIC BLOCKADE IN ASTHMATICS

1.4.1 Szentivanyi's Theory

Both sympathetic and parasympathetic mechanisms are important in the asthmatic but adrenaline is more effective treatment in an attack than atropine. Therefore, a theory of asthma based on the malfunction of the beta adrenergic system, first proposed by Szentivanyi in 1962, excited considerable interest. He postulated that the underlying mechanism of bronchial hyperactivity in the asthmatic was an autonomic imbalance caused by a defect of beta adrenergic receptor function.

Two animal models of atopy or allergy had been important in the development of this concept. The first was the hypothalamically imbalanced anaphylactic guinea pig. Szentivanyi had previously found that ablation of the tuberal area of the hypothalmus which controls the parasympathetic nervous system protects the animal against anaphylaxis : 17 :

and histamine shock (Szentivanyi and Szekely, 1956; Szentivanyi and Filipp, 1958; Filipp and Szentivanyi, 1958; Szentivanyi and Szekely, 1958). The guinea pig's reactivity to otherwise non-toxic concentrations of mediators could be increased by stimulating or destroying different areas in the hypothalmus making it pharmacologically and immunologically similar to the atopic patient. However, Szentivanyi discarded this model as surgical intervention was artificial, anaphylaxis was not an ideal model for asthma, and the important role of respiratory infection as a trigger in the asthmatic could not be explained.

The Bordetella pertussis-vaccinated mouse which showed increased sensitivity to histamine was first described by Parfentjen and Goodline in 1948. Szentivanyi and his co-workers developed this model and used it extensively (Fishel et al., 1962; Fishel and Szentivanyi, 1963; Szentivanyi et al., 1963; Townley et al., 1967). After injection with living or dead organisms, animals responded abnormally to numerous stimuli. They were hypersensitive to various mediators which are, or may be, important in asthma - histamine, serotonin, bradykinin and in one strain, acetylcholine. They proved sensitive to non-specific respiratory irritants and cold, they had enhanced antibody formation which was of the reaginic (IgE) type, and they had eosinophilia. They had a reduced response to beta adrenergic stimulants and most importantly all these abnormalities could be reproduced by treating unimmunized mice with a beta adrenergic blocking drug (Fishel et al., 1962).

In his review article in 1968, Szentivanyi noted various points which any acceptable theory of the aetiology of asthma must explain and discussed them in terms of his hypothesis of reduced beta adrenergic function. The bronchial smooth muscle spasm and possibly the increased mucous secretion characteristic of an asthmatic attack was due to relatively unopposed alpha adrenergic activity. The mouse model, like the asthmatic, was hypersensitive to histamine. Zaid and Beall (1966) had shown that asthmatic patients were more sensitive to methacholine and histamine after being given the beta adrenergic antagonist propranolol. This supported Szentivanyi's hypothesis; increasing beta blockade would make a partial beta adrenergic defect worse. The high level of reaginic or IgE antibody seen in atopic subjects was mirrored in the Bordetella pertussis- immunized mouse model. Beta adrenoceptors in different human organs : 18:

could be affected differently; thus those in the lung might be normal while those in nasal mucosa or skin partially defective resulting in hay fever or eczema.

Szentivanyi had discarded his earlier model of atopic disease because it was unaffected by infection, frequently an important cause of an exacerbation in the asthmatic. The Bordetella pertussis immunization of mice led to reduced beta adrenergic function; infection itself could 'presumably cause an increase in beta adrenergic blockade.

Eosinophilia is common in asthmatics and is unexplained; beta adrenergic stimulants normally cause a fall in eosinophils; logically a partial defect might produce a chronic eosinophilia Therapy could be explained rationally: adrenaline partly overcame the beta blockade, and drugs which by-pass the beta adrenoceptor but increase cyclic AMP levels, such as phosphodiesterase inhibitors, or steroids which improve receptor function, should be active. Finally, cyclic AMP had been shown to mediate the effects of beta adrenoceptor stimulation within the cell (Sutherland and Robison, 1966); Szentivanyi claimed that the susceptibility of asthmatics to a wide variety of precipitating factors was due to the importance and ubiquitous nature of this second messenger which acted as the final common pathway.

The beta adrenergic theory of asthma did produce a unifying hypothesis which attempted to explain some of the puzzling features of the disease. It stimulated a considerable amount of research and a large number of papers were published which appeared to support the concept of a beta adrenoceptor defect in these patients.

1.4.2 SUPPORTING EVIDENCE

(a) in vivo: Beta Adrenergic Blockade in Asthmatics and Normals. Metabolic and Cardiovascular Studies

Some investigators tried to show that beta adrenergic blockade in normals or in atopic but non-asthmatic subjects could precipitate asthma. Jones (1972) found some normals treated with propranolol had a lability index of more than 20 percent, which was comparable to asthmatics. Several groups challenged patients with allergic rhinitis before and after propranolol with antigen (Ouellette and Reed, 1967) or methacholine : 19:

(McGeady et al., 1968). Measurements of airways obstruction showed a significant increase and some patients actually developed wheeze.

However, beta adrenergic blockade in normal subjects has very little effect and does not produce bronchoconstriction even after exercise (Zaid at al., 1968). Therefore, many workers studied metabolic or cardiovascular responses to beta adrenergic stimulants in asthmatics and normals to see if there was a difference in the two groups.

Cookson and Reed (1963) infused three doses of isoprenaline in normals and asthmatics. They claimed that the latter group had a significantly smaller fall in diastolic blood pressure, dicrotic wave (a measure of peripheral resistance) and rise in blood glucose. These workers were the first to mention the possibility that long-term treatment with beta adrenergic bronchodilators might influence beta adrenoceptor function. However, they dismissed this possibility as all their patients had discontinued sympathomimetic drugs for 18 hours. For the next 10 years treatment with these drugs was mentioned only to be discarded as a possible factor.

Various other authors (Lackey et al., 1967; Inoue, 1967; Middleton and Finke, 1968; Makino at al., 1970; Bernstein et al., 1972; Fireman at al., 1970) confirmed the significantly reduced rise in blood sugar in asthmatics after different regimes of intravenous or subcutaneous adrenaline. Several (Lackey at al., 1967; Fireman at al., 1970) examined the hyperglycaemic response to glucagon and showed that this was normal; the defect was specific to the beta2 adrenoceptor. Other workers (Lecks et al., 1968; Grieco et al., 1968; Kirkpatrick and Keller, 1967) using infusions of adrenaline or isoprenaline, could not show an impaired rise in blood glucose. And there was more dispute about other metabolic variables. Some claimed a difference in the rise of free fatty acids - an alpha adrenergic response, most did not; a few found a significant difference in the rise of pyruvate or lactate. Makino et al. (1970) could not confirm Cookson and Reed's findings on blood pressure, although they found a significantly reduced hyperglycaemic response.

Such metabolic studies are particularly difficult to control and to interpret because many of the variables being studied are inter-related. : 20

The subject's own hormonal controls may or may not be stimulated and could respond in an individual way. In addition different routes of administration, doses, and sampling times were used by different investigators. It is perhaps not surprising that there was a considerable amount of disagreement as to which beta adrenergic responses were abnormal in asthmatics. Maselli et al. (1970) compared asthmatics and normal subjects given propranolol. The response of blood glucose and lactic acid to an intravenous infusion of adrenaline was the same in both groups, thus parelleling Szentivanyi's work in mice.

Although not all the metabolic and cardiovascular studies were in agreement, the majority did favour the hypothesis of defective beta adrenergic function in asthmatics. Data showing a reduced eosinopenic response to adrenaline in these patients (Makin et al., 1970; Reed et al., 1970) also supported Szentivanyi's theory. Other groups (Bernstein et al., 1972; Schwartz and White, 1973) analysed cyclic AMP in urine; following subcutaneous adrenaline the rise was significantly reduced in asthmatics as compared to normal subjects.

(b) in vitro Studies

Studies in intact man can be difficult to interpret, therefore many investigators used isolated cells and tissues which have the advantage of eliminating circulating catecholamine and other hormonal and nervous influences. The biochemical abnormality could also be examined with more precision than estimating cyclic AMP in the urine over a two-hour period. Amongst the tissues used were human lymphocytes or mixed leucocytes, platelets and skin.

Carr et al. (1973) chose skin because it is easily obtained and frequently affected in atopic disease. In normal skin DNA synthesis is inhibited by - 1 -6 - 1 catecholamines between 10- TO moles. litre and lo moles. litre in a dose-related fashion. These workers found that skin from atopic subjects did not show the normal catecholamine inhibition of DNA synthesis and, therefore, postulated a defect of receptor function. Unfortunately, their data had a large standard error, were largely not tabulated and statistical significance was not mentioned. This model has not been pursued further. : 21 :

Platelet aggregation was first examined by Fishel and Zwemer (1970) who compared the pertussis-vaccinated mouse model to atopic subjects and normals. After ADP induced aggregation, beta adrenoceptor agonists will inhibit platelet disaggregation; incubation with propranolol increases disaggregation (Bucher and Stucki, 1969). Fishel and Zwemer (1970) showed that platelets from atopic patients and from Bordetella pertussis- immunized mice disaggregated more rapidly than normal as though a beta adrenergic blocker was present in the system. Solinger et al. (1972) examined platelet aggregation in the presence of adrenaline. Platelets from many of the patients with asthma or allergic rhinitis showed an abnormal response to beta adrenergic stimulation. McDonald et al. (1974) however, were unable to show consistent or significant differences of platelet aggregation between asthmatics and normals.

Platelets were also used to examine an alpha adrenergic function - ATPase activity. Coffey and Middleton (1975) noted a large variation but claimed a significant increase in ATPase activity in asthmatics not taking corticosteroids.

The skin and platelet studies appeared to support the concept of an underlying beta adrenergic defect and possibly increased alpha adrenergic activity in atopic subjects. There was, however, a considerable spread in the data, so much so that statistical significance was rarely shown. As in many of the metabolic studies, the possible contribution of long term treatment in the asthmatic was not examined.

The leucocyte or lymphocyte has been studied frequently and has the advantage of being relatively easily obtained regardless of the patient's clinical state. The number of cells can be readily quantitated thus avoiding some of the difficulties inherent in using akin biopsies.

Leucocytes have been shown to possess alpha and beta adrenoceptors (Coffey et al., 1972) and receptors to other mediators important in the allergic response such as histamine (Bourne et al., 1971), prostaglandins (Bourne and Melmon, 1971) and acetylcholine (Hadden, 1975), as well as to insulin and growth hormone (Gavin et al., 1972).

Mixed leucocyte preparations from allergic individuals release histamine 22:

(Lichtenstein and Margolis, 1968) and SRS-A (Grant and Lichtenstein, 1974) when challenged in vitro with the appropriate antigen. Histamine release can be prevented by beta adrenergic stimulants or other agents which raise intracellular cyclic AMP levels (Bourne et al., 1972).

The disadvantage of using mixed preparations is that the proportion of different cells may vary. For this reason, attempts have been made to isolate single cell types. It is possible to isolate a mononuclear cell preparation containing approximately 90 percent lymphocytes. The response of these cells to beta adrenergic stimulation is quantitatively greater than that of the neutrophil although the results obtained with both white cell preparations are broadly similar.

Smith and Parker (1970) first suggested measuring the cyclic AMP response of leucocytes as a convenient way of evaluating Szentivanyi's hypothesis. Mixed leucocytes from normal subjects, atopic individuals with hay fever and patients who had had a recent attack of severe asthma, were incubated 2 -1 with high concentrations of isoprenaline 10 moles. litre , prostaglandin E1 and hydrocortisone. The cells from asthmatic subjects showed a poor cyclic AMP response to all the drugs except PGE1.

A larger study by the same workers (Parker and Smith, 1973) showed that patients with active asthma had a significantly reduced leucocyte cyclic AMP response to isoprenaline compared to normal subjects (p 10.001). Asthmatics in remission occupied an intermediate position between these two groups. A similar pattern was seen with adrenaline which was also studied over a wide dose range. They postulated that beta adrenoceptor responsiveness varied with the activity of the disease at the time of study.

The possibility that bronchodilator therapy might affect cyclic AMP response was explored by Parker and Smith (1973). "Tedral" - a combination of ephedrine 24 mg, theophyllin 130 mg and phenobarbitone 8 mg - in a dose of four tablets a day, was taken by five normal volunteers for two weeks. No significant reduction in lymphocyte cyclic AMP production -2 -1 -1 with isoprenaline 10 moles. litre or adrenaline 106 moles. litre was found in cells obtained more than 12 hours after the last dose of bronchodilator. The amount of drug taken by these subjects was moderate, :23:

although it was known that asthmatics were prone to use much larger amounts during an acute attack (Speizer et al., 1968).

Parker and Smith (1973) also studied several patients with severe asthma after stopping beta adrenergic medication for two to seven days. The cyclic AMP response to beta adrenoceptor stimulation was still poor; they suggested, therefore, that it was not related to therapy. No other studies seriously tried to eliminate or evaluate the effect of beta adrenergic stimulants in vivo on leucocyte beta adrenoceptor responsiveness; the problem was discussed but was generally dismissed.

Using mixed leucocyte preparations, other investigators (Logsdon et al., 1972; Patel at al., 1974) showed a depression of beta adrenergic responsiveness in asthmatic subjects. However, Gillespie at al. (1974) were unable to confirm a statistically significant difference between cells incubated with concentrations of isoprenaline between 10 moles. -1 -2 litre and 10 moles. litre from normals and asthmatics.

Steroids were known to be an effective treatment and were thought to improve beta adrenoceptor responsiveness. Leucocytes from patients on steroids were shown to have a significantly increased response to isoprenaline compared to cells from patients not on these drugs (Logsdon et al., 1972). In another study, hydrocortisone intravenously or in vitro (10- 6 moles. litre-1).Improved cyclic AMP responsiveness to beta adrenergic stimulation (Parker et al., 1973).

Some workers studied alpha adrenergic responses in these cells to see if they were exaggerated in asthmatic patients. Coffey and Middleton (1975) showed that leucocyte ATPase activity, which is stimulated by alpha adrenergic agonists, was significantly greater in asthmatics not on corticosteroids as compared to that in normal subjects. The group of patients on steroids had greater enzyme activity than normals but not significantly. No other treatment was mentioned in this short report.

Alston et al. (1974) confirmed Parker and Smith's (1973) findings that leucocyte beta adrenoceptor response was poor in asthmatics during an exacerbation of their disease, and improved with the clinical state. Thymoxamine or phenotolamine, alpha adrenergic blocking drugs, partially :24:

restored beta adrenergic responsiveness towards normal levels. This group postulated that defective beta adrenergic response in asthmatics had given rise to increased alpha adrenergic activity and bronchial hyper- reactivity. In their paper they claimed bronchodilator treatment had no effect, but did not present any data to support this statement.

1.5 POSSIBLE TOLERANCE TO BETA ADRENERGIC AGENTS: THE INCREASE IN ASTHMA DEATHS

Between 1961 and 1967, the death rate from asthma rose three-fold in England and Wales. An excess of 3,500 deaths occurred over that predicted from previous mortality figures, mostly in the group aged between five and 34. This was not an artifact due to a change in either incidence of the disease or the classification (Speizer at al., 1968a).

The rise in mortality occurred at the same time as the introduction of convenient pressurized aerosols containing isoprenaline or other sympathomimetics for the treatment of asthma. Speizer et al. (1968) in a survey of a series of asthma deaths noted that 86 percent of the patients had used inhalers, many excessively. They also commented that 77 percent had received little or no corticosteroids during their terminal illness.

Several papers appeared subsequently supporting the indictment of pressurized aerosols. Inman and Adelstein (1969) noted that the curve for asthma mortality resembled the curve for aerosol sales; by then both were declining and aerosols were available only on prescription. However, mortality was falling faster than aerosol usage; an important factor here was medical awareness of the need for further treatment including steroids and hospitalization in an asthmatic attack. Fraser et al. (1971) investigated the treatment before death of a series of young people in 1968-69. They felt that excessive inhalation of bronchodilators might have accounted for about one-third of these deaths, which was approximately the excess mortality at that time.

Several reports of patients made worse by pressurized aerosols of beta adrenergic bronchodilators also appeared. Three patients studied on numerous occasions consistently showed an increase in airway resistance after using isoprenaline inhalations. They responded well, however, to sublingual isoprenaline and the author (Keighley, 1966) postulated that 25:

they might be experiencing a rebound swelling of the bronchial mucosa due to local application of drug such as that seen in rhinitis medicamentosa. Van Metre (1969) reported that 30 patients with severe refractory asthma improved only after stopping isoprenaline. On subsequent challenge with normal doses all but one of these patients had a good response to isoprenaline. Also, of his 17 patients who had died nine had used beta adrenergic bronchodilators excessively. The data was consistent with the hypothesis that large amounts of isoprenaline could induce or maintain intractable asthma though therapeutic doses were quite k/ko safe. Reisman (1970) reported 12 of 30 severe asthmatics had had no improvement in respiratory function tests with isoprenaline. Nine of these patients overused their aerosols and seven improved on discontinuing them.

A similar conclusion could be drawn from a paper by Paterson et al. in 1971. They compared salbutamol and isoprenaline by constructing cumulative dose-response curves with graded infusions of both drugs. After discontinuing the isoprenaline infusion four out of 15 patients showed a serious deterioration in their asthmatic state necessitating active intervention. The authors postulated that the drug might have produced resistance in beta adrenoceptors which reduced the patients normal response to endogenous catecholamines. Rebound bronchoconstriction occurred in only a few patients and was not seen in any in a subsequent study (McEvoy et al., 1973); however at least some of the patients were on steroids. Other workers (Marlin and Turner, 1975) have also found some rebound bronchoconstriction after beta adrenergic agonists in asthmatics on various treatment regimes.

Not all workers agreed with the hypothesis that mortality was directly linked with the use of pressurized aerosols. Although a decline in mortality had been seen in Australia after a warning about pressurized aerosols in 1964 (McManis, 1964), this was not as clear a trend in other countries (Herxheimer, 1972, a and b). Another argument is that patients and doctors had become too complacent about asthma with the availability of aerosol bronchodilators. On several, occasions Herxheimer (1968, 1972a, 1972b) pointed out that tolerance to isoprenaline developed in severe asthmatic attacks. The patient who took increasing amounts of the drug obtained less and less relief and was thus basically without effective :26 :

treatment - in particular corticosteroids which he needed. The decline in asthma mortality was almost certainly due, at least in part, to an appreciation of this. When more patients were treated aggressively with steroids and admitted to hospital, the death rate declined.

The circumstantial evidence against beta adrenergic aerosols outlined above led to a considerable amount of research on how their use and abuse could cause death. Greenberg and Pines (1967) suggested that excessive beta adrenergic stimulation could cause arrhythmias in hypoxic patients, but autopsies had shown that patients died with the pathological features of asthma (Speizer at al., 1968). Furthermore, the greatest increase in mortality was in the young who are least likely to have premature ventricular ectopic beats and in whom the myocardium would be anticipated to have most resistance to arrhythmias.

The fluorocarbon aerosol propellants came under suspicion for two reasons. Firstly, they are contained in glue, and several glue sniffers died at around the same time (Bass, 1970). Secondly, fluorocarbons share with chloroform the ability to provoke halothane- induced arrhythmias. However, studies by Dollery et al. (1970) showed that doses high enough to sensitize the heart to arrhythmias required the subject to use his aerosol for every breath for between two and three minutes.

Another suggested mechanism was the production of an active metabolite of isoprenaline which is a beta adrenergic blocking agent (Paterson et al., 1968). However, this is a weak beta antagonist and only a small amount is formed from the inhaled dose (Blackwell et al., 1970). Orciprenaline, another beta adrenergic bronchodilator incriminated in the increased asthma deaths (Inman and Adelstein, 1969), does not have a comparable metabolite.

1.6 EVIDENCE OF TOLERANCE TO BETA ADRENERGIC STIMULANTS IN ANIMALS AND MAN

Tolerance to these medications has been shown in several systems. As early as 1946 Herxheimer demonstrated that use of ephedrine for three to four days could produce tachyphylaxis to its bronchodilating effect in some patients. This drug acts partially by releasing noradrenaline so :27:

tachyphylaxis may be due to depletion of stores (Goodman and Gilman, 1975). The general phenomenon was investigated more extensively after a link was postulated between the abuse of adrenergic aerosols and increased mortality from asthma. Cardiovascular responses, bronchodilation and other beta adrenergic effects such as tremor, have been studied. Workers investigated models of asthma such as sensitized guinea pig lungs and histamine mortality in this species to examine the effect of excessive beta adrenergic drugs.

In 1968 Paterson et al. showed that five chronic bronchitics taking over 20 puffs (8.8 mg) of isoprenaline from a Medihaler-Iso-Forte pressurized aerosol per day, showed no increase in heart rate when inhaling therapeutic doses. Normal subjects developed a tachycardia after one (0.44 mg) to three puffs (1.32 mg), while these patients required eight puffs (3.52 mg) or more; they also required a larger intravenous dose of isoprenaline to produce tachycardia.

Conolly et al. (1971) demonstrated the development of isoprenaline resistance after low dose infusions of isoprenaline lasting 15-45 minutes. Similar results were obtained in dogs; both tachycardia and fall in diastolic blood pressure were significantly reduced showing an effect on both beta and beta adrenoceptors by pretreatment in these animals. In 1 2 addition, they showed that a cross tolerance could be produced using other sympathomimetic agents, terbutaline and isoetharine. Mortality from histamine-induced bronchospasm in guinea pigs which had been pretreated with beta adrenoceptor stimulants was also studied. Intramuscular injections of isoprenaline, salbutamol or terbutaline were given at 25-40 minute intervals over a five-hour period. These animals and a saline-injected control group were challenged two hours later with a standard dose of histamine. The treated animals had a significantly higher mortality than their controls. In their report of these experiments they proposed that tachyphylaxis to beta adrenergic stimulants might result from excessive use of beta adrenoceptor bronchodilators. This might have been important in the increased asthma mortality of the 1960s.

Bouhuys et al. (1972) confirmed the guinea pig mortality experiments with isoprenaline and also looked at an in vitro preparation of tracheal strip : 28 :

from the same species. In the latter, pre-incubation with isoprenaline initially prevented histamine contraction; however, after one hour, histamine susceptibility returned but not the protective effect of isoprenaline. The tissue would not relax unless a much larger dose of isoprenaline was used.

Izard et al. (1971) sensitized guinea pigs, daily injected one group with adrenaline and the control group with saline for three weeks and then challenged the animals with antigen. When adrenaline was given with the antigen challenge it failed to protect the previously treated animals; and there was a significant increase in mortality. Again this suggested that tolerance to a beta adrenoceptor stimulant had been produced by prior exposure to the drug.

More recently, isolated guinea pig lung has been studied to determine bronchoconstrictor responses in a similar situation. Benoy et al. (1975) pretreated normal animals with isoprenaline three times daily in doses calculated to approximate that which an asthmatic might take during an attack. The isolated lungs were then studied to determine the effect of adrenaline on histamine-induced bronchoconstriction. Lungs from pretreated animals did not respond to adrenaline as well as control lungs and this was related to both the length of the pretreatment and to the dote used. Cross tachyphylaxis between adrenaline, isoprenaline, terbutaline and aminophylline was demonstrated.

The intact animal and isolated organ studies supported the concept that tolerance to beta adrenoceptor stimulants could develop in patients on these drugs. Nelson et al. (1975) examined the cardiovascular and metabolic responses of seven normal men to a four-hour infusion of adrenaline 7.3 pg per minute before and after a one week course of ephedrine sulphate (100-150 mg per day). Two control infusions at 24-hour intervals gave statistically similar results before the treatment period. After ephedrine for one week the response to infused adrenaline was reduced. The rise in blood glucose and in pulse rate was significantly lower, mean blood pressure rose rather than fell, and the fall in eosinophils and in urinary cyclic AMP was less after the treatment period, though not significantly. The results were similar to previous studies on asthmatics and on normals treated with beta :29 :

adrenergic antagonists. During the second post-treatment infusion (approximately 38 hours after the last dose of ephedrine) two of these parameters - pulse and blood pressure - appeared to be returning to normal.

These workers pointed out the importance of their findings in relation to previous studies supporting Szentivanyi's theory of partial beta adrenergic blockade in asthmatics: most of the patients previously studied had been on adrenergic bronchodilators. Nelson had shown that these drugs effect beta adrenergic responsiveness of metabolic and cardiovascular parameters in normal non-asthmatic subjects.

Fireman et al. (1970) had examined the hyperglycaemic response to subcutaneous adrenaline before and after a 30-day course of ephedrine but had not shown any depression of response following medication. His protocol, however, was quite different with a shorter sampling period, different route of adrenaline administration and discontinuation of drug up to 24 hours prior to the repeat study.

A study (Jenne et al., 1977) with terbutaline 15 mg daily for three weeks in nine asthmatics and 10 bronchitics, who had not had medication for the previous two weeks, showed the development of significant tachyphylaxis in measurements of blood pressure, rise in glucose, plasma cyclic AMP, serum lactate, and fall in eosinophils in response to a 5 mg challenge dose of the drug in both groups. A significant reduction in the rise of FEY, following terbutaline was also found in these subjects. Nelson et al. (1977) have published an abstract claiming there is reduction in bronchodilator response to albuterol after a few days of regular therapy.

The possibility of desensitization to the effect of adrenergic bronchodilators in the airways is particularly crucial to the asthmatic. Most studies on the long-term effect of these drugs have not demonstrated the development of such tolerance. In one study oral salbutamol (4 mg four times daily) maintained its effectiveness for four weeks as measured by peak expiratory flow rate (Parker at al., 1971); however, these patients were on corticosteroids. Metaproterenol (orciprenaline) maintained its effect on FEV for two to three months (Hurst, 1973; Sackner, at al., 1976) 1 and ephedrine (75 mg daily) or terbutaline (15 mg dailyr for up to one :30 :

year (Wilson et al., 1976). Szedmyr et al. (1976) treated patients with terbutaline (5 mg three times daily) and reported that bronchodilation following isoprenaline was maintained though tremor, another beta2 effect, was reduced after chronic treatment.

In contrast, Holgate et al. (1977) using a more sensitive test of bronchodildtation, change in specific conductance, in normal subjects inhaling increasing doses of salbutamol (up to koo pg four times daily) over a four week period showed a progressive reduction in response to this drug which could be restored to normal by intravenous hydrocortisone.

One group has published results in abstract form suggesting that bronchodilator treatment in vivo may change adrenoceptor responsiveness of leucocytes in vitro. Morris et al. (1974) gave subcutaneous injections of adrenaline (5 pg/kg) to 10 normal subjects, isolated leucocytes at times up to two hours later, re-exposed the cells to the same beta adrenergic stimulant in vitro and assayed cyclic AMP. The responsiveness of the cells declined progressively over the two-hour period. Another abstract (Morris et al., 1976) states that ephedrine, terbutaline and carbuterol taken by asthmatics over several weeks or days reduces leucocyte cyclic AMP response to adrenaline in vitro.

There is a suggestion from the work of Holgate et al. (1977) and that of Jenne et al. (1977) and Nelson et al. (1977) that tolerance to beta adrenergic stimulants may develop in the airways of asthmatics, though this is still uncertain. Various data presented above show that tolerance to the metabolic and cardiovascular effects of these drugs probably does occur. Two preliminary in vitro studies have shown an effect of treatment, though earlier work (Parker and Smith, 1973) did not.

1.7 IN VITRO PRODUCTION OF TOLERANCE TO BETA ADRENOCEPTOR AGONISTS Numerous workers have produced and studied desensitization to beta adrenoceptor agonists in vitro. It was well known that continued exposure to these agents for an hour or more resulted in an initial rise and then slow fall in the amount of cyclic AMP produced. Investigators have exposed isolated cells or tissues to repeated catecholamine : 31

stimulation either adding more drug cumulatively to the incubation media or washing the cells before re-exposure to drug. A diminished response to beta adrenergic stimulation can be found in either of these circumstances and considerable interest has centred on the mechanism(s) involved.

Increased activity of PDE, causing increased destruction of cyclic AMP, has been postulated as one possibility. This has been shown in brain slices (Kakiuchi and Rall, 1968; Schultz and Daly, 1973) and C6 astrocytoma cells (Browning et al., 1974; 1976) following prolonged exposure to beta adrenergic stimulants. However, increased activity of this enzyme has not been found in all of the cells and tissues studied (DeRubertis and Craven, 1976; Franklin and Foster, 1973; DeVellis and Brooker, 1974; Smith et al., 1976).

Adipose tissue and isolated adipocytes have the advantage that both cyclic AMP production and glycerol can be measured after catecholamine stimulation, i.e., both the level of second messenger and the effect. Ho and Sutherland (1971) found an antagonist of cyclic AMP production in the incubation media; adipocytes which had not previously been exposed to adrenaline but were incubated in media that previously contained cells plus drug, were resistant to the action of the hormone. The cells did not respond to ACTH or glucagon, hormones acting through other receptors to raised cyclic AMP levels and increase lipolysis. Thus, in the fat cell, tolerance to a wide range of hormones stimulating cyclic AMP could be induced by exposure to one of these.

Manganiello et al.(1971) postulated that a stimulant of PDE might be produced although they were unable to show this. Schimmel (1974) used intact tissue as well as adipocytes. He was unable to show an antagonist in the media from the tissue, as opposed to that from the cells, but the experiments were short term and diffusion may have a limiting factor.

Explants of fat have been cultured by Smith et al. (1976) with noradrenaline for 12-48 hours, carefully washed and re-exposed to beta adrenoceptor agonist for two hours. Cells previously exposed to hormone did not respond with increased cyclic AMP, and showed reduced lipolysis. Incubation of fresh cells in the culture media which had contained : 32 : noradrenaline blunted their subsequent response to this drug; so these workers, like Ho and Sutherland (1971), postulated that an antagonist had been formed during the initial culture period. Addition of a phosphodiesterase inhibitor partly restored the lipolytic response but assay of phosphodiesterase activity showed no increase. Neither ACTH o_Ori glucagon were used in these studies, so there is no information as to the specificity of the observed desensitization. Fat cells seem to be unusual in producing an antagonist which is present in the incubation media. As yet there are no data to indicate what this antagonist is or how it works. If the non-specific desensitization shown by Ho and Sutherland (1971) is confirmed this is also unusual.

Cultured fibroblasts have also been studied to demonstrate tachyphylaxis. Franklin and Foster (1973) produced desensitization to isoprenaline or PGE by pre-incubation with the appropriate hormone for up to two and one 1 half hours. This desensitization was specific, pre-incubation with isoprenaline had no effect on response to PGE1 and the pre-incubation media did not contain an antagonist to subsequent cyclic AMP production. PDE activity was examined in one experiment but no increase was observed. In contrast to this, however, an earlier paper (Manganiello and Vaughn, 1972) had shown increased phosphodiesterase activity in fibroblasts exposed to PGE for 24 hours and rendered resistant to subsequent incubation with 1 this agent. Pre-incubation of human fibroblasts with salbutamol and other beta adrenergic agonists also produced desensitization to subsequent incubation with isoprenaline (Franklin et al., 1975). Thus cross- tachyphylaxis occurs between the different stimulants of the same receptor. Recovery required approximately 24 hours and was prevented by inhibitors of protein synthesis suggesting that new receptors had to be produced.

Several studies have suggested that desensitization depends on the dose of drug. Remold-O'Donnell(1974) incubated monolayers of guinea pig peritoneal macrophages for two hours with either adrenaline or PGE2 and showed a poor response of adenylate cyclase in cell homofgenates stimulated with the same hormone. The degree of desensitization to PGE2 was dependant on the dose used in the two-hour incubation. Makman (1971) incubated small lymphocytes recovered from rat or mouse lymphoid tissue with different concentrations (8 x 107 moles. litre-1 to 2 x 105 moles. litre-1) of adrenaline for three hours. The adenylate cyclase activity • 33 •

of homogenates from these cells was reduced on repeated exposure to adrenaline in a dose-dependant fashion. Again this was specific as the response to fluoride ion was unchanged. He noted several sources of variation: thymocytes had more activity than lymphocytes from splenic tissues, adenylate cyclase activity was higher in cells of fetal and newborn mice than in older animals, and different incubation media affected adenylate cyclase activity.

Peripheral blood cells have been studied infrequently in vitro despite their ready availability. The frog erythrocyte beta adrenoceptor can be desensitized by pre-incubation with isoprenaline 10 moles. litre 1 (Mickey et al., 1975) for between 10 and 24 hours. Adenylate cyclase activity in the membrane fraction is significantly less responsive to subsequent isoprenaline, but cyclic AMP response to PGE1 or fluoride ion remains undiminished.

These authors have also developed a technique for ennumerating receptors using radioactive alprenolol, a beta adrenergic blocking drug. After pretreatment the desensitized cells show fewer alprenolol binding sites, which is taken to indicate fewer beta adrenoceptors. Mukherjee et al. (1975) have reproduced this effect in vivo by injecting frogs every six hours with noradrenaline or isoprenaline over a 24 hour period. The red cell membrane preparations from these animals bind less alprenolol and are postulated to have a reduced number of beta adrenoceptors. Human red cells, however, possess no adrenoceptors (Williams et al., 1976) and therefore, cannot be used for such studies.

Kalisker and Middleton (1975) have reported in an abstract that monolayers -1 of human lymphocytes exposed to isoprenaline 105 moles. litre for one hour respond poorly to re-stimulation with the same dose of drug, but normally to PGE1. Thus there is specific desensitization of beta adrenoceptors by the beta adrenergic agonist. They also pre-incubated lymphocytes with PGE1 for up to two and a half hours but this did not produce any change in cyclic AMP response to subsequent incubation with either isoprenaline or PGE1. As with many of the in vitro studies, no figures on statistical significance are mentioned.

Investigators of the desensitization phenomenon, which has been shown after : 34 :

as little as 10 minutes exposure to drug in some systems, have discussed and excluded receptor occupancy as the cause. Basal unstimulated activity of adenylate cyclase is not affected by pre-incubation with beta adrenergic stimulants as would be expected if isoprenaline remained at the receptor. Cells are washed thoroughly as Franklin et al. (1975) have shown that picomolar amounts of isoprenaline can delay the recovery of beta adrenergic responsiveness in human fibroblasts. In the frog erythrocyte binding studies isoprenaline added to the blood in vitro immediately prior to preparation of membranes for the adenylate cyclase assay did not affect the number of receptors found; the isoprenaline was effectively washed from these preparations. Most importantly, remaining beta adrenoceptor agonists should act competitively with the beta adrenergic blocking agent alprenolol. Binding curves would then shift to the right but show the same maximal effect at a higher concentration of agonist. However, this has not been found - maximal binding is lower in desensitized cells no matter how high the ligand concentration. Adenylate cyclase assays show the same phenomena; the diminished response in desensitized cells is not overcome by increasing the concentration of the beta adrenergic agonist.

The possibility that reduced ATP is important has been examined indirectly by some investigators. Increasing adenosine does not improve the response of desensitized cells (Schultz et al., 1972), nor does the addition of glucose to the media (Manganiello et al., 1971) which is supposed to prevent intracellular ATP depletion.

The cause of beta adrenoceptor desensitization remains to be determined. Loss of receptors seems probable and this possibility is being investigated by several workers. PDE activity may be increased but this is certainly not the only mechanism as inclusion of an inhibitor of this enzyme does not restore beta adrenoceptor responsiveness to normal.

1.8 POSSIBLE PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPORTANCE OF TOLERANCE

The normal physiological importance of desensitization to beta adrenoceptor stimulation was discussed by Romero and Axelrod (1974). Pineal gland cells have a diurnal variation in the amount of N-acetyl transferase - an enzyme whose concentration is controlled by beta : 35 :

adrenergic stimulation and this beta adrenoceptor is more sensitive in the presence of low levels of neurotransmitter (Deguchi and Axelrod, 1973). Glands removed early in the dark period, when noradrenaline traffic is low, and enzyme activity high, show a greater response to isoprenaline in vitro than glands removed at the end of the dark period when the opposite conditions obtain. These workers (Romero et al., 1975) also demonstrated a change of alprenolol binding under the same conditions as reduced responsiveness to isoprenaline. Thus the pineal cell has been shown to change its responsiveness because of a change in endogenous beta adrenoceptor stimulation. The change in receptor sensitivity - possibly increase in adrenoceptor numbers - amplifies the effect of the neurotransmitter greatly; a small increase in concentration of noradrenaline resulted in a forty-fold increase in enzyme activity.

Endogenous catecholamine activity could be important in regulating other beta adrenoceptors such as those on leucocytes, bronchial smooth muscle and mast cells. Likewise treatment with beta adrenergic stimulants such as isoprenaline or salbutamol could affect receptor responsiveness.

It is possible that a degree of tachyphylaxis to beta adrenergic bronchodilators might develop in the respiratory system of some asthmatic patients because of receptor desensitization. This phenomenon is probably not common, as these medications continue to benefit most patients. However, as the dose of any drug increases so does the risk of this effect.

The use of corticosteroids seems to restore or maintain beta adrenergic function in a clinical setting (Ellul-Micallef and Fenech, 1975) and patients with severe asthma are now almost invariably on steroids as well as on adrenergic bronchodilators possibly in large dose.

However, this was not the case in the early 1960s when there was an increase in mortality from asthma. At that time patients took excessive amounts of beta adrenergic bronchodilators and on a much greater scale, and doctors were unaware that steroids were needed when the former drags produced no response. Receptor desensitization due to drugs and possibly also to increased endogenous catecholamines may have contributed to the clinical tolerance that was observed. : 36:

1.9 PURPOSE OF THE PRESENT STUDY

As beta adrenergic stimulants are widely used long term in the treatment of asthma their effects in this situation should be studied and understood, even more so because of the possibility that these drugs may have contributed to the increase in asthma deaths in the 1960s. An understanding of their effects is also important in evaluating the data supporting Szentivanyi's hypothesis of an underlying partial beta adrenergic blockade in bronchial asthma.

The present thesis describes a series of experiments designed to investigate the effect of beta adrenergic medication, in normal or excessive doses, on beta adrenoceptor responsiveness in normal subjects and asthmatics. Peripheral blood mononuclear cells have been studied, as they have the advantage of being easily and repeatedly obtainable in relatively pure suspension even from a seriously-ill patient, criteria which lung tissue does not satisfy. Basophils would be the most pertinent white blood cell to study in the asthmatic as they are similar to the tissue mast cell, but it is virtually impossible to isolate them in pure suspension. Burnet (1975) believes that the basophil and the mast cell are derived from lymphocytes, and Iskizaka et al. (1976) have shown that mast cells develop from rat thymus in culture conditions. The possibility that lymphocytes and mast cells may be closely related gives some justification for the use of these cells in studies on asthmatic subjects. Nevertheless, an essential preliminary to this thesis was to establish a degree of identity between the beta adrenoceptors of the lymphocyte and of the lung.

The general methods and materials used throughout the work presented in this thesis are described in Chapter II. Chapter III reports experiments on the similarity between the lung and lymphocyte beta adrenoceptor, and the pattern and variability of response to beta adrenoceptor stimulation in mononuclear cells. Results from cells isolated from asthmatic subjects on various regimens of beta adrenergic bronchodjiAtors are presented in Chapter IV, and the study of mononuclear cells from normal subjects given similar drugs is described in Chapter V. Finally, lymphocytes were cultured with beta adrenergic stimulants in vitro to see if significant desensitization developed in this situation (Chapter VI). The conclusions and their implications are discussed in the last chapter. 37

CHAFMR II

GENERAL MATERIALS AND METHODS 38 :

2.1 INTRODUCTION

In all the experiments throughout this thesis the changes in the levels of cyclic 3' 5' adenosine monophosphate (cyclic AMP) have been used as an index of beta adrenoceptor response. This compound was measured in purified extracts of mononuclear cells and lung tissue by a modification of Gilman's (1970) technique.

Isolation of the mononuclear cells, purification of cyclic AMP, preparation of the binding protein, and conduct of the assay are described in separate sections of this chapter. Methods relevant to only one portion of the thesis and subjects for the experiments are described in the appropriate chapters.

2.2 MATERIALS, DRUGS AND CHEMICALS

The following materials were used in the steps described in this chapter:-

2.2.1 Preparation of Assay Binding Protein

Cyanogen bromide (finely divided) Koch Light Protamine Boehringer Ingelheim Sepharose 6B Pharmacia Fine Chemicals AB, Upsala, Sweden EDTA BDH Limited, Poole, Dorset Unicam SP 500 spectrophotometer Bovine serum albumen Sigma Chemical Co. Ltd., Kingston-upon-Thames

2.2.2 Cell Separation

Hanks balanced salt solution (HBSS) Burroughs Wellcome Ltd. Lyrnphoprep Nyegaard & Co., Oslo, Norway Ficoll Pharmacia Fine Chemicals AB, Upsala, Sweden Sodium diatrizoate (hypaque sodium) Winthrop Laboratories, Surbiton Fetal calf serum Burroughs Wellcome Ltd.

:39:

2.2.3 Incubation of Lymphocytes

Trizma base Sigma Chemical Co. Ltd., Kingston-upon-Thames Theophylline BDH Limited, Poole, Dorset Isoprenaline sulphate Sigma Chemical Co. Ltd. Kingston-upon-Thames Ascorbic acid BDH Limited, Poole, Dorset

2.2.4 Purification of Cyclic AMP

Formic acid (analar grade) BDH Limited, Poole, Dorset Dowex AG1 x 8 formate form 200 to 400 mesh Bio-rad Laboratories, Richmond, California, U.S.A.

2.2.5 Cyclic AMP Assay

3H cyclic AMP New England Nuclear Corporation, Frankfurt, West Germany Unlabelled cyclic AMP Sigma Chemical Co. Ltd., Kingston-upon-Thames Cellulose filters, 0.45 p pore size HAWP 02400 Millipore, London 2-ethoxyethanol BDH Limited, Poole, Dorset Instagel Packard Instrument Co. Inc., Downers Grove, Ill., U.S.A. Packard 3375 spectrometer Phosphodiesterase Boehringer Mannheim, London

2.2.6. Cell Strains

Giemsa (modified formula) Raymond A. Lamb, 6 Sunbeam Road, London, N.W.10

2.3 PREPARATION OF CHROMATOGRAPHY MATERIALS

2.3.1 Dowex AG1 x 8 Resin The Dowex resin was prepared initially and recycled after each use. One pound of resin was equilibrated with distilled water, placed in a large column and washed with 3 litres of 0.5N NaOH, 3 litres of distilled water, 4 litres of 2N HCl, 4 litres of distilled water, 2 litres of 2N formic acid and a further 4 litres of distilled water. By this procedure the resin was uniformly freed of residual cyclic AMP and the :40 :

elution pattern for the nucleotides remained constant.

2.3.2 Protamine - Sepharose Affinity Chromatography Column

Five hundred millilitres of Sepharose 6B was first washed with water and filtered in a Buchner funnel. It was transferred to a large beaker, 500 ml water added and then 50 g of finely divided cyanogen bromide. During this reaction the pH was kept at 11 by adding 5N NaOH and the temperature at 20°C by adding ice. The reaction was completed when the pH became constant; excess ice was then added. The mixture was washed with 10-20 volumes of ice-cold 0.1M NaHCO (pH 9.0) under suction in a 3 Buchner funnel and resuspended in NaHCO pH 9.0. Protamine 2.0 g was 3' added and the resulting resin stirred overnight in a cold room. Sodium azide (1:500) was added to stop fungal growth. The resin was stored at 4°C and, after washing, re-used over a three-year period for the preparation of several batches of binding protein. A comparable yield was obtained on each occasion.

2.4 PREPARATION OF BINDING PROTEIN

A protein binding assay modified from that of Gilman (1970) was chosen for these studies in preference to methods which depend on the conversion of tritiated adenosine to 3H cyclic AMP as the latter methods give no data on basal levels of cyclic AMP.

The binding protein was extracted from rabbit skeletal muscle and prepared by affinity chromatography (Cuatrecasas et al., 1968). The animal was killed by injection of air, the skeletal muscle rapidly excised, chopped into 1 cm cubes and placed on ice. It was put through a 2 -1 meat grinder and then homogenized in 4 x 10 moles. litre potassium -1 phosphate buffer with 4 x 103 moles. litre EDTA at pH 6.5. All further steps were done at 4°C. The homogenized material was centrifuged at 10,000 RPM for 30 minutes and the supernatant applied to the column of protamine sepharose which had been previously washed and 2 -1 equilibrated with 2 x 10 moles. litre potassium phosphate buffer with -1 2 x 103 moles. litre EDTA at pH 6.5. The column was initially washed with 8 litres of buffer at a flow rate of 6 ml/minute until the absorbance at 280 nm was less than 0.06. The binding protein was then eluted with a 0-2 molar gradient of NaC1 in potassium phosphate buffer at : 41

a flow rate of approximately 3 ml/minute. Twenty millilitre fractions were collected and both optical density at 280 nm and capacity for binding tritiated cyclic AMP (Figure 1) were measured. The fractions with the highest binding capacity were pooled and bovine serum albumen (1 mg/ml final concentration) was added. This material was dialysed against 14 litres of 5 x 10-3 moles. litre-1 potassium phosphate buffer 1 with 2 x 103 moles. litre EDTA at pH 4 overnight to remove the salt, centrifuged and then frozen at -20°C in 1 ml aliquots. Under these conditions it showed no loss of cyclic AMP binding capacity for over two years.

Unlike the enzyme described by Gilman, this binding protein, was very pH sensitive in its ability to bind cyclic AMP (Figure 2). Therefore, the pH at which binding of cyclic AMP reached a plateau was determined for each batch of binding protein and assays were done in a phosphate- citrate buffer adjusted to the appropriate pH.

Two other characteristics of each batch of binding protein were determined prior to assay. Firstly, as albumein enhances cyclic AMP binding by an unknown mechanism, the most favourable ratio of binding protein to added bovine serum albumen was established. Secondly, the amount of this mixture able to bind about one-third of the labelled cyclic AMP in the absence of unlabelled nucleotide was determined. This quantity of binding protein and albumen was freshly prepared immediately prior to each assay so that a 50 )1l aliquot contained the requisite amount for each assay tube.

2.5 CELL SEPARATION AND VIABILITY

Lymphocytes were prepared from heparinized venous blood by a modification (Harris and Ukaejiofo, 1970) of Boyum's Technique (1968). All centrifuging was done in a MSE Mistral 4L refrigerated centrifuge at 15°C as the response of the beta adrenoceptor in cells prepared at uncontrolled temperatures was poor. Sixty millilitres of blood was centrifuged at 250 g for 20 minutes and the platelet-rich plasma discarded. This was replaced with an equivalent volume of HBSS with 10% fetal calf serum previously gassed with 95% 02/5% CO2 and adjusted to pH 7.4. Fetal calf serum was used to prevent cell clumping and loss due to increased adhesiveness. Twelve millilitre portions of this diluted

Figure 1 The isolation of protein kinase from rabbit skeletal muscle

Absorbance at 280 nm counts per minute 1.5 — - 7000

- 6000

1.0 -

- 4000

O. 5 - Cyclic AMP Binding - 2000

Absorbance

10 20 30 40 50 60 70 80 90 100

Tube Number • k3•

Figure 2 Influence of pH on cyclic AMP binding

of protein kinase from rabbit skeletal muscle

• •

•

0 I • 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

pH : 44 :

"blood" were layed onto 10 ml of "Lymphoprep" or a mixture of sodium diatrizoate (Hypaque) 240 grams per litre and ficoll 90 grams per litre of specific gravity 1.077. This was centrifuged for 25 minutes to provide an estimated 400 g at the interface. Mononuclear cells accumulated just below the interface between the two liquids. They were harvested, diluted with HBSS, recentrifuged at 1000 g for 20 minutes to remove residual lymphoprep and the washing fluid discarded. The cell pellet was resuspended in a small amount of HMS by gentle shaking.

The cells were counted in a Coulter cell counter and diluted with further HBSS plus fetal calf serum and the buffer TRIS HCI at pH 7.35 (5 x 103 moles. litre-1 final concentration) to produce a final suspension of 4 million cells/ml.

The isolation procedure took approximately three hours and was standardized. Nevertheless, cell yields varied from the expected yield of mononuclear cells which was assumed to be 35% of the total white cell count. Differential white cell counts performed on some samples did not explain or improve this variation in mononuclear cell yields.

A smear was prepared from each final cell suspension and stained with Giemsa (modified formula) 1% stain for 30 seconds. Examination of these smears showed that 80-95%, usually 790%, of the white blood cells were lymphocytes, the majority of the remainder were monocytes with only occasional granulocytes. Red cell contamination varied but was usually less than 1-2 RBC/lymphocyte. Trypan blue exclusion tests on the final cell suspensions showed greater than 95% viability.

2.6 INCUBATION

Duplicate and triplicate samples of cells were incubated at 37°C with - 2 -1 theophylline 10 moles. litre dissolved in HESS, and isoprenaline 4 0-10 moles. free base litre-1. Ascorbic acid (1 mg/m3) was used as an antioxidant in making up all of the isoprenaline solutions. This concentration of ascorbic acid had no effect on the level of cyclic AMP 6 produced by 4 x 10 lymphocytes in the presence of theophylline. The total incubation volume was 2.1 ml for 4 million cells, or, if the cell yield was poor, 1.05 ml for 2 million cells. Under these conditions cyclic AMP production reached a plateau after 10 to 15 minutes. All incubations

: 45:

were terminated after 15 minutes by placing the tubes in boiling water for 3 five minutes. Then 0.18 picomoles H cyclic AMP (5000 cpm) was added to permit the estimation of recovery after the subsequent purification.

2.7 CYCLIC AMP PURIFICATION

The cell suspensions were homogenized, centrifuged and the supernatant applied to a 2.0 x 0.5 cm column of Dowex AG1 x 8 (formate form). Ten millilitres of distilled water was applied to the column to elute adenosine, adenosine triphosphate and other nucleotides, and then discarded. The cyclic AMP was then eluted with 12 ml of 2N formic acid and collected. Cyclic GMP remained on the column but could be eluted with 4N formic acid (Figure 3)-

After ion exchange chromatography the eluate was lyophillized and the residue dissolved in 1 ml or 2 ml phosphate-citrate buffer (pH 5.5 - 6.5 depending on the enzyme used in the subsequent assay). Recovery was estimated by counting a 50 pl aliquot of this material. Further 50 pl aliquots were used in the cyclic AMP assay described below.

2.8 ASSAY OF CYCLIC AMP

All saturation binding assays depend on the competition between the unlabelled compound in the experimental sample and a known amount of its labelled analogue for a limited number of binding sites. Labelled cyclic AMP (0.5 picomoles, 14000 cpm in 10 pl of water) was added to each assay tube except the first and last which received only 10 pl of water to act as background samples, For the calibration curve known amounts of unlabelled cyclic AMP or the lyophyl/ized samples dissolved in phosphate- citrate buffer at the appropriate pH were then added to the tubes in a 50 pl volume. The binding protein - album6 mixture (50 pl) was added and the material thoroughly mixed (vortex mixer). It was then allowed to equilibrate for three hours at 0°C.

Each duplicate sample was passed through a Millipore filter to adsorb the protein and the,cyclic AMP bound to it. The unbound cyclic AMP was -2 -1 rinsed through h10 ml of ice cold 2 x 10 moles. litre phosphate buffer (pH 6.0). The filter was dissolved in 1 ml 2-ethoxyethanol in a scintillation vial, and, after the addition of 10 ml Instagel, the Figure 3 Elution pattern of 0owex AG 1 x 3

Separation of cyclic AMP and cyclic GMP

3.16 .> 6* C1 4.05

2.0

.3

cyclic GMP cyclic AMP

•1CCO

ash 300

300 0.4

0.2 - 100 .

2N formic acid 4N formic acid 4 12 20 23 Millilitres eluted samples were counted. As counting efficiency was very similar for all samples, calculations were based on counts per minute without correction for quenching.

The sensitivity of the assay permitted measurements of cyclic AMP levels as low as 0.1 picomoles per assay tube. Using known amounts of unlabelled cyclic AMP, the assay was found to be accurate to within + 10%. Other nucleotides were tested to see if they interferred with binding of cyclic AMP to the protein. Adenosine triphosphate and adenosine diphosphate were without effect. Cyclic GNP in concentrations 100 times higher than cyclic AMP interferred with binding (Figure 4). However, levels of cyclic GMP in mammalian cells are considerably lower than cyclic AMP (Goldberg et al., 1969) and in any case the anion-exchange chromatography step separated these two cyclic nucleotides. The media and drugs used in the cell separation and incubation were also tested and showed no interference with the binding assay.

Some previously assayed samples were treated with beef heart PDE and assayed again. A 500 pl volume of cell homogenate or cyclic AMP was - 1 - 1 incubated with 1000 pl 10 moles. litre TRIS HCl (pH 8.5), 200 pl of 4 ) SO 4 x 2 x 10 moles. litre-1 (NH4 2 4' 200 p1 of 105 moles. litre I Mg SO4, and 100 pl beef heart PDE in water for three hours at 370C. The reaction was terminated by placing the tubes in boiling water for two 3 minutes. Recovery H cyclic AMP was added, the sample purified by anion exchange chromatography and assayed as usual. After treatment with PDE there was no assayable cyclic AMP.

In summary, the assay as described was accurate and specific for cyclic AMP; under the conditions of the experiments there were no interfering substances.

2.9 CALCULATION OF RESULTS

Estimation of cyclic AMP content of the unknown samples was determined according to the method of Gilman (1970). Standard curves were obtained using known quantities of unlabelled cyclic AMP; the total amount of cyclic AMP (labelled and unlabelled) in each assay was plotted on the abpissa, the counts per minute minus the background on the ordinate. This generated a straight line on a logarithmic plot (see Figure 5), and : 48 :

. 3 Figure 4 Displacement of 2 picomoles or H cyclic AMP by

10, 000— ATP, ADP, cyclic GMP and cyclic AMP

...... Control . . - •-• • • • • ..7, 1, 000 — ADP ATP a. _

>4C.) S. N. cn O. s

cyclic GMP c

L 61 Q. 100 Cyclic AMP

10 0.01 0.10 1.0 10 100

Nucleotide Concentration (pmols) 49

Figure 5

Example plot of standard curve of cyclic AMP binding for assay

counts per minute

10, 000 —

1, 000

100

10 100. 0 0.1 1.0 10.0 pmols cyclic AMP in sample :50 :

the amount of cyclic AMP in unknown samples could then be readily calculated. Recovery of cyclic AMP in unknown samples was estimated by counting a 50 pl volume in 10 ml of Instagel. The proportion of added 'recovery' 3H cyclic AMP remaining in the sample could then be determined. This varied between 60% and 100%.

All calculations pertaining to the assay were performed on a PDP8/1 Digital Computer. A copy of the programme which was written by Mr. E. Emons appears in Appendix I.

The duplicate and triplicate control levels - results from cells not exposed to isoprenaline - were averaged and this value was taken as the basal unstimulated amount of cyclic AMP per 4 million mononuclear cells. This basal amount of cyclic AMP was subtracted from all of the subsequent samples which had been exposed to increasing concentrations of isoprenaline. Thus a figure for the absolute increase in cyclic AMP produced by the cells and for a percentage increase could be obtained. The average of the duplicate and triplicate samples were calculated to give a dose response curve of the cells to isoprenaline.

2.10 STATISTICAL METHODS

The mean dose response curves for different subject populations obtained by these calculations were compared by a two-way analysis of variance with replication (Snedecor and Cochran, 1967). This allowed one dose response carve - such as that for normal subjects - to be compared with another one - such as that for asthmatics taking large doses of beta adrenergic stimulants. As the data did not conform to a normal distribution, the values are shown graphically as median and interquartile ranges, or full ranges when few subjects were studied. :51:

CHAPTER III

CHARACTERISTICS OF THE LYMPHOCYTE BETA ADRENOCEPTOR 52:

3.1 INTRODUCTION The leucocyte has been used extensively as an in vitro model for the study of beta adrenergic receptor function in asthmatics since it was first suggested by Smith and Parker (1970). The method based on it has the advantage of removing many of the variables inherent in studying the response of the whole patient. Though simpler than the human subject, this still involves a complex system.

Cells have been incubated with isoprenaline, with or without a PDE inhibitor, and the amount of cyclic AMP formed has been assayed. A reduction in the amount of cyclic AMP, the 'second messenger' produced by beta adrenoceptor activation, can be explained in several ways. It could be the result of deformity and/or loss of beta adrenoceptors, impaired coupling of the receptor to adenylate cyclase, malfunction of the catalytic sub-unit of the enzyme, lack of a co-factor or of the precursor ATP, or increased destruction of cyclic AMP. A reduction in cyclic AMP from whatever cause has been shown to be correlated with increased mediator release in lung tissue (Orange et al., 1971) and nasal polyps (Kaliner et al., 1973). It is therefore an index of a function that is highly relevant to the asthmatic.

The first section of this chapter presents data on cyclic AMP accumulation in lymphocytes from normal subjects. A normal dose response curve to isoprenaline has been constructed. The variations between and within subjects will be illustrated and discussed.

The lymphocyte beta adrenoceptor has been used to study asthmatics though it should be regarded as a valid model only if it is similar to the beta adrenoceptors of respiratory tissues. The following sections of this chapter characterize the lymphocyte beta adrenoceptor according to Lands' et al. (1967) classification. The last section presents similar data on lung parenchyma. :53:

3.2 METHODS

3.2.1 Lymphocytes: Response to Isoprenaline and to Salbutamol

Eleven normal subjects (nine men and two women) were studied on 28 occasions. Mononuclear cells were separated and incubated with 2 theophylline 10 moles. litre 1 and a range of concentrations of isoprenaline as described in Chapter II. Purification and assay of cyclic AMP was also described in the previous chapter.

Lymphocytes obtained from two normal subjects were incubated with salbutamol and isoprenaline.

Salbutamol (a gift from Allen and Hanburys) was dissolved in 1 mg/ml ascorbic acid as was the isoprenaline.

Subsequent steps were as in Chapter II.

3.2.2 Response to Isoprenaline in the Presence of Beta Adrenoceptor Antagonists

Four normal subjects (two men and two women) of whom two had participated in the initial experiments, provided blood samples.

On one occasion, lymphocytes from a patient with chronic lymphatic leukaemia were studied. They were obtained from a cell separator, washed twice with phosphate-buffered saline, re-suspended in Hanks Balanced Salt Solution (HBSS), and then incubated in the same way as normal cells.

For each experiment the beta adrenoceptor blocking agents propranolol or practolol (gifts from ICI) were dissolved in distilled water and added in a 100 pl volume. The total incubation volume was 2.2 ml being 1 ml cells, 1 ml theophylline, 100 pl of isoprenaline in 1 mg/ml ascorbic acid and 100 pl of the beta adrenoceptor antagonist. An attempt was made to calculate the pA2 values (Schild, 1947; Arunlakshana and Schild, 1959) for the two beta adrenergic antagonists to characterize further the lymphocyte beta adrenoceptor. 3.2.3 Lung Tissue Fragments: Response to Isoprenaline and Salbutamol Lung tissue was obtained from four patients at operation for carcinoma of the bronchus. None had a history of respiratory disease or was on any drugs pre-operatively.

Immediately after resection the tissue was placed in previously prepared HBSS. Normal tissue was separated from any that was grossly diseased, dissected free of visible bronchi and pleura, and chopped into small pieces. Fragments totalling approximately 200 mg wet weight were added to each tube containing 1 ml buffered HBSS (without fetal calf serum).

Duplicate samples were incubated for 15 minutes at 37°C with a range of 2 concentrations of. isoprenaline or salbutamol and theophylline 10 moles. litre-1. The tubes were then placed in boiling water for eight minutes to terminate the reaction. The tissue was homogenized and centrifuged and cyclic AMP in the supernatant was purified and assayed.

The protein content was measured by the method of Lowry et al. (1951).

3.2.4 Lung Tissue Fragments: Response to Isoprenaline in the Presence of Propranolol and Practolol

Lung tissue was obtained from four patients operated on for carcinoma of the bronchus. Pre-operatively they had not been taking any drugs. The fragments were incubated with isoprenaline with and without propranolol or practolol. After incubation the samples were treated as described above.

3.3 RESULTS 3.3.1 Lymphocytes: Normal Dose Response Curve to Isoprenaline

There was considerable variation in the amount of cyclic AMP found in unstimulated mononuclear cells and in their responsiveness to isoprenaline. Table I shows results from 11 normal subjects whose cells were examined on 28 occasions. These are expressed as the increase (in picomoles cyclic AMP per 4 million cells) above the basal level, produced by each concentration of isoprenaline. In some studies insufficient cells were isolated to examine the full range of concentrations. Figure 6 shows these data plotted as a log dose response curve. Cyclic AMP levels :55:

TABLE I

Normal Subjects - Lymphocyte Response to Isoprenaline Increase in cyclic AMP above baseline (all values picomoles/4 x 106 cells)

Baseline Molar Concentration of Isoprenaline Name Sex Unstimulated Cyclic AMP 10-8 10-7 10-6 10-5 10-4

DSD M 13 11 48 70 70 74 CTD M 25 11 39 77 81 87 29 16 57 126 - 16o 61 0 84 152 - 139 GL M 28 0 64 58 55 139 MEC 1,1 25 9 26 72 121 101 34 123 179 220 372 43 108 269 321 318 335 135 88 296 846 774 837 92 - 246 436 304 276 JKG F 82 51 235 317 - - 8o 37 236 292 - 34 1 19 116 231 - 26 7 116 203 - 43 34 104 17o 171 210 PS M 63 4 57 148 168 165 83 48 176 203 258 234 RM M 50 75 273 275 291 324 50 129 249 290 321 33o 13 15 44 72 8o 69 37 19 73 loo 97 93 RH F 51 36 194 181 222 238 HF M 52 6 65 98 100 114 52 27 62 94 105 136 MRB M 9 4 9 12 15 13 17 11 27 58 39 48 JCM M 55 34 144 259 26o 323 22 25 79 94 73 83

Median 47 19 82 150 168 160 IQR 26-61 7-37 57-194 94-259 80-258 87-324 : 5 6 :

Figure 6 Absolute increase in lymphocyte cyclic AMP

in response to isoprenaline pmols/ 4 x 106 cells (Median IQR)

250

200

AMP 100 ic l c cy in ase re 50 inc

8 7 6 5 -4 10 10 10 10 10 molar concentration of isoprenaline

:57:

(expressed as picomoles per 4 million cells) increased from a basal level of 47 (Interquartile range (IQR) 26 - 61) to 160 (IQR 87 - 324) in 4 the presence of 10 moles. litre-1 isoprenaline.

The cells with the lowest basal levels of cyclic AMP had the lowest increase in absolute amounts of cyclic AMP, and vice versa. Table II shows the same data expressed in terms of percent increase over the unstimulated level and Figure 7 the median plus interquartile ranges. This form of analysis reduces the between-subject variation. Thus, at 10 4 moles. litre-/ isoprenaline, the increase in cyclic AMP varies between 144% and 779% - approximately a six-fold range - whereas in terms of absolute increase of cyclic AMP at the same isoprenaline concentration the range spans 13 picomoles to 837 picomoles - approximately a 70-fold range. The pattern is similar regardless of which method is used to express the data.

3.3.2 Lymphocytes: Response to Salbutamol Lymphocytes incubated with this selective beta2 adrenoceptor agonist showed a dose-related increase in cyclic AMP as did cells incubated with isoprenaline. Figure 8 compares data from two normal subjects, each studied on two occasions, expressed as percentage increase (median values and range) of cyclic AMP with isoprenaline and with salbutamol stimulation. Salbutamol was not as potent as isoprenaline; its dose response curve was further to the right and where the curves are roughly parallel, isoprenaline is about 9-15 times more potent than salbutamol. Salbutamol was only a partial agonist in these experiments producing a 255% (range 250% - 403%) increase in cyclic AMP as compared with 464% (range 332% - 519%) with isoprenaline.

3.3.3 Lymphocyte Response to Isoprenaline in the Presence of Beta Adrenoceptor Antagonists

Lymphocyte response to isoprenaline was studied with the non-selective beta adrenoceptor antagonist propranolol and the selective betal adrenoceptor blocking agent practolol. Cells were incubated with isoprenaline either alone or with various concentrations of the beta adrenoceptor antagonist. Figure 9 shows a graph of the cyclic AMP produced by stimulation with isoprenaline 10-5 moles. litre-1 in the presence of the antagonists. Each dose response curve is the median (range) : 58 :

TABLE II

Normal Subjects - Lymphocyte Response to Isoprenaline Data expressed as percent increase in cyclic AMP above baseline

Baseline Molar Concentration of Isoprenaline Name Sex Unstimulated -8 -7 -6 -4 Cyclic AMP 10 10 10 10-5 10

DSD M (13) 85 369 538 538 569 CTD M (25) 44 156 308 324 348 (29) 55 197 434 - 552 (61) 0 138 249 - 228 GL M (28) 0 229 207 196 496 MEC M (25) 36 104 288 484 - (101) 34 122 177 218 368 (43) 251 626 747 740 779 (135) 65 219 626 572 619 (92) - 267 474 33o 500 JKG F (82) 62 287 387 - - (80) 46 295 365 - (34) 3 56 341 679 - (26) 27 446 781 (43) 79 242 395 398 488 PS M (63) 6 90 235 267 262 (83) 58 212 245 311 283 RM M (50) 150 546 550 582 648 (5o) 258 498 580 642 660 (13) 115 338 554 615 531 (37) 51 197 270 262 251 RH F (51) 71 380 355 435 467 HF M (52) 12 125 188 192 219 (52) 52 119 181 202 262 MRB M (9) 44 100 133 167 144 (17) 65 159 341 229 282 JCM M (55) 62 262 471 473 .-- 587 (22) 114 359 427 332 377

Median (47) 55 224 360 332 377 NR (26-61) 36-79 138-338 249-474 262-572 282-569 : 59 :

Figure 7 Percentage increase in lymphocyte cyclic AMP

in response to isoprenaline • (Median ÷ IQR)

500

400

100

8 7 6 5 4 10 10 10 10 10 molar concentration of iscprenaline : 60 :

Figure 8 Response of lymphocyte cyclic AMP

to isoprenaline or salbutamol

ISOPRENALINE

400 P 300 AM

SALBUTAMOL lic c cy in e as e 200 cr in t en rc e p r 100

4 10 10 10 10 10

MOLAR CONCENTRATION OF AGONIST : 61 :

Figure 9 Percentage increase in lymphocyte cyclic AMP -5 -1 in response to isoprenaline 10 moles. litre

in the presence of propranolol or practolol

percent inhibition in response (Median + range)

C cS3 C

C

C PRACTOLOL r 0 0.1 L

50

U

C 0

C

PROPRANOLOL

8 5 4 -3 10 10 10 10 10 10

MOLAR CONCENTRATION OF ANTAGONIST

: 62:

of four experiments. The amount of cyclic AMP was progressively reduced by increasing amounts of propranolol. However, practolol below doses of 105 moles.litre-1 had no effect on isoprenaline-stimulated cyclic AMP production. Although no valid dose ratio can be calculated in these circumstances as the dose response curves were never parallel, propranolol appears to be approximately five thousand times more potent than practolol in this system.

In order to characterize the lymphocyte beta adrenoceptor more precisely in terms of receptor affinity an attempt was made to derive pA2 values for propranolol and practolol from Schild plots (Figure 10). In these experiments cells were obtained on several occasions from three normal subjects and on one occasion from a patient with chronic lymphatic leukaemia. The calculated pA2 values are shoWil on Table III and range from 8.32 to 8.38 for propranolol and 3.89 to 4.0 for practolol. Values for the slopes of these lines are given beside the pA2 values; they are all greater than unity.

3.5.4 Lung Experiments: Incubation with Isoprenaline and Salbutamol

The median results from lung parenchyma which had been incubated with isoprenaline and salbutamol are shown in Figure 11. Both compounds stimulated cyclic AMP formation but isoprenaline was a more potent agonist. At 10 6 moles. litre-1 isoprenaline there was a median rise of 377% (range 170 - 715) in cyclic AMP; the same dose of salbutamol 4 produced little response 2C (range 0 - 66). Isoprenaline 10 moles. litre-1 produced a mean increase in cyclic AMP of 580% (range 380 - 794); the same concentration of salbutamol only increased cyclic AMP by 131% (range 21 -188). Salbutamol was only a partial agonist and the higher -1 concentration of 103 moles. litre did not increase further cyclic AMP levels.

3.3.5 Lung Experiments: Incubation with Isoprenaline and Propranolol or Practolol

Lung tissue incubated with isoprenaline 10 7 moles. litre-1 with and 6 without the beta adrenoceptor antagonists propranolol (109 to 10 moles. -1 6 litre ) and practolol (10 to 103 moles. litre-1) responded in a similar way to the mononuclear cell preparation (Figure 12). The data for

: 63 :

Fgure 10 RillId oicts showing affect of orcoranolol and d rectziol

on lympnorite cyclic AMP resconse to isoorenaline

5 I

- 4 _ 0 A11 t I SE. I DO OG l

• I

-3 7 10 a 10 10 MCLAR CCNCENTRATI CN CF PROPRANOLCL 0 - I Ail SE It O D I IOC

-a 10 10 3

MOLAR CONCENTRATI ON CF RRACTOLCL : 64 :

TABLE III

pA2 Values for Propranolol and Practolol Observed in Human Lymphocytes

Sub'ect Propranolol (Slope) Practolol (Slope)

1 - Normal 8.32 (1.8) 4.o (3.2)

2 - Normal 8.34 (2.0)

3 - Normal 3.89 (2.6)

4 - Leukaemic 8.38 (2.2)

Mean 8.35 3.95 : 65 :

Figure 11 Cyclic AMP response of human lung parenchyma

to isoprenaline and salbutamol

percent increase over baseline (Median + range)

800

II SOP RENA L INE 600 .....----- U U U U C.) cZ 400 8-3

200 1.,------1 i --SALBUTAMOL mT- t -8 5 10 10 10 10 . 10 4

MOLAR CONCENTRATION OF AGONIST : 66 :

Figure 12 Propranolol or practolol inhibition of cyclic AMP response

to isoprenaline in human lung parenchyma

percent inhibition of response (Median)

0 —

20 —

40 —

Cel C C 0. S._ o_ PROPRANOLOL 60 U ›-•U U V "5 C . 80

C

0 100 -9 -8 -7 -6 -5 -4 3 10 10 10 10 10 10 10 MOLAR CONCENTRATION OF ANTAGONIST : 67 :

four experiments are plotted as median percentage increase in cyclic AMP. Propranolol at low concentrations antagonized the effect of isoprenaline; practolol did so at concentrations approximately 1,000 times as greif.

3.4 DISCUSSION

3.4.1 Classification of the Lymphocyte Beta Adrenoceptor

The division of beta adrenoceptors into two subgroups (Lands et al., 1967) has not been fully accepted because results from some tissues do not fit neatly into these categories (Bristow, et al., 1970; Lefkowitz, 1975). Nevertheless, it has been a useful concept clinically. In particular asthmatic patients respond to selective beta2 adrenoceptor stimulants without the degree of tachycardia produced by isoprenaline and are relatively unaffected by low doses of selective betal adrenergic antagonists (Ryo and Townley, 1976). The human respiratory system, therefore, seems to possess beta2 adrenoceptors. Any model used to study beta adrenoceptor responsiveness in asthmatics must also possess a beta2 adrenoceptor. the beta adrenoceptor of The data presented above show thatlthe peripheral blood lymphocyte has similar characteristics to that of lung parenchyma. It is an adrenoceptor stimulated by salbutamol, a selective beta2 agonist. The drug is about one-tenth as powerful as isoprenaline in stimulating cyclic AMP production in lymphocytes. This is similar to the comparative potencies found by Cullum at al. (1969) studying the beta2 adrenoceptor in guinea pig tracheal chain and very different from the 2000:1 potency ratio that these authors found for cardiac (beta ) receptors in the same 1 species.

The data on selective and non-selective beta adrenergic antagonists supports the hypothesis that the beta adrenoceptor on the lymphocyte is beta in type. Practolol does not block the action of isoprenaline in 2 raising cyclic AMP levels in lymphocytes. The attempted pA2 values calculated from normal and leukaemic cells are similar to those found in other tissues possessing beta2 adrenoceptors. Table IV gives pA2 values in different tissues investigated by other workers for comparison. :68:

TABLE IV

pA2 Values for Propranolol and Practolol Obtained in Other Laboratories

Tissue Propranolol Practolol Reference Guinea pig atria 8.70 Blinks, 1967 8.02 Moore and O'Donnell, 1970 7.98 Bassett, 1971

8.71 Conolly, 1972 8.32 6.49 Barrett et al., 1973

Guinea pig trachea 8.02 Moore and O'Donnell, 1970 7.93 Bassett, 1971 8.46 4.26 Barrett et al., 1973

Human bronchus 6.65 4.29 Hedges and Turner, 1971 :69:

The slopes of the Schild plots are all greater than one (the defined value for competitive antagonism). The reason for this is not clear. Williams et al. (1976) have demonstrated competitive antagonism between isoprenaline and the radiolabelled beta adrenoceptor antagonist, 3H alprenolol, in binding studies on human lymphocytes. The cells in their study were homogenized in a Tris HCl buffer rather than in HBSS, but it seems unlikely that diffusion or protein binding could have affected the slopes in this way. Propranolol is highly lipid soluble, practolol more soluble in aqueous media but in both cases the slopes were over one. One possible explanation for this phenomenon is uptake of the antagonist by the cell (Jellettand Shand, 1973; McDevitt et al., 1976).

3.4.2 Variability of Lymphocyte Cyclic AMP Response to Isoprenaline

There is considerable variation of the lymphocyte cyclic AMP response to isoprenaline as shown in Table I. Other workers have noted the same effect. Gillespie et al. (1974) using different incubation conditions found a 20-fold variation in picomoles of cyclic AMP produced; Smith and Parker (1973) noted approximately a seven-fold variation. Our range of between 12.5 picomoles and 837.2 picomoles per 4 million cells spans almost a 70-fold range.

Inter-individual variation is greater than intra-individual variation. Cells from MEC, studied on five occasions show at the maximum concentration of isoprenaline only a three-fold variation which is considerably less than for the group as a whole. We attempted to control the subject's basal state as much as possible by taking blood samples at the same time of day, prohibiting coffee or tea, and noting sexual activity or excessive exercise. However, the variation was unaffected and these restrictions were subsequently relaxed. Parker and Smith (1973) noted greater fluctuations in women and postulated that this was due to a hormonal effect presumably correlated with the menstrual cycle. JKG was studied on five occasions at different times in the menstrual cycle, but her results were no more variable than those of her male colleague, MEC. Furthermore, there was no obvious correlation between high and low levels of cyclic AMP and time of the cycle. Pregnant women have also been studied. Their lymphocyte cyclic AMP response to beta adrenergic stimulation was similar to that of normal subjects (data presented in Chapter V); therefore, hormonal differences do not seem to be an : 70 :

important determinant of variability.

Parker and Smith (1973) suggested that the proportion of T and B cells in ficoll-h4que lymphocyte isolates could explain some of the variability of this preparation. They found that cells passed through a nylon fibre column (i.e., T lymphocyte enriched) have relatively poor isoprenaline responsiveness. Bach (1975) studying sub-classes of lymphocytes from mice, found similar differences, and confirmed that B lymphocytes, those with immunoglobulin determinants on the cell surface, produced more cyclic AMP with isoprenaline stimulation than did thymus- dependent lymphocytes.

It was not possible to obtain information on the relative numbers of T and B lymphocytes in our preparations. Simultaneous determination of our normal dose response curve and the proportions of lymphocyte sub-classes would have necessitated taking an unacceptably large amount of blood from each subject. Vaughn-Smith and Thompson(to be published)have noted that T lymphocytes may be selectively lost during the ficoll-hypaque isolation procedure. Some of our cell yields were very low (15% maximum possible yield) but these preparations were very responsive to isoprenaline stimulation. This would be compatible with a selective T lymphocyte loss.

Smears of the final cell preparations were stained and examined on numerous occasions. The number of monocytes - usually between 5 and 15% but occasionally outside this range - did not correlate with cyclic AMP response to isoprenaline. However, Zucher-Franklin (1974) has shown that a large and variable (23.9%-76.6%) number of mononuclear cells isolated on a ficoll-hypaque gradient phagocytize latex particles and, therefore, may be classified as monocytes despite their appearance on routine staining. It is possible, therefore, that the cell preparations used in this work were more varied than was apparent.

It is also possible that cell viability may not be identical in all studies. Trypan blue exclusion tests always showed a greater than 95% viability but this is a crude test of a cell's ability to carry on all its metabolic functions (Yuhas et al., 1974; Lee et al., 1975). : 71 :

Lymphocytes show considerable variability in other systems as well. Intra-individual variations in the response to phytohaemagglution have been noted (Yu and Clements, 1976) as well as fluctuations in absolute T and B cell numbers (Steel et al., 1974). Lichtenstein and Margolis (1968) commented on the variable concentrations of adrenaline which inhibited 50% of the histamine release from sensitized leucocytes from day to day and donor to donor. The activities of the enzymes phosphoglucomutase and glucose-6-phosphate dehydrogenase differ by a factor of 15 in normal cells (Monahan et al., 1975). By analogy variability could be expected in the activity of the beta adrenoceptor adenyl cyclase complex.

The considerable inter- and intra-individual variability makes it preferable to examine the increases in cyclic AMP in percentage rather than absolute terms. This reduces the inter-individual range considerable, although it does not eliminate it: a six-fold range is still seen. Thus, individual experiments have only limited usefulness, but the use of percentage increase allows easier comparison between experiments by reducing the variation of the model. Therefore, data in this thesis will be presented in terms of percentage increase; however, the absolute amounts of cyclic AMP will also be given for reference.

Despite the difficulties of day-to-day and inter-individual variation, the lymphocyte is an easily accessible cell which can be investigated repeatedly. It possesses a beta2 adrenoceptor which can be studied relatively conveniently and used as a model for similar receptors on other cells. : 72:

CHAPTER IV

LYMPHOCYTE BETA ADRENOCEPTOR RESPONSE IN ASTHMATICS : 73 :

4.1 INTRODUCTION

Several groups (Parker and Smith, 1973; Logsdon et al., 1972; Alston

• et■•■ ••■•al., 1974) have reported that following isoprenaline stimulation cyclic AMP production is reduced in leucocytes or lymphocytes of asthmatic patients. This poor responsiveness appeared to wax and wane with the severity of the disease (Parker and Smith, 1973) and could be corrected by treating the patient with steroids (Logsdon et al., 1972). However, Gillespie at al. (1974) could not confirm a significant difference between asthmatics and normals.

The leucocyte data have been used by some workers to support Szentivanyi's theory of an inherent defect in beta adrenoceptor function in atopic patients including asthmatics. Since virtually all asthmatics are on some adrenergic medication, orally or by inhalation, or both, some workers (Parker and Smith, 1973; Parker et al., 1973; GillespieGillespie et al., 1974) have considered the possibility that this might affect leucocyte beta adrenoceptor responsiveness. However, they thought it to be unlikely.

Recently, as discussed in the introduction, there have been further studies suggesting that beta adrenoceptor function is influenced by prior exposure to beta adrenergic stimulants. To consider this possibility in asthmatics systematically, we divided patients into groups depending on their intake of these drugs. The first group consisted of patients admitted to hospital with severe asthma who were judged to be using their beta adrenergic medications excessively. The second comprised asthmatic patients who were receiving exclusively non-adrenergic drugs such as disodium cromoglycate or beclomethasone diproprionate by inhalation. Some patients (the third group) were studied serially - initially while using beta adrenoceptor agonists and subsequently after stopping these drugs for varying periods of time.

4.2 SUBJECTS AND METHODS

4.2.1 Asthmatics on Large Doses of Beta Adrenoceptor Medication Seven asthmatic patients were studied at the end of a period (up to 10 days) of heavy use of adrenergic bronchodilators, either inhaled isoprenaline (up to 1500 pg/day) or salbutamol (2500-4000 pg/day). Two

74:

patients also took salbutamol orally in a dose of 16 mg/day. Simple tests of airways obstruction, a recording of forced expiratory volume in one second (FEV ) and vital capacity or peak expiratory flow rate in 1 litres per minute (PEFR), were made before the blood was taken. In one instance this was impossible as the patient was on a respirator. Clinical details are given in Table V.

4.2.2 Asthmatics on Non-Adrenergic Medication Twelve asthmatics being treated with non-adrenergic anti-asthmatic drugs (cromoglycate or beclomethasone by inhalation) were studied. None had received adrenergic drugs for at least three weeks. The patients were comparable to the first group in that all had asthma by clinical evidence. Five had a previous history of severe resistant asthma requiring admission to hospital and six had required at least one course of systemic steroids in the recent past. Clinical details are given in Table VI. Airways obstruction was measured by tests as in Group I.

4.2.3 Asthmatics Studied Serially, Initially on Large Doses of Beta Adrenergic Bronchodilators and Subsequently on Other Anti- Asthmatic Drugs Five patients were studied. Three (Subjects 2, 6 and 7) were part of the first group and were subsequently weaned off all adrenergic medication. Their lymphocytes were examined up to two months after being on their new regime. The other two subjects were relatively mild asthmatics with only marginally abnormal spirometry.

Subject 17 had become psychologically dependent on his salbutamol aerosol and was taking approximately 30 inhalations (3000 pg) of salbutamol a day. Two months later after he had been taking normally prescribed doses of salbutamol (800 pg daily by inhalation), his cells were studied again.

Subject 18 attempted to commit suicide by taking salbutamol 16 mg by mouth and 4 mg by inhalation. Blood was taken 20 hours after the event when the pharmacological effects of the drug had subsided. Her cells were restudied two weeks later.

Lymphocytes from all the patients were isolated from heparinized blood and incubated with theophylline and various doses of isoprenaline. Cyclic TABLE V

Patients on High Doees of Beta Adrenergio Bronchodilatore

Spirometry Patient Asthmatic Ago Sex Medication at Time of Study No. Type Beat Worst At Time of Study

1 62 M Intrinsic PEFR 370 Go On Isoprenaline 3000 pg/24 hr respirator Adrenaline 300 pg + 700 pg s.c. Aminophylline 1000 mg Hydrocortisone 750 mg

2 63 Intrinsic FEV 1.0 o.4 Isoprenaline 1000-1500 pg/24 hr F 1- 0.35 — 1.5 0.75 1.0 (aerosol) FVC Prednisone 15 mg

3 63 H Intrinsic Not Not 0.35 Salbutamol 2500 pg/24 hr (nebulizer) recorded recorded 0.95 Hydrocortisone 800 mg Aminophylline 1.0 g

4 26 F Extrinsic FEV1 2.6 22 9A Salbutamol 4000 pg/24 hr (aerosol) Kb 1.0 1.0 Prednisone 40 mg FVC Cromoglycate (apinhaler) Aminophylline suppository 5 47 F Intrinsic FEV1 2.0 1.0 1.0 Salbutamol Woo pg/24 hr (aerosol) 37 2.5 2.5 FVC

F Intrinsic FEV 0.6 0.6 Salbutamol 4000 pg/24 hr (aerosol) 6 53 1 1:-.2 3.0 1.2 1.2 and 16 mg (tablets) FVC 7 47 F Intrinsic FEV 2.6 1.0 1.0 Salbutamol 2000 pg/24 hr (aerosol) 1 - and 16 mg (tablets) FVC 3.3 .17 TX FEV1

Forced expiratory volume in 1 second (litreo) PEER Peak expiratory flow rate (litree/min) FVC Forced vital capacity (litres) TABLE VI

Patients on Non-Adrenergic Medication

Spirometry Patient Asthmatic Age Sex Medication at Time of Study No. Type ' Best Worst At Time of Study

2 63 F Intrinsic FEV1 1.0 0.35 9,1 Beclomethasone 300 pg/day (aerosol) 0.75 FVC 1.5 1.1 Prednisone 10 mg

6 53 F Intrinsic FEV 1...1 0.6 1.1 Beclomethasone 400 pg/day (aerosol) 1 FVC 3.0 1.2 2.1

7 47 F Intrinsic FEV 2.6 1.0 Beolomethasone 400 pg/day (aerosol) 1 - a/ FVC 3.X3 T 3.1

8 53 F Intrinsic PEFR 120 80 85

23 H Extrinsic FEV IA 2.0 9 1 W- Cromoglycate (20 mg) 2 daily FVC 5.3 .4-: 5.3 10 36 M Extrinsio FEV 2.0 1.1 2.2 1 3.9 3.0 3.7

H Intrineio FEV 11 70 1 1.6 Q 2.z.Z Cromoglycate (20 mg) 6 daily FVC 3.3 1.35 1.35 12 52 F Intrinsic PEFR 330 240 310 Cromoglycate (20 mg) 3 daily

13 60 F Intrinsic PEFR 390 210 390 Beclomethaeone 300 pg/day (aerosol)

14 20 M Extrinsic FEV 1,2 1 1.2 FVC 3.6 t41

15 29 M Intrinsic FEV1 2,2 0.6 1.8 Cromoglycate (20 mg) 2 daily FVC- 5.0 1.1 3:3

16 13 F Extrinsic PEFR 400 130 400 Cromoglycate (20 mg) 4 daily :77:

AMP was assayed using the methods described in Chapter II.

4.3 RESULTS

4.3.1 Asthmatics on Large Doses of Beta Adrenergic Bronchodilators

Lymphocytes isolated from these patients had a baseline unstimulated level of cyclic AMP which was not significantly different from that of cells from normal subjects. Median baseline cyclic AMP was 47 (IQR 26 - 61) in the latter as compared to 27 (range 12 - 63) in the asthmatics.

Lymphocytes from these patients showed a marked and significant depression of response to isoprenaline compared to cells from normal subjects (Figure 13). The cells from these asthmatics responded to isoprenaline 4 -1 10 moles. litre with an increase of only 63% (range 9 - 145) compared to 577% (IQR 282 - 569) in normals (see Chapter III). The data are shown in Table VII in percentage terms and in Table VIII as absolute 6 increase on cyclic AMP (picomoles/4 x 10 cells). The results are significantly different (p< 0.001) from normal cells whichever way they are analysed.

4.3.2 Asthmatics on Non-Adrenergic Medication

Lymphocytes from asthmatics on non-adrenergic drugs had a significantly lower level (1)40.05) of basal unstimulated cyclic AMP than normal subjects. The figures were median 20 (IQR 16 - 38) as compared to 47 (IQR 26 - 61).

The cells from these asthmatics responded similarly in percentage terms to cells isolated from normals. The maximal increase in cyclic AMP was 4 383% (IQR 327 - 560) after incubation with isoprenaline 10 moles. -1 litre compared to 377% (IQR 282 - 569) in normal cells. Figure 14 shows that the two dose response curves are almost identical. The data are shown in Table IX.

The absolute amounts of cyclic AMP produced are shown in Table X. When looked at in detail all of these absolute values fall within the range spanned by normal subjects (see Table VIII), but as a group they are significantly lower (p4c0.001) than those for normals. The maximal increase in cyclic AMP response was 92 (IQR 58 - 101) in this group with : 78

Figure 13 Cyclic AMP response of lymphocytes from asthmatic patients

on large amounts of beta adrenergic bronchodilators (Median + range)

and normal subjects (Median 4- I4R) percent increase over baseline

500

400 NORMALS MP A lic c 300 cy in

ase e r inc t

en 200 rc e p

100 ASTHMATICS

7 -15 -4 10 10 10-5 10

molar concentration of isoprenaline : 79

TABLE VII

Asthmatic Patients on Large Doses of Beta Adrenergic Bronchodilators: Lymphocyte Cyclic AMP Response to Isoprenaline

Percentage increase over baseline unstimulated level

Baseline Molar Concentration of Isoprenaline Patient Unstimulated -8 -6 -5 -4 No. Cyclic AMP 10 10-7 10 10 10

1 18 0 22 - 22 -

2 63 0 13 60 157 143 3 12 0 42 - 158 - 4 16 - 13 63 81 63 5 52 4 90 113 - 100 6 27 - 22 67 19 26 7 55 20 25 53 47 9

Median 27 0 22 63 64 63

Range 12-63 0-20 13-90 53-113 19-158 9-143

Normal Subjects

Median 47 55 224 360 332 377 WI 26-61 36-79 138-338 249-474 262-572 282-569 : 80:

TABLE VIII

Asthmatic Patients on Large Doses of Beta Adrenergic Bronchodilators: Lymphocyte Cyclic AMP Response to Isoprenaline

Data given as baseline unstimulated level and increase above this level (picomoles cyclic AMP/4 x 106 cells)

Patient Baseline Molar Concentration of Isoprenaline No. Unstimulated 8 7 6 -5 -4 Cyclic AMP 10 10 10 10 10

1 18 0 4 - 4 -

2 63 0 8 38 99 90

3 12 0 5 - 19 - 4 16 - 2 10 13 10

5 52 2 47 59 - 52 • 6 27 - 6 18 5 7

7 55 11 14 29 26 5

Median 27 0 6 29 16 10

Range 12-63 0-11 2-47 10-59 4-99 5-90

Normal Subjects

Median 47 19 82 150 168 160

IQR 26-61 7-37 57-194 94-259 80-258 87-324 _f : 81 :

Figure 14 Cyclic AMP response of lymphocytes from asthmatic patients

on non-adrenergic drugs and normal subjects

percent increase over baseline (median ÷ IQR)

500

400

AMP 300 ic l c cy in

ease r c 200 in t en rc e

p • Normals

100 ■ Asthmatics

6 5 4 10 8 10 10 10 10-

molar concentration of isoprenaline : 82

TABLE IX

Asthmatic Patients on Non-Adrenergic Drugs: Lymphocyte Cyclic AMP Response to Isoprenaline

Percentage increase over baseline unstimulated level

Baseline Molar Concentration of Isoprenaline Patient Unstimulated No. - 8 -7 -6 -5 -4 Cyclic AMP lo 10 10 10 10

2 38 95 182 297 324 259 6 17 - 253 440 635 594 7 66 0 58 135 92 97 8 15 27 13 120 213 327 9 11 9 36 182 418 445 10 20 45 265 500 530 725

11 10 50 270 33o 490 56o 12 24 - 17 79 225 383 13 37 19 114 222 354 257 14 75 84 236 351 364 457 15 17 206 647 753 16 31 294 431 494 363 16 41 93 327 393 507 -

Median 20 45 209 330 418 383

IQR 16-38 27-93 58-265 182-400 324-507 327-560 :83:

TABLE X

Asthmatic Patients on Non-Adrenergic Drugs: Lymphocyte Cyclic AMP Response to Isoprenaline

Data given as baseline unstimulated level and increase above this level (picomoles cyclic AMP/4 x 106 cells)

Baseline Molar Concentration of Isoprenaline Patient Unstimulated No. -8 -7 -6 -5 -4 Cyclic AMP 10 10 10 10 10

2 38 36 69 113 123 98 6 17 - 43 68 108 101 7 66 0 38 89 61 64 8 15 4 2 18 32 49 9 11 1 4 20 46 49 10 20 9 53 100 106 145 11 10 5 27 33 49 56 12 24 - 4 19 54 92 13 37 7 42 82 131 95 14 75 63 177 263 273 343 15 17 35 110 128 16 5 47 69 79 58 16 41 38 134 161 208 - . . Median 20 7 43 82 106 92

IQR 16-38 5-36 27-53 33-110 54-128 58-101 . , : 84 :

4 -1 isoprenaline 10 moles. litre as compared to 160 (IQR 87 - 324) in normals.

Figure 15 shows the dose response curves in percentage terms for asthmatics on large doses of beta adrenergic bronchodilators and for patients not on these drugs. The reduced response of the former group is appreciable (p<0.001).

In terms of absolute amount of cyclic AMP (picomoles per 4 million cells), asthmatics on non-adrenergic medication have a significantly better response to isoprenaline (p40.001) than patients on large doses of beta adrenergic stimulants. When the data are analysed in this form, they occupy an intermediate position between asthmatics on large doses of beta adrenergic bronchodilators and normals and are significantly different (1040.001) from both these groups (Figure 16).

4.3.3 Asthmatics Studied Serially, Initially on Large Doses of Beta Adrenergic Bronchodilators and Subsequently,on Other Anti- Asthmatic Drugs

Five patients were studied while taking excessive amounts of beta adrenergic stimulants and after discontinuing these drugs, and one patient (Subject 17) was studied after the amount was reduced to the normally recommended dose (800 pg salbutamol by pressurized aerosol per day).

Lymphocyte beta adrenoceptor responsiveness was depressed initially in all the patients in both proportional and absolute terms. The results for the group as a whole are shown in Figure 17. After patients discontinued or markedly reduced the use of beta adrenergic bronchodilators, lymphocyte cyclic AMP response was significantly improved (p<0.001) increasing -4 -1 at isoprenaline 10 moles. litre from a median response of 26% (range 0 - 138) to 258% (range 94 - 754). The data are given in proportional terms in Table XI and in absolute terms in Table XII. Figures 18 and 19 show the lymphocyte cyclic AMP response in two of the severe asthmatic subjects (2 and 6) individually with relevant clinical data.

Although cyclic AMP responsiveness in lymphocytes improved after 85 :

Figure 15 Cyclic AMP response of lymphocytes from asthmatic patients on large amounts of beta adrenergic bronchodilators ( 2) (median + range) and patients on other drugs ( •) (median IQR) percent increase' over baseline

500

400 P AM

lic c 300 cy in rease t inc 200 rcen e p

100

-8 6 5 -4 10 10 10 10 10 molar concentration of isoprenaline : 86 :

Figure 16 Absolute increase in lymphocyte . cyclic AMP in response to isoprenaline: asthmatics on large amounts of beta adrenergic stimulants (ii ) (median + range), other medication ( • )

(median ÷ IQR) and normal subjects (median + IQR) (0) 6 pmois cyclic AMP 14 x 10 cells 259 ; 258 324 i 4

200 —

-8 7 -5 -4 10 10 1c 6 10 10

molar concentration of isoprenaline : 87 :

Figure 17

Serial study of lymphocyte cyclic AMP response in asthmatic patients initially on large amounts fo beta adrenergic bronchodilators u) and subsequently on other drugs (•) percent increase over baseline (median 4- range)

500

400

300

C cn CD CD 200

Q.

100

-8 7 6 5 4 10 10 10 10 10 molar concentration of isoprenaline : 88 :

TABLE XI

Five Asthmatic Patients Studied Before and After Changing from Large Doses of Adrenergic Bronchodilators to Non-Adrenergic Drugs

Percent increase in cyclic AMP above baseline levels

Baseline Molar Concentration of Isoprenaline Patient Unstimulated -, No. -8 -6 -5 -4 Cyclic AMP 10 10-7 10 10 10

BEFORE

2 (65) 0 12 58 152 138

6 (27) - 22 67 19 26

7 (55) 20 25 53 47 9

17* (69) 0 25 65 101 93 18* ( 8) 13 0 25 25 0

Median (55) 7 22 58 47 26

AIM=

AFTER

2 (38) 95 182 297 324 258

6 (17) - 253 400 655 594

7 (68) 0 56 131 90 94

17* (101) 156 480 531 708 754

18* (41) 5 41 202 215 151

Median (41) 50 182 297 324 258

*Mild asthmatics : 89

TABLE XII

Five Asthmatic Patients Studied Before and After Changing from Large Doses of Adrenergic Bronchodilators to Non-Adrenergic Drugs

Increase in cyclic AMP (picomoles/4 x 106 cells)

Baseline Molar Concentration of Isoprenaline Patient Unstimulated No. 8 6 -4 Cyclic AMP 10 10-7 10 10-5 10

BEFORE

2 65 0 8 38 99 90

6 27 - 6 18 5 7

7 55 11 14 29 26 5

17* 69 0 17 45 70 64

18* 8 1 0 2 2 0

Median 55 1 8 29 26 7

AFTER

2 38 36 69 113 123 98

6 17 - 43 68 108 101

7 68 0 38 89 61 64 17* 101 158 485 536 715 762

18* 41 2 17 83 88 62

Median 41 19 43 89 108 98

*Mild asthmatics : 90 :

Figure 13 Percentage increase in ciclic AMP in lymphocytes from patient 2, initially on large Oases of beta adrenergic 7timulants and subsequently cn other therapy

• Day 30 Prednisone 3maiday Beclomethasone 300 .4/day

P normal

AM subjects

ic l

c (median + IQR) cy In

e s 300 ea cr In A PUM Day 1 0 — Prednisone 10 mg/day 0 Isoprenaline 1C030 by aerosol .0

3 7 6 3 4 10 10 1.0 10 10 - _ — molar concentration of iscoranaline : 91 ►

F7,gure 19 Percentage increase in cyclic AMP in lymodocytes from oatient • 6 initially on large poses of beta adrenergic stimulants and subsequently on other therapy

700 Pt. 5. F. 53 yrs.

Day 30 X PEFR 270 Ilmin On beclomethasone c1COuglday 600

Normal suojects • (median IOR) P AM

ic l c cy In

e s a cre 300 Day 11 PEFR 300 limin On salbutamol 4C0 Aicay 'oeclamethasone 300 ugiday prednisone 20 mgrday 2C0

100 Day 1 A PEFR. ?CO Irmin • On salbutamol aerosol 3C00ugiday tablets 16 mg/day A

a 4 -3 10 to-5 10 5 0 10

molar concentration of isoorenaline : 92 :

discontinuing excessive beta adrenergic medication, measurements of pulmonary function in this group were little changed. The two patients with mild asthma (Subjects 17 and 18) had virtually normal spirometry on both occasions. The most severe asthmatic (Subject 2) showed marked improvement of lymphocyte responsiveness but no corresponding improvement in airways obstruction. Both Subject 6 and Subject 7 were studied on admission to hospital and then some time later. After the introduction of steroids Patient 6 improved clinically and her lymphocytes became more responsive to beta adrenergic stimulation. When studied again one month later (Figure 19) her lymphocyte beta adrenoceptor response was well within the normal range but her spirometry had not improved further.

4.4 DISCUSSION These studies demonstrate that lymphocytes from asthmatic patients who are taking excessive amounts of beta adrenergic bronchodilators respond poorly to isoprenaline when compared with cells from asthmatics treated exclusively with non-adrenergic medication (p<0.001) or from normal subjects (p<0.001).

The serial studies further support the conclusion that therapy affects beta adrenoceptor responsiveness. A significant improvement (p<0.001) in cyclic AMP response was found in cells studied after patients had discontinued, or in one case substantially reduced, adrenergic bronchodilators.

Other workers (Parker and Smith, 1973) have suggested that cyclic AMP response waxes and wanes with the patients' clinical state. This was not true in the case of the five patients studied on several occasions. Their asthma as measured by simple tests of airways obstruction changed little, whereas their lymphocyte beta adrenoceptor responsiveness improved markedly after discontinuing beta adrenergic stimulants.

There is some evidence that corticosteroids improve beta adrenoceptor response (Logsdon and Coffey, 1972; Parker et al., 1973; Holgate et al., 1977). However, the one patient on continuous prednisolone (Subject 2) had a markedly reduced lymphocyte cyclic AMP response while using her isoprenaline inhaler excessively. The other patient studied after being on corticosteroids for 11 days showed only partial return of :93:

beta adrenoceptor responsiveness and this normalized after both beta adrenergic bronchodilators and systemic corticosteroids were discontinued.

The difference between asthmatics on adrenergic bronchodilators and normal subjects has been observed before and used as evidence of an inherent beta adrenoceptor defect in asthmatics. However, the data presented here lend little support to this hypothesis. Lymphocytes from patients on non-adrenergic drugs behaved almost identically (Figure 14) to cells from normal subjects when the results were analysed in percentage terms which seems preferable for the reasons given in Chapter III.

When the data presented in this chapter is analysed in terms of absolute increase in cyclic AMP (Figure 16), there is a significant difference (p.40.001) between the results for normals and for asthmatics on non- adrenergic treatment. This could be evidence of a relatively minor inherent defect in beta adrenoceptor function as postulated by Szentivanyi.

Also cells from asthmatics on non-adrenergic medication had a significantly (p<0.05) reduced baseline level of cyclic AMP compared to normals. Lower control levels have been found in asthmatic's cells by some other investigators (Gillespie et al., 1974; Parker and Smith, 1973) but not by all (Makin et al., 1977). Again, this could conceivably reflect an inherent defect in asthmatic beta adrenoceptors.

A possible explanation of the phenomena found may be increased adrenergic drive and catecholamine production in asthmatics causing a hormone-induced desensitization of beta adrenoceptor response. However, data in support of this hypothesis is not clear. In one study Morris et al. (1972) found that urinary adrenaline was significantly higher in asymptomatic asthmatic children than in normals. Mathe- andKnapp (1969) noted no difference between two groups of young adults, one normal subjects and the other asthmatics, but found that under stress, the asthmatics excreted less adrenaline in the urine.

Our results show that large amounts of beta adrenergic bronchodilators significantly depress lymphocyte beta adrenoceptor responsiveness in :94: asthmatics. It is possible that this phenomenon was peculiar to these patients, or that their cells were particularly sensitive. To substantiate this possibility we studied the cells from normal subjects "treated" with various regimens of beta adrenergic stimulants. These data are presented in Chapter V. : 95

CHAPTER V

NORMAL SUBJECTS "TREATED" WITH BETA ADRENERGIC STIMULANTS :96:

5.1 INTRODUCTION Data in the previous chapter showed that lymphocytes from asthmatics taking large doses of beta adrenergic bronchodilators have a depressed cyclic AMP response to isoprenaline. If the asthmatic beta adrenoceptor is abnormal, as postulated by Szentivanyi, it could be particularly susceptible to the development of drug-induced desensitization. However, if lymphocytes from normal subjects show a similar desensitization it could mean that the diminished beta adrenoceptor response demonstrated in asthmatics is due to drug therapy rather than to the atopic diathesis.

Tolerance to the effect of beta adrenergic stimulants in normal subjects has been shown in several systems by different workers. Conolly et al. (1971) demonstrated tachyphylaxis of the cardiac beta adrenoceptors in man after isoprenaline infusions. Nelson and his co-workers (1973 and 1975) showed tolerance in normal subjects to some of the metabolic effects of adrenaline after treatment for one week with usual doses of ephedrine alone or of the combination product "Tedral" (ephedrine hydrochloride 24 mg, theophylline 130 mg, phenobarbital 8 mg). Holgate et al., (1977) recently reported that in normal subjects chronic administration of inhaled salbutamol (up to 400 ylg four times a day for four to five weeks) induced tolerance to its effect as demonstrated by the SGaw, the reciprocal of pulmonary resistance. However, other workers using dogs (Minatoya and Spilker,1975), cats (Atkinson and Rand, 1968) and man (Kingsley et al., 1972; Pun et al., 1971) have been unable to produce beta adrenoceptor tachypylaxis in intact subjects.

Parker and Smith (1973) studied leucocytes from five normal subjects who had taken four "Tedral" tablets daily for two weeks. They were unable to show any diminution of beta adrenoceptor function as measured by cyclic AMP production.

In the experiments described in this chapter, three groups of normal subjects were studied before and after taking beta adrenoceptor stimulants and a fourth group who had phaeochromocytomata. One group took salbutamol, orally 16 mg daily, a dose commonly used clinically. The second group inhaled four times the recommended daily dose of salbutamol (i.e. 3.0 mg) an amount which could easily be taken by an asthmatic during an acute attack. The third group consisted of four obstetric patients :97: given large doses of the beta adrenoceptor agonist isoxsuprine by infusion to prevent the onset of labour after intra-uterine transfusion of the foetus.

The fourth group of non-asthmatic patients studied had phaeochromocytomata. If excess exogenous beta adrenergic stimulation in the form of bronchodilator treatment depresses lymphocyte beta adrenoceptor function as measured by cyclic AMP production, patients with increased endogenous catecholamine production may also have impaired beta adrenoceptor responsiveness. This might revert to normal after removal of the catecholamine-producing tumour. Blood from four patients with phaeochromocytomata was obtained and lymphocyte cyclic AMP production was assayed. One of them was re-studied after removal of the tumour when plasma noradrenaline was normal.

5.2 SUBJECTS AND METHODS

5.2.1 Normal Subjects on Oral Salbutamol 12-16 mg/day Three normal subjects, two men and one woman, were studied. Samples of blood were taken on several occasions for control dose response curves. Subjects then took salbutamol 12-16 mg in divided doses for 10 to 12 days. The morning dose of drug was taken as usual two to three hours before the study.

One subject's cells were studied once at the end of this regime, one subject three times and one four times.

5.2.2 Normal Subjects on Excessive Salbutamol by Inhalation 3 mg (30 metered doses) Daily Five normal men participated in this study, two of whom had taken part in the first experiment. A similar protocol was followed with control dose response curves before the study began and further blood samples were taken at the end of a seven to 12 day period of "treatment". The subjects took 30 metered doses (normal recommended dose eight inhalations) during a 24- hour period. One subject took six inhalations six hourly, another two inhalations an hour, while awake.

:98:

5.2.3 Obstetric Patients Given Prolonged Infusions of Isoxsuprine

Four obstetric patients were studied before and after a 48-hour infusion of isoxsuprine (5 mg/hour). This was required to prevent labour following an intra-uterine transfusion for rhesus incompatibility. During the second 24-hour period, they were also given intramuscular injections of isoxsuprine 30 mg at eight-hour intervals. The patients were between 29 and 32 weeks pregnant and none went into labour on this regime. None of them had a history suggestive of asthma.

5.2.4 Patients with Excess Endogenous Catecholamine Production Due to Phaeochromocytomata

Four patients with proven phaeochromocytoma were studied. Cells from one (Subject 8), were obtained on two occasions before removal of his tumour and once three months following the operation. At the time of the last blood sample both urinary vanillyl mandelic acid and plasma noradrenaline were at the upper end of the normal range. Two other patients (Subject 5 and Subject 6) were only studied pre-operatively, both returning abroad after the operation. The fourth patient (Subject 7) had a malignant phaeochromocytoma partially removed in 1966. His urinary and plasma catecholamines remained elevated. Clinical details are given in Table XIII.

Plasma noradrenaline was determined by the method of Henry et al. (1975) and urinary vanillyl mandelic acid by that of Pisano et al. (1962).

Lymphocytes were isolated, incubated with a range of concentrations of isoprenaline in the presence of theophylline, and the cyclic AMP was purified and assayed as previously described (Chapter II). The results before and after salbutamol or isoxsuprine were compared statistically by two-way analysis of variance with replication (Chapter II). The results of these patients have been compared statistically to the mean dose response curve for normal subjects (Chapter III) as only one patient's cells were examined after resection of the phaeochromocytoma, when catecholamine production had returned to normal. :99:

TABLE XIII

Clinical Details: Patients with Phaeochromocytomata

Plasma Noradrenaline Patient (normal range No. Drugs 0.1-0.7 ng/ml) 5 None 5.2 ng/ml lying

6 None 2.27 ng/ml lying 2.48 ng/ml standing

7 - malignant phaeochromocytoma B.P. normal None 1.42 ng/ml lying 1.65 ng/ml standing

8 - pre-op. 11.2.75 None 2.54 ng/ml lying

- post-op. 25.6.75 Bendrofluazide 10 mg daily 0.819 ng/ml lying 100 :

5.3 RESULTS 5.3.1 Normal Subjects on Oral Salbutamol 12-16 mg Daily

The dose response curve obtained from lymphocytes of three normal subjects before taking salbutamol is shown in Figure 20. The relevant data in terms of proportional increase are given in Table XIV; Table XV shows the absolute increase (picomoles of cyclic AMP per 4 million cells) for reference. There is an extensive inter- and intra-individual variation similar to that found in the results presented in Chapter III. The present data fall within the range spanned by that of the larger group of normal subjects.

The results after these subjects took oral salbutamol for 10 to 12 days are shown on the same figure and tables. The percentage increase in cyclic AMP formed with isoprenaline stimulation was modestly but significantly reduced (1)40.05) in lymphocytes isolated at the end of 4 the "treatment" period. Isoprenaline 10 moles. litre-1 produced a median of 486% (IQR 328 - 517) increase in cyclic AMP over basal levels as compared to 531% (IQR 368 - 648) before salbutamol.

5.3.2 Normal Subjects Taking Excessive Salbutamol by Inhalation

The cyclic AMP response to isoprenaline in lymphocytes from normal subjects before and while taking large amounts of salbutamol by metered dose aerosol is shown in Tables XVI and XVII and Figure 21. There was a significant reduction (p4.0.01) in beta adrenergic responsiveness in percentage terms after bronchodilator use. At the maximal dose of isoprenaline used 4 -1 (10 moles. litre ), lymphocytes isolated pre-"treatment" showed a median 330% (IQR 262 - 471) increase in cyclic AMP. After the "treatment" period cyclic AMP increased by only 189% (IQR 154 - 331) over the unstimulated level.

5.3.3 Obstetric Patients Given Prolonged Infusions of Isoxsuprine Figure 22 and Table XVIII show the results for four obstetric patients as percentage increase in cyclic AMP after incubation of their lymphocytes with isoprenaline. The control dose response curve before the patients received any isoxsuprine is not significantly different from that of normal subjects (Chapter III). Cyclic AMP produced by stimulation with -4 -1 isoprenaline 10 moles. litre was 4410 (range 380 - 589) above the 101 :

Figure 20 Effect of oral salbutamol (12 - 16 mg/day for 10 days) on lymphocyte responsiveness in 3 normal subjects (median values plus interquartile range) 648 642 600 — e pre salbutamol o post salbutamol ..••••111

0

500 — P < 0.05 0

•

400 7" 0

t-) >, (-)

3G0

Si or ,/ 0 O it U S- 0- 200—

I

10-8 10 -7 lo ° 10-5 104 Molar concentration of isoprenaline : 102 :

TABLE XIV

Normal Subjects Taking Oral Salbutamol (12-16 mg/day): Lymphocyte Cyclic AMP Response to Isoprenaline Percentage increase over baseline unstimulated level

CONTROL STUDIES

Baseline Molar Concentration of Isoprenaline Name Unstimulated 8 -6 Cyclic AMP 10 10-7 10 10-5 10-4

MEC' ( 25) 36 104 288 484 (101) 34 122 177 218 368 ( 43) 251 626 747 740 779 (135) 65 219 627 573 620 ( 93) - 265 469 327 297 JKG ( 82) 62 287 387 - - ( 80) 46 295 365 - _ ( 34) 3 56 341 679 - ( 26) 27 446 781 - ( 43) 79 242 395 398 488 RM ( 50) 150 546 550 582 648 ( 50) 258 498 580 642 660 ( 13) 115 338 554 615 531 ( 37) 51 197 270 262 251

Median ( 47) 62 276 432 573 531 IQR (34-82) 36-115 197-446 341-580 398-642 368-648

AFTER SALBUTAMOL

NEC ( 47). 104 226 360 515 517 ( 50) 68 622 632 1076 692 ( 24) 29 342 113 488 508 JKG ( 67) 33 300 270 282 328 (170) 78 195 135 ( 36) 22 222 275 317 297 ( 14) 57 293 457 45o 486 RM ( 26) 15 85 142 185 181

Median ( 42) 33 260 273 384 486 IQR (26-50) 29-68 222-300 195-360 282-488 328-517 : 103 :

TABLE XV

Normal Subjects Taking Oral Salbutamol (12-16 mg/day): Lymphocyte Cyclic AMP Response to Isoprenaline Data given as baseline unstimulated level and increase above this (picomoles cyclic AMP/4 x 106 cells)

CONTROL STUDIES

Baseline Molar Concentration of Isoprenaline Name Unstimulated -b -4 Cyclic AMP 10 107 10-6 105- 10

MEG 25 9 26 72 121 - 101 34 123 179 220 372 43 108 269 321 318 335 135 88 296 846 774 837 93 - 246 436 304 276 JKG 82 51 235 317 - - 8o 37 236 292 - - 34 1 19 116 231 - 26 7 116 203 - - 43 34 104 170 171 210 RM 50 75 273 275 291 324 5o 129 249 290 321 330 13 15 44 72 8o 69 37 19 73 100 97 93

Median 47 34 179 239 231 324 IQR 34-82 15-75 73-249 116-317 171-318 210-335

AFTER SALBUTAMOL

MEC 47 49 106 169 242 243 5o 34 311 316 538 346 24 7 82 27 117 122 JKG 67 22 201 181 189 220 170 133 332 23o 36 8 8o 99 114 107 14 8 41 64 63 68 RM 26 4 22 37 48 47

Median 42 8 94 134 153 122 IQR 26-50 8-34 80-133 64-181 114-230 107-243

: 104 :

Figure 21 Effect of salbutamol inhalations (30 doses = 3.0 mg/day for 10 days) on lymphocyte responsiveness in 5 normal subjects (median values with interquartile range)

500 oPre inhalation o Post inhalation

P < 0.(.31

NINO •••■ ••■• IM■

1 _7 I _6 -8 -5 10 10 10 10 10 Molar concentration of isoprenaline : 105 :

TABLE XVI

Normal Subjects Taking Large Amounts of Inhaled Salbutamol (30 inhpiations/day): Lymphocyte Cyclic AMP Response to Isoprenaline

Percentage increase over baseline unstimlated level

CONTROL STUDIES

Baseline Molar Concentration of Isoprenaline Unstimulated Name -8 --47 Cyclic AMP 10 10 10- 6 10-5 10

HF ( 52) 12 125 188 192 219 ( 52) 52 119 181 202 262 MRB ( 9) 44 100 133 167 144 ( 17) 65 159 341 229 282 EEC ( 59) 78 302 681 937 800 JCM ( 55) 62 262 471 473 587 ( 22) 105 359 427 332 377 RM ( 24) 75 383 458 488 471

Median ( 38) 64 211 384 281 330 IQR (22-52) 52-75 125-302 188-458 202-473 262-471

AFTER SALBUTAMOL

HF ( 13) 15 108 138 185 154 ( 19) 32 116 121 121 189 MRB ( 9) 67 289 533 467 433 ( 14) 5o 264 479 543 600 MEC (290) 50 58 86 34 101 JCM (121) 50 141 291 364 331 ( 80) 30 50 108 125 163 RM ( 36) 31 172 425 325 328 (110) 14 0 20 38 13

Median ( 36) 32 116 138 185 189 IQR (14-110) 30-50 58-172 108-425 121-364 154-331 _ _. : 106 :

TABLE XVII

Normal Subjects Taking Large Amounts of Tnhaled Salbutamol (30 inhalations/day): Lymphocyte Cyclic AMP Response to Isoprenaline

Data given as baseline unstimulated level and increase above this (picomoles cyclic AMP/4 x 106 cells)

CONTROL STUDIES

Baseline Molar Concentration of Isoprenaline Unstimulated Name -8 -7 -6 -5 Cyclic AMP 10 10 10 10 10

RF 52 6 65 98 100 114 52 27 62 94 105 136 ' MRB 9 4 9 12 15 13 17 11 27 58 39 48 MEC 59 47 178 402 553 472 JCM 55 34 144 259 260 323 22 23 79 94 73 83 RM 24 18 92 110 117 113

Median 38 21 72 96 103 114 IQR 22-52 11-27 62-92 94-110 73-117 83-136

AFTER SALBUTAMOL

HF 13 2 14 18 24 20 19 6 22 23 23 36 MRB 9 6 26 48 42 39 14 7 37 67 76 84 MEG 290 145 169 248 100 294 JCM 121 60 171 352 44o 401 80 24 40 86 100 130 RM 36 11 62 153 117 118 110 15 0 22 42 14

Median 36 11 37 67 76 84 IQR 14-110 6-24 22-62 23-153 42-100 36-130 : 107 =

Figure 22 Lymphocyte responsiveness in 4 obstetric patients before (4) and • after (o) a 48 hr infusion of isoxsuprine (S mgih) (median ÷ range)

500

400 P M A ic l c cy in

se 300 rea inc t rcen Pe 200

100

0. 1 -8 1_7 1 -6 1 -3 1 -4 10 10 10 10 10 Molar concentration- of isoprenaline : 108 :

TABLE XVIII

Obstetric Patients Studied Before Isoxsuprine Percent increase in cyclic AMP

Baseline Molar Concentration of Isoprenaline Subject Unstimulated No. 8 -7 -6 -4 Cyclic AMP 10 10 10 lo-5 10

1 (51) 71 38o 355 435 467 2 (78) 114 324 340 413 415 3 (18) 44 189 406 222 589 4 (10) 5o 200 38o 37o 38o

Median 35 61 262 368 392 441

Same Patients Studied After Isoxsuprine for 48 hours Percent increase in cyclic AMP

Molar Concentration of Isoprenaline Subject Baseline Unstirnulated -8 -4 No. Cyclic AMP 10 10-7 10-6 10-5 10

1 (36) 94 150 175 158 (10) 70 90 380 150 280 2 (15) 60 8o 120 193 160

3 (36) 6 33 69 89 103 4 (15) 13 27 107 173 187

Median 15 37 80 120 173 160 : 109 :

unstimulated level as compared to that 377% (IQR 282 - 569) in men and non-pregnant women. A large degree of inter-individual difference (almost ten-fold) is seen in these four patients similar to that seen in normal subjects.

After treatment with isoxsuprine, cyclic AMP production in response to isoprenaline stimulation fell significantly (p40.001) in both percentage and absolute (picomoles cyclic AMP) terms. Lymphocytes incubated with 4 -1 isoprenaline 10 moles. litre showed an increase in cyclic AMP of only 160% (range 103 - 280). These results are shown in the same tables and figures. The basal levels of cyclic AMP produced by unstimulated cells (Table XIX) are lower after isoxsuprine treatment but the difference is not significant.

In one instance (Patient 3), cells were incubated with isoprenaline in 2 -1 concentrations up to 10 moles. litre to determine whether the higher dose could improve lymphocyte cyclic AMP response. This did not make an appreciable difference either in the pre-infusion study or after isoxsuprine. Cells isolated after the 48-hour infusion showed an increase -1 -2 -1 of 90% at 10-3 moles. litre and 81% at 10 moles. litre . This was slightly less than the increase seen at the standard maximal dose of 4 -1 10 moles. litre (105%). Thus, after prolonged treatment of the patients with a beta adrenoceptor stimulant the dose response curve of cyclic AMP production with isoprenaline stimulation shifted to the right and the maximal effect obtainable was reduced.

5.5.4 Patients with Phaeochromocztomata Data from cells of four patients with proven phaeochromocytomata are shown in Tables XX and XXI and Figure 23. Results from the 11 normal subjects (see Chapter III) are included on the figure and at the bottom of the tables for comparison. The percentage increase in cyclic AMP was significantly lower in patients with excess endogenous catecholamines (1)40.001) than in the group of normals, and reachola median of 263% (range 123 - 356) as compared to 377% (IQR 282 - 569).

One subject (Patient 8) was also studied after removal of his tumour. His two dose response curves are shown in Figure 24; post-operatively his : 110 :

TABLE XIX

Obstetric Patients Studied Before Isoxsuorine Absolute increase in cyclic AMP per 4 million cells

Baseline Molar Concentration of Isoprenaline Subject Unstimulated - No. -8 -7 -6 -4 Cyclic AMP 10 10 10 10-5 10

1 51 36 194 181 222 238 2 78 89 253 265 322 324

3 18 8 34 73 40 106 4 10 5 20 38 37 38

Median 35 22 114 127 131 172

Same Patients Studied After Isoxsuprine for 48 hours Absolute increase in cyclic AMP per 4 million cells

Molar Concentration of Isoprenaline Subject Baseline No. Unstimulated -8 -7 6 -4 Cyclic AMP 10 10 10- 10-5 10

1 36 34 54 63 57 10 7 9 38 15 28 2 15 9 12 18 29 24 3 36 2 12 25 32 37 4 15 2 4 16 26 28

Median 15 6 12 25 29 28 : 111 :

Figure 23. Lymphocyte responsiveness in normal subjects (e) and oatients with phaeochrompcytoma (o) (Median ÷ range) 600 —

•••••11*

500 —

Molar concentration of isoprenaline : 112 :

TABLE XX

Patients with Phaeochromocytomata: Lymphocyte Cyclic AMP Response to Isoprenaline

Percentage increase over baseline unstimulated level

Baseline Molar Concentration of Isoprenaline Subject Unstimulated No. -8 -4 Cyclic AMP 10 10-7 10 10-5 10

5 (16) 31 213 363 356 356 6 (16) 0 0 38 194 263

7 (26) 4 50 96 188 123 8 (37) 27 92 138 211 200 (25) -8 24 268 404 320

Median (25) 4 50 138 211 263

Normal Subjects

Median (47) 55 224 360 332 377 IQR (26-61) 36-79 138-338 249-474 262-572 282-569 : 113 :

TABLE XXI

Patients with Phaeochromocytomata: Lymphocyte Cyclic AMP Response to Isoprenaline

Data given as baseline unstimulated level and increase above this (picomoles cyclic AMP/4 x 106 cells)

Baseline Molar Concentration of Isoprenaline Subject Unstimulated No. 8 Cyclic AMP 10 10-7 10-6 105 10 . - 5 16 5 34 58 57 57 6 16 0 0 .6 31 42 7 26 1 13 25 49 32 8 37 10 34 51 78 74 25 -2 6 67 101 80

Median 25 1 13 51 57 57

Normal Subjects

Median 47 19 82 150 168 160

IQR 26-61 7-37 57-194 94-259 80-258 87-324 U increase in cyclic AMP 800 400 600 200 Figure 24LymphocytecyclicAMPresponseinapatientwith phaeochromocytoma beforeandafteroperation. percent increaseoverbaseline 10 molar concentrationofisoprenaline 7

: i lk : 10 -

10 -

10 -4 : 115 :

lymphocyte cyclic AMP response was considerably improved rising to 868% at the maximal dose of isoprenaline as compared with a mean of 260% in two pre-operative studies.

5.4 DISCUSSION

The results from the first three groups of normal subjects show that lymphocyte beta adrenergic responsiveness as measured by cyclic AMP production is reduced by prolonged exposure to beta adrenergic stimulants. Subjects given a normally recommended dose of oral salbutamol (16 mg daily), showed a modest but significant reduction (p40.05) in response to isoprenaline stimulation. Large amounts of salbutamol by pressurized aerosol (3 mg daily) also produced a significant reduction (104.0.01) in beta adrenoceptor responsiveness. This dose, which exceeds the manufacturer's instructions by a factor of four, was chosen because a mild asthmatic (Patient 17, Chapter IV) had shown depressed lymphocyte cyclic AMP response to isoprenaline while taking approximately this amount and it is a realistic quantity of drug for an asthmatic to take during an exacerbation.

The obstetric patients received large amounts of the beta adrenergic stimulant isoxsuprine during the treatment period. Initially they complained of palpitations and postural hypotension; after 48 hours this had entirely disappeared in three of the four patients. The response of lymphocyte cyclic AMP to isoprenaline showed a highly significant reduction (p4:0.001) after the infusion in both percentage and absolute (picomoles cyclic AMP) terms.

The unstimulated basal levels of cyclic AMP found in these three groups of patients were not significantly different before and after treatment. Lower basal levels in asthmatic subjects on non-adrenergic drugs has been noted by some workers (Gillespie et al., 1974; Parker and Smith, 1973) and one group of asthmatics studied in Chapter IV (those on non-adrenergic drugs) showed a significantly (1)4:0.05) lower basal level. The obstetric patients who had the most depression in beta adrenoceptor responsiveness apparently had lower levels of baseline cyclic AMP than normals, and similar results were found in patients with phaeochromocytoma. However, the use of beta adrenergic agonists did not result in a statistically higher or lower basal level of cyclic AMP. : 116

Treatment with exogenous beta adrenergic stimulants reduces beta adrenoceptor responsiveness in normals. Elevation of endogenous catecholamines had a similar depressant effect on the cyclic AMP response in four patients with phaeochromocytomata. Cells from these patients responded less well to isoprenaline than lymphocytes from normals (p40.001); in the one patient studied post-operatively, his cyclic AMP responsiveness improved. These data are in accord with work by Smith et al. (1975) which showed beta adrenoceptor desensitization in fat ■ •••■ NowWIN cells of patients with phaeochromocytomata.

Our data show conclusively that lymphocyte beta adrenoceptor function in non-asthmatic subjects is depressed by "treatment" with bronchodilators, other beta adrenergic stimulants or by increased endogenous catecholamines. Moreover, asthmatics are not peculiarly susceptible or sensitive to this desensitization, since obstetric patients also showed a very significantly reduced cyclic AMP response to isoprenaline when given large amounts of beta adrenergic stimulants.

After obtaining these results, the next step was to confirm that this desensitization occurred in cells from both normal subjects and asthmatics in a more controlled in vitro environment where the mechanism(s) could be studied. Those experiments are reported in the next chapter. 117

CHAPTER VI

PRODUCTION OF BETA ADRENOCEPTOR DESENSITIZATION IN VITRO 118 :

6.1 INTRODUCTION

Many workers using various cell types in vitro have produced desensitization to beta adrenoceptor agonists. Human fibroblasts (Franklin and Foster, 1973; Franklin et al., 1975) and adipocytes (Smith et al., 1976), rat macrophages (Remold -01 Donne11,1974), pineal tissue (Deguichi and Axelrod, 1973), lymphoid cells (Makman, 1971) and C6 astrocytoma cells (Browning et al., 1976) all show desensitization to beta adrenergic agents after exposure to these drugs as discussed in Chapter I.

Kalisker and Middleton (1975) incubated monolayers of human lymphocytes -1 with isoprenaline 105 moles. litre for one hour. Subsequent stimulation with isoprenaline resulted in a lower cyclic AMP response as compared with that of untreated cells; the response to prostaglandin El (PGE 1) however, was unaffected. They also incubated monolayers with PGE -5 -1 1 10 moles. litre for two-and-a-half hours but found no changes in the cyclic AMP response to either PGE or to isoprenaline. These experiments used relatively short-term incubations and a single high dose of isoprenaline to produce desensitization and did not explore the mechanism. In contrast to Kalisker and Middleton's results, Franklin and Foster (1973) using human diploid fibroblasts showed desensitization to PGE 1 after exposure to this compound, as well as reduced responsiveness to isoprenaline after pre-incubation with isoprenaline.

The mechanism of desensitization has been investigated by some workers. In fibroblasts (Russell and Pastan, 1974), lymphoma tissue (Bourne et al., 1973), brain tissue of guinea pigs (Schultz, 1975) and rabbits (Kakiuchi and Rall, 1968), and C6 astrocytoma cells (Schwartz and Passonneau, 1974; Browning et al., 1976) PDE activity was found to be increased after incubation with various catecholamines. Manganiello and Vaughn (1972) showed increased PDE activity in mouse fibroblasts treated with PGE for 24 hours. However, other workers using different cells, 1 species or incubation times have not shown a change in PDE activity following exposure to agents causing increased cyclic AMP (DeRubertis and Craven, 1976; Franklin and Foster, 1973; Smith et al., 1976).

This chapter describes experiments designed to investigate whether the cyclic AMP response of the lymphocyte beta adrenoceptor can be : 119:

desensitized in vitro, and if so, whether this is a dose-related phenomenon and whether it is due to increased PDE activity. Experiments on lymphocytes from normal subjects cultured for 24 hours with isoprenaline 8 -1 6 -1 10 moles. litre to 10 moles. litre and subsequently incubated with isoprenaline or prostaglandin El are described initially. In the second group of studies cells were cultured with PGE 1 for 24 hours and then exposed to isoprenaline or PGE1. Lymphocytes from asthmatic subjects were cultured with isoprenaline to see if they behaved differently from normal cells (experiment 3). In the last group of experiments, PDS activity was assayed in cells cultured with isoprenaline or PGE1.

6.2 MATERIALS AND METHODS FOR IN VITRO STUDIES

In this chapter "culture" always refers to the 24-hour pre-incubation.

6.2.1 Special Materials used in the in vitro Studies

Cell isolation and culture

TC 199 Media single strength (with penicillin and streptomycin) Burroughs Wellcome Ltd., Beckenham Sterile sodium bicarbonate 4.4% Burroughs Wellcome Ltd. Isoprenaline sulphate B.P. 50 micro- grams in 2 millilitres MacCarthys

Incubation

Prostaglandin El Gift from Dr. J.E. Pike, The UpJohn Company, Kalamazoo, Michigan, U.S.A.

Phosphodiesterase experiments

Dowex AG1 x 2 (200-400 mesh) Bio-rad Laboratories 14c adenosine Radiochemical Centre, Amersham 5-nucleotidase grade IV Sigma Chemical Co. Ltd., Kingston-upon-Thames

6.2.2 Cell Separation

Sterile syringes, needles, pipettes and test tube6 were used throughout the separation procedure. One hundred millilitres of single strength 120:

TC 199 Media with penicillin and streptomycin plus 1 ml of 4.4% sterile sodium bicarbonate and 10 ml fetal calf serum (FCS) was used as suspending media throughout. The heparinized blood was centrifuged at 250 g, plasma removed and the same volume replaced by the TC 199 mixture. This diluted 'blood' was layered over lymphoprep or previously autoclaved ficoll-hypaque as in the usual isolation procedure (see Chapter II). Following centrifuging at 400 g at the interface, the mononuclear cells were harvested, washed once with the buffered TC 199 mixture and re-suspended in this medium. An aliquot of the cell suspension was taken for estimation of cell numbers (Coulter Counter). 6 Further medium was added to yield a final suspension of 4 x 10 cells per millilitre.

6.2.3 Lymphocyte Culture for 24 hours with and without Isoprenaline and Prostaglandin E 1

The cell suspension was divided equally between the number of doses of isoprenaline and prostaglandin E1 to be used in the experiment and a control. Sterile isoprenaline, or prostaglandin El was diluted with the buffered TC 199 plus fetal calf serum to give final concentrations of -8 -1 -6 -1 isoprenaline 10 moles. litre to 10 moles. litre and of PGE -6 -1 -7 -1 1 2.8 x 10 moles. litre or 2.8 x 10 moles. litre . The dose of isoprenaline or PGE1 was added in a 10 p.1 volume to the cell suspension which was then divided into 1 ml aliquots in loose-capped tubes. Thus a number of tubes at each dose of isoprenaline was prepared. A flow chart of the culture procedure is shown in Figure 25.

The tubes were placed in an incubator in an atmosphere of 95% air and 5% CO for 24 hours at 37°C. At the end of this culture period the 2 cells were gently re-suspended, the replicates were pooled and HESS pH 7.35 was added to wash the cells. The suspension was centrifuged at 480 g, the supernatant discarded, and the cells then re-suspended in the original volume of HBSS plus FCS. This cell suspension was again divided into 1 ml aliquots and returned to the incubator to equilibrate with the mixture for 45 minutes at 37° 95% air and 5% CO2 C prior to the 15 minute incubation.

: 121 :

Figure 25 Flow chart of in vitro culture method

Isolate mononuclear cells (30 ml 4 x 10 cells per ml)

10 ml 10 ml 100 ml

'Blank' Culture Isoprenaline Cultures 4 x 106 cells/m1 TC199 4 x 106 cella/m1 TC199 with isoprenaline doses

Split into 1 ml aliquots 111111 1 W Ill 1111

24-hour culture at 37°C in 95% air + 5% CO2

II [HI 1P111111elli Ili 11111 it

1 1

Wash in EBSS, centrifuge, resuspend

Check viability

Split into 1 ml aliquots

1111

Incubate with theophylline 10 -6 -3 -4 -1 alone or with isoprenaline 10 , 10 , 10 moles. litre for 15 minutes at37°C in 95% air 5% CO2 1'31 1 III

'Pool duplicates I I Lij ILU I Liu.

Add recovery cyclic AMP purify and assay cyclic AMP

: 122:

6.2.4 Isoprenaline Remaining in the Culture Media after 24 hours The well known susceptibility of catecholamines to auto-oxidation made it necessary to estimate the likely loss of isoprenaline during the culture Period. This was performed by a method developed in this laboratory (Price, E., Davies, D.S. and Reid, J.L., unpublished). The method is 3 based on conversion of isoprenaline (in the presence of a H-methyl donor) to 3H 3 -0 -methylisoprenaline by catechol-O-methyltransferase. The radiolabelled product is isolated and then assayed by liquid scintillation counting.

Samples of media containing an initial isoprenaline concentration of -1 10-6 moles. litre with and without cells, were assayed before and after the 24-hour incubation period. There was a fall of about 65% in the concentration of isoprenaline at the end of this period in both cell-free and cell-containing samples.

6.2.5 Cell Viability After the 24-hour Culture Cell viability using the Trypan blue dye exclusion test was determined for cells cultured with and without isoprenaline after they had been re-suspended in HBSS. Ten microlitres of cell suspension were mixed with 10 pl of dye, left for five minutes and then examined under a microscope. If the cell suspensions cultured at different isoprenaline concentrations did not have similar viabilities, the experiment was discarded. The viabilities varied between experiments but were all above 70 percent.

6.2.6 Incubation Drug solutions for the 15 minute incubation were made up in 1.1 ml of HBSS 2 and gave a final incubation concentration of theophylline 10 moles. -o -4 -1 -7 -1 moles. litre or PGE 5 x 10 to litre and isoprenaline 10 to 10 1 1 10 5 moles. litre . The drugs were equilibrated for 45 minutes in the incubator with 95% air and 5% CO2 at 37°C and then mixed with the cell suspensions at time zero. The incubation lasted for 15 minutes at 37°C and was terminated by placing the tubes in boiling water for five minutes.

: 123 :

6.2.7 Addition of Recovery Cyclic AMP

Replicate tubes were pooled (see flow chart Figure 25). This was done because preliminary experiments indicated that the levels of cyclic AMP in cultured cells were very low. Pooling produced enough cyclic AMP in each sample to allow reliable assay. Labelled cyclic AMP, 0.18 picomoles (5000 cpm) was added after pooling to provide a measure of recovery at the end of the purification procedure.

The purification and assay of cyclic AMP has been described in Chapter II.

6.2.8 Subiects

For the initial experiments culturing cells with different concentrations of isoprenaline, six men and one woman donated blood on several occasions. None were on any medication.

Cells from four asthmatic patients were also studied. All were on beta adrenergic bronchodilators and one (Subject 2) took excessive amounts of isoprenaline by inhalation. Two were on systemic steroids in small dose and three on beclometbnsone by inhalation. Clinical details are given in Table XXII.

Lymphocytes from six normal subjects and from the asthmatic (Subject 2) who took excessive isoprenaline were cultured with PG 1. PDE activity was studied in the cultured lymphocytes of three normal men and one woman, none of whom were on any drugs.

6.2.9 Assay of PDE Activity Cells which had been cultured with and without isoprenaline or PGE1 were examined for PDE activity by a modification of the technique of Thompson and Appleman (1971). Tritiated cyclic AMP was converted to 5' adenosine monophosphate by cellular PDE and this product in turn was converted to adenosine by snake venom 5-nucleotidase added in excess. The rate of production of adenosine thus provided a measure of cellular PDE activity. -2 -1 The reaction mixture contained, in a 200 pl volume, 5 x 10 moles. litre 4 Tris HCl buffer at pH 8.5, 3H cyclic AMP (400,000 dpm), 4 x 10 moles. 1 -1 litre unlabelled cyclic AMP, 4 x 10 3 moles. litre MgC12, 0.2 units of 5-nucleotidase and 100 pl of lymphocyte homogenate. The latter was : 124:

TABLE XXII

Clinical Details of Patients with Active Asthma at the Time of Lymphocyte Study

Patient Spirometry No. Sex (FEV/FVC) Therapy 1 F 1.1/1.7 Beclomethasone 200 pg tds Salbutamol aerosol 400 pg tds

2 F 0.9/1.1 Prednisone 5 mg bd Beclomethasone 200 pg tds Isoprenaline aerosol 1200 pg/day

3 M 1.75/3.1 Prednisolone 5 mg alt. days Beclomethasone 200 pg tds Salbutamol aerosol intermittently

4 M 2.8/3.7 Salbutamol aerosol 600-1000 pg/day : 125 :

6 prepared from a suspension of 40 x 10 cells/ml in 5 x 10-2 moles. litre-1 Tris HC1, pH 8.5 by rapidly freezing and thawing the sample twice followed by homogenization in a tightly-fitting ground-glass homogenizer. The samples were incubated for 15 minutes at 37°C and the reaction terminated by placing the tubes in ice.

14 Fifty microlitres (5000 dpm) of C adenosine was added to quantitate recovery which varied between 80 and 95;5. Two millilitres of Tris HC1 buffer at pH 9 was added and the reaction mixture placed on a 4 cm column of Dowex AG1 x 2 (200-400 mesh, chloride form, Bio-rad Laboratories). The initial eluate and that obtained after a further 2 ml buffer (pH 9) had been applied to the column were discarded. Nine millitres of the same buffer was then added to the column and the entire fraction collected into a scintillation vial. Figure 26 shows the chromatographic separation of cyclic AMP and adenosine obtained by this method. At pH 9 cyclic AMP is not eluted from the column.

After the addition of 10 ml Instagel (Packard Instrument Company) the sample was counted in a Packard 2650 liquid scintillation spectrometer with automatic quench correction.

In three experiments PDE activity in the cytosol and cell membrane (particulate) fractions was measured separately.

After correction for losses during the purification, PDE activity was expressed in terms of nanomoles of cyclic AMP converted into adenosine during the 15 minute incubation. Results obtained from cells incubated with isoprenaline or with FGE1 were expressed as a percentage of the values obtained in cells incubated with buffer alone.

6.2.10 Statistical Analysis

The calculation of results was as described in Chapter II. The corrections for cell number due to pooling varied. A correction was also made for the viability of each cell suspension.

The data are presented as percent reduction in cyclic AMP formation or percent increase in PDS activity. Graphical presentation is of the median values and the interquartile ranges. Tests for significance between : 126 :

14 3 Figure 26 Elution profile foror C adenosine and H cyclic AMP using Dowex AG 1 x 2 (fraction volume 4 ml)

3 140 H CPN1 x 1000

14 120 C CPM x 1000 5 100 Adenosine cyclic AMP

•■•■■=0

4 80

3 60

2 40

1 20

0 FT; 2N HCI ' SIHCI TRISIHCI 2N HCI pH 9.0 pH 9.0 : 127 :

groups of observations was by the binomial sign test and analysis of the data relating to the dose ranging study of desensitization in normal subjects was by the non-parametric Spearman's Rank Sum Test.

6.3 RESULTS

6.3.1 Cyclic AMP Response to Lymphocytes from Normal Subjects Cultured with or without Isoprenaline

Lymphocytes cultured for 24 hours without isoprenaline had a low basal unstimulated level of cyclic AMP. They responded to isoprenaline 104 moles. litre-1 with a median increase of 384% (IQR 235 - 472) which was similar to the results of freshly isolated cells (median 377%. (IQR 282 - 569). This increase in cyclic AMP was significantly reduced in -6 -1 lymphocytes which had been cultured with isoprenaline 10 moles. litre during the 24-hour period (median 141%tigR 100 - 159)) p40.01. These results are shown in Table XXIII and Figure 27.

The desensitization of lymphocyte beta adrenoceptor function after culture with isoprenaline was dose related. There appeared to be a reduced responsiveness of cells cultured at 10 8 moles. litre-1, but this did not attain statistical significance in the numbers studied (n = 5). Cells cultured at the two higher doses of isoprenaline showed a significantly -1 reduced cyclic AMP response; those cultured at 10§ moles. litre - responded significantly worse (1340.05) than those cultured at 10 7 moles. -1 litre (n = S). The downward trend in responsiveness with increasing concentrations of isoprenaline was significant (134.0.001).

Figure 28 shows the results of 14 individual experiments plotted graphically. Increases in cyclic AMP found in cells cultured at different doses of isoprenaline for the 24-hour culture period have been joined for each individual experiment. The graph shows the dose related isoprenaline desensitization of cyclic AMP formation to subsequent isoprenaline stimulation which was seen in all exmeriments.

The intra- and inter-individual variation found when using this technique is also illustrated by this figure. One subject (MEC) was studied on five occasions; his control cells (i.e., cells not exposed to isoprenaline in the 24-hour culture) responded with an increase of 123% to 785% in : 128 :

Figure 27

Dose - related reduction in cyclic AMP response in lymphocytes from normal subjects cultured for 24 hours with isoprenaline and then incubated with isoprenaline 10-4 moles. Titre-1 n = 14 percent increase over baseline 500 (median + IQR or range)

* P< 0.05

400 * * P< 0.01

P n =5 Range AM

lic

c 300 n = 5 • Range cy in

e n = 9 as * * Incre t T n 200 — rce Pe

100 —

NIL 10 -8 10-7 10-6 Molar Concentration of I soprenaiine in Culture medium

: 129 :

TABLE

Percent Increase over Baseline in Cyclic AMP Response to Isoprenaline 10-4 soles. litre-1 in Lymphocytes Cultured with and without Isoprenaline for 24 hours

Molar Concentration 15 minute Incubation of Isoprenaline in -4 Donor 24-hour Culture Baseline Isoprenaline 10 o.4 MEC Nil-8 775% 23.9.75 10 0.7 545% MEC Nila 6.8 123% 14.10.75 lo 7.1 86% RS Nil-8 3.5 283% 7.11.75 10 3.5 251% 405% RP Nil-8 1.9 1.12.75 10 1.7 271% KG Nil 2.3 23% 3.12.75 10-7 2.9 90% 1.8 MEC Nil-7 15.12.75 10 2.2 54 10-6 2.5 92% KG Niles 3.7 303% 7.1.76 10 3.7 235% 10-6 2.5 196% Nil 3.6 489% RP -7 15.1.76 10-6 4.1 10 4.1 .T: 411% MEC Nil-7 1.9 27.1.76 10 1.9 232% 10-6 2.2 186% 424% CD Niles 6.3 24.2.76 10 6.8 1% s..7 Nil 20.2 362% 16.3.76 14.2 177% 1(0)-1 14.9 150% SJ Niles 6.3 195% 25.5.76 10 5.1 133% MEC N116 5.6 182% 1.7.76 10 3.7 159% sm Nil 6.9 472% 8.7.76 10- 6.9 100% Median a = 14 Nil 3.7 384% ma 1.9-6.3) (Ig2 235-472) -8 n = 5 10 3.5 251% n = 5 10-7 2.9 177% a = 9 10-6 4.1 141% (ic,2 2.5-6.8) (NeR 1o9-159) 130 :

Figure 28

Individual results of lymphocytes cultured with increasing concentrations of isoprenaline for 24 hours and then incubated with isoprenaline 10 4 moles. litre-1 percent increase in cyclic AMP over baseline 800 --t

600 P M A ic l c cy in se a re c in ■••• • •••••... •••••• ▪ 1.4„.••••■ • ••:N• ' ...... ••••■••-••••••• 200 j ... y . ...am, • !U.* • •■• • ••••• ••••• ••■•• • ••••• • •••• o .... • mime • ■•• • • •, ...... 0 • • • ••

1 -8 -7 NIL 10 10 10' Concentration of isoprenaline in culture medium ( moles I litre ) 131 :

cyclic AMP on incubation with isoprenaline 1 x 10-4 moles. litre-1. This is a 6.4-fold difference and represents the variation, in the group as a whole. His cells cultured with isoprenaline always responded less well, but again to a variable extent.

-1 The absolute amount of cyclic AMP in picomoles litre produced by cultured cells is shown in Table XXIV. The basal unstimulated level of cyclic AMP was not affected by isoprenaline in the culture medium. Some variation was seen but this was not consistent and did not attain statistical significance.

In five experiments cells which had been cultured with and without isoprenaline were incubated for 15 minutes with PGE1 5 x 107 moles. -1 -1 4 -1 litre or 10-5 moles. litre and with isoprenaline 10 moles. litre for the same period. The results are shown in proportional terms in Table XXV and Figure 29. As in freshly isolated cells (Parker, Huber and Baiiiann, 1973), incubation with PGE produced more cyclic AMP than 1 incubation with isoprenaline. Cells cultured with isoprenaline showed a depressed cyclic AMP response to isoprenaline as has been previously discussed, but there was no significant difference in the response of the cells to PGE1.

6.3.2 Cyclic AMP Response of Lymphocytes from Normal Subjects and One Asthmatic Patient Cultured with and without PGE1 -6 The results of cells cultured for 24 hours with PGE 2.8 x 10 moles. litre-1 or 2.8 x 10-7 moles. litre-1 and then incubated with either PGE or isoprenaline are shown in percentage terms in Table XXVII and 1 Figure 30. Culture with PGE1 for 24 hours significantly reduced (p<0.02) subsequent response to PGE1 from a median of 1424% (range 722 - 2109) to 486% (range 216 - 914) in those cells previously cultured with the 4 -1 drug. It also reduced the response to isoprenaline 10 moles. litre (p40.05). The data in picomoles of cyclic AMP produced is given in Table XXVIII. : 132 :

TABLE =Iv

Cyclic AMP Response to Isoprenaline 10-4 moles. litre-1 in Lymphocytes Cultured with and without Iscnrenaline for 24 hours piccmolea/4 x 106 cells increase over baseline level Molar Concentration 15 minute Incubation of Isoprenaline in Donor 24-hour Culture Baseline Isoprenaline 10-4 MEC Nil (control) 0.4 3.1 23.9.75 10-8 0.7 2.4 MEC mils 6.8 '8.4 14.10.75 10- 7.1 6.1 Ra Nil 3.5 9.9 7.11.75 10-8 3.5 8.8 RP Nil 1.9 7.7 1.12.75 10-8 1.7 4.6 KG Nil 2.3 5.4 5.12.75 10-7 2.9 2.6 MEC Nil, 1.8 10.0 15.12.75 10 2.2 3.1 10' 2.5 2.3 KG Niles 3.7 11.2 7.1.76 10 _6 3.7 8.7 10 2.5 4.9 212 Nil 3.6 17.6 15.1.76 10 4.1 9.5 4.1 5.8 MEC Nil., 1.9 7.8 27.1.76 10-' 1.9 4.4 10-6 2.2 4.1 CD Nil 6.3 26.7 24.2.76 10-6 6.8 0.1 s..7 Nil, 20.2 73.1 16.3.76 10... 14.2 25.1 10 14.9 22.3 SJ Nil6 6.3 12.3 25.5.76 10 5.1 6.8 MEC Niles 5.6 10.2 1.7.76 10 3.7 5.9 sm mils 6.9 32.6 8.7.76 10 . 6.9 6.9 Median n m 14 Nil 3.7 10.1 (NR 1.9-6.3) (IQR 7.8-17.6) -8 n = 5 lo 3.5 6.1 n = 5 107 2.9 4.4 n = 9 10-6 4.1 5.8 (IQ./ 2.5-6.8) (I;11 4.1-6.8) : 133

Figure 29

Response of lymphocytes from normal subjects to isoprenaline -4 -1 -5 -1 10 moles. litre and to PGE1 10 moles. litre after 24 hour -1 culture with isoprenaline 10 moles. litre percent increase over baseline (median ÷ range) PGE 1 n = 5 NS

1500

in . Isoprenaline se

ea n = 5 r

c p < 0.02 in t en c

r 500— Pe

-71.5%

NIL -6 10-6 10 Molar Concentration of Isoprenaline in Culture Medium : 134 :

TABLE XXV

Percent Increase in Cyclic AMP Response to Isoprenaline 10-4 moles. litre-1 or PGE1 10-5 moles. litre-1 in Lymphocytes Cultured with and without Isoprenaline for 24 hours

Molar Concentration 15 minute Incubation of Isoprenaline in -4 PGE 5 Donor 24-hour Culture Baseline Iso411560 --1--10 MEC Ni.]. 1.9 27.1.76 107 1.9 23 1642% 10- 6 2.2 160C%

CD Ni16 6.3 424% 714% 24.2.76 10 6.8 1% 812%

SJ Nil 20.2 36% 1197% 16.3.76 10-7 14.2 177% 82k% 10-6 14.9 150% 155

MEC Nil_ 5.6 182% 875% 1.7.76 10 3.7 159% 1211%

SM Ni16 6.9 472% 2109% 8.7.76 10 6.9 100% 2225%

Median Nil 6.3 411% 1197% lo-6 6.8 15o% 1552% :135:

TABLE XXVI

Cyclic AMP Response to Isoprenaline 10-4 moles. litre-1 or PGE1 10-5 moles. litre-1 in Lymphocytes Cultured with and without Isoprenaline for 24 hours 6 picomoles/4 x 10 cells increase over baseline level

Molar Concentration 15 minute Incubation of Isoprenaline in 4 Donor 24-hour Culture Baseline Iso 10 PGE101 5 MEC Nil (control) 1.9 7.8 30.8 27.1.76 101 1.9 4.4 31.2 10 2.2 4.1 35.2

CD Nil 6.3 26.7 45.0 24.2.76 10-6 6.8 0.1 55.2

SJ Nil 20.2 73.1 241.7 16.3.76 10-7 14.2 25.1 117.0 10-6 14.9 22.3 231.2

NEC Nil-6 5.6 10.2 49.o 1.7.76 10 3.7 5.9 48.5

SM Nil-6 6.9 32.6 145.5 8.7.76 10 6.9 6.9 153.5

Median Nil 6.3 26.7 49.o 6 10 6.8 5.9 55.2 136 :

Figure 30

Response of lymphocytes from normal subjects to isoprenaline -4 -1 -5 -1 10 moles. litre and to PGE1 10 moles. litre after 24 hour -6 -1 culture with PGE1 2.8 x 10 moles. litre percent increase over baseline (median ÷ range) PGE 1 n =6

2000 —

* p< 0.05

* * p< 0.01

1500 —

%-

0 0 >, (..) C a.) LEI 2 1000 --1 ,_0 C I soprenaline C n = 6 a) 0 * * s- a.) C.-

-68.25 500 — T *

-73.5%

[....,1 NIL 2.8x10-6 NIL 2.8x10-6 Molar Concentration of PGE1 in culture medium : 137

TABLE XXVII

Percept Increase in Cyclic AMP Response to Isoprenaline 10". moles. litre-1 or PGE1 10-5 moles. litre-1 in Lymphocytes Cultured with and without PGE1 for 24 hours

15 minute Incubation Molar Concentration Molar Concentration of Agonist of PGE1 in 4 -5 Donor 24-hour Culture Baseline Iso 10 PGE lo

CD Nil -6 7.0 319% 1859% 23.3.76 2.8 x lo 4.7 34% 457%

NEC Nil -6 13.3 226% 722% 25.3.76 2.8 x lo 9.7 178% 514%

MEC Nil 9.4 443% 1547% 22.4.76 2.8 x 10 5.5.88 45o% 914%

MEC Nil -6 184q 875% 1.7.76 2.8 x 10 6.2 65% 216%

SM Nil -6 6.9 472% 2109% 8.7.76 2.8 x 10 17.5 194% 391%

306% 1300% MEC Nil -6 3.1 29.9.76 2.8 x 10 4.6 61% 613%

Median Nil 7.o 313% 1424% -6 2.8 x 10 6.o 122% 486% :138:

TABLE XXVIII

Cyclic AMP Response to Isoprenaline 10-4 moles. litre-1 or PGE1 10-5 moles. litre-1 in Lymphocytes Cultured with and without PGE1 for 24 hours 6 picomoles/4 x 10 cells increase over baseline level

15 minute Incubation Molar Concentration Molar Concentration of Agonist of PGE1 in -5 Donor 24-hour Culture Baseline Iso 10-4 PGE1_____ lo CD Nil 7.0 22.3 130.1 23.3.76 2.8 x 10 4.7 1.6 21.5

NEC Nil 13.3 30.0 96.o 25.3.76 2.8 x 10 9.7 17.3 49.9

NEC Nil 9.4 41.6 145.4 22.4.76 2.8 x 10 5.8 26.1 53.o

NEC Nil 5.6 10.2 49.0 1.7.76 2.8 x 10 6.2 4.o 13.4 sm Nil -6 6.9 32.6 145.5 8.7.76 2.8 x 10 17.5 33.9 68.5

MEC Nil 3.1 9.5 40.3 29.9.76 2.8 x 10 4.6 2.8 28.2

Median Nil 7.o 26.2 113.1 -6 2.8 x lo 6.o 10.7 39.1 : 139:

6.3.3 Cyclic AMP Response of Lymphocytes from Asthmatic Subjects Cultured with and without Isoprenaline or PCE -6 After exposure to 10 moles. litre-1 isoprenaline for 24 hours lymphocytes from four asthmatic patients also showed a significantly reduced (p40.05) cyclic AMP response to this drug. The results are shown in Table XXIX in percentage terms and in Table XXX in terms of picomoles of cyclic AMP produced. The response of lymphocytes not exposed to isoprenaline for the 24-hour culture period was lower than that of cells from normal subjects, but this was not significant (p>0.05, unpaired T test). After culture with isoprenaline the cells from asthmatics responded significantly less well than those from normal subjects (p40.025). Nevertheless, as shown in Figure 31 the impairment of response in cells from asthmatics and normals was very similar in percentage terms (68.2% to 68.7%). Cells from the asthmatic who took excessive amounts of inhaled isoprenaline (Subject 2) were also after culture with isoprenaline (Table XXXI). The incubated with PGE1 cyclic AMP response to PGE1 was within the range spanned by the normal subjects, the response of her control cells to isoprenaline was poor.

6.3.4 Assay of Phosphodiesterase Activity in Lymphocytes Cultured with Isoprenaline or PGE1 Lymphocytes cultured with and without isoprenaline 10 6 moles. litre-1 6 1 and PGE 2.8 x 10 moles. litre were assayed for total and for 1 membrane-bound PEE activity. After culture with PGE1, total PDE activity increased 23.8% (median value) (1)40.02) whereas the change in activity after isoprenaline culture was extremely variable and did not achieve statistical significance (Table XXXII). PDE activity in the membrane fraction was not increased significantly by either drug (Table XXXIII).

6.4 DISCUSSION The above results show that lymphocyte beta adrenoceptor cyclic AMP response can be desensitized by exposure to isoprenaline in vitro over a 24-hour period. This is a concentration-related phenomenon (p 40.001). 8 The lowest concentration used, 10 moles. litre-1 which is approaching "physiological" levels, produced a suggestive although insignificant effect. The effect was probably insignificant due to the small amount of isoprenaline remaining in the culture after 24 hours and to the small number of subjects. Therefore, it seems possible that a degree of desensitization : 140 :

TABLE XXIX

Percent Increase in Cyclic AMP Response to Isoprenaline 10-4 moles. litre-1 in Lymphocytes from Asthmatic Subjects Cultured with and Without Isoprenaline for 24 hours

Molar Concentration 15 minute Incubation of Isoprenaline in -4 Donor 24-hour Culture Baseline Isoprenaline 10

1 Nil 4.4 248% 10-6 4.7 85%

2 Ni16 4.1 168% 10 3.3 61%

3 Nil 4.6 120% 10 4.4 66% lo-6 4.0 38%

4 Nil 5.2 162% -7 9.o 49% 10 6 lo 6.5 23%

Median Nil 4.5 165% 10-6 4.4 50% : 141 :

TABLE XXX

Cyclic AMP Response to Isoprenaline 10-4 moles. litre-1 in Lymphocytes from Asthmatic Subjects Cultured with and without Isoprenaline for 24 hours 6 picomoles/4 x 10 cells increase over baseline level

Molar Concentration 15 minute Incubation of Isoprenaline in Donor 24-hour Culture Baseline Isoprenaline lo 1 Nil (control) 4.4 10.9 10-6 4.7 4.o

2 4.1 6.9 Nil-6 10 3.3 2.0

3 Nil 4.6 5.5 -7 4.4 10-6 2.9 10 4.o 1.5

Nil 5.2 8.4 -7 10- 9.o 4.4 10 6 6.5 1.5

Median Nil 4.5 7.7 -6 10 4.4 1.8

142 :

Figure 31 Cyclic AMP response of lymphocytes from asthmatics and from normal subjects to isoprenaline 10-4 moles. litre-1 after culture with and without isoprenaline 10 6moles. litre-1 for 24 hours percent increase over baseline (median + range) Nor mals 500 —1 n = 9

400 — P < 0.05

P * P<0.01 AM

ic l c cy

in 300 —

se Asth manes rea n = 4 t Inc n ce Per 200 — * *

-68.25

100 —

-68.'77,

NI L NIL 10-6 10' Molar Concentration of I scprenaiine in Culture Medium TABLE XXXI

Cyclic AMP Response to Isoprenaline or PGE1 of Lymphocytes from Asthmatic Subject No. 2: 6 cells) Cells Cultured with and without Isoprenaline or PGE1 for 24 hours (picomoles/4 x 10

15 minute Incubation

Molar Concentration Molar Concentration of Agonist Isoprenaline or PGE1 4 -6 Isoprenaline 10 PGE 5 x 10 PGE10-5 in 24-hour Culture Baseline 1 1 Nil 4.1 6.4 28.8 79.4 -6 Isoprenaline 10 3.3 2.1) 27.8 92.2

PGE 2.8 x 10-7 6.1 2.7 10.0 21.2 1 -6 PGE 2.8 x 10 10.7 0 0 18.4 144:

TABLE XXXII

Effect of 24-hour Culture in the Presence of Isoprenaline or PGE, on Phosphodiesterase Activity in Total Lymphocyte Homogenate '

Drug-free Isoprenaline Prostaglandin Culture Culture Culture

Subject PDE PDE % PDE % No. Activity* Activity* Increase Activity* Increase 1 1.50 1.34 -10.7 1.34 -10.7 2 2.46 4.09 +66.3 2.47 +0.3 3 6.46 2.3o -64.4 8.10 +25.4 4 4.49 4.50 0 5.67 +26.3 5 1.72 1.71 -0.5 2.13 +23.8 6 9.96 10.30 +3.o 12.8o +28.5 7 0.88 1.70 +94.o 0.99 +12.9 8 2.19 2.18 -0.5 2.97 +35.6 9 3.51 3.18 -10.0 3.6o +2.5

*PDE activity expressed as nanomoles cyclic AMP hydrolysed/15 minutes/4 x 106 cells : 143 :

TABLE XXXIII

Effect of 24-hour Culture in the Presence of Isoprenaline or PGE on Phosphodiesterase Activity in Lymphocyte Membrane 1

Drug-free Isoprenaline Prostaglandin Culture Culture Culture

Subject PDE PDE % PDE % No.,16 Activity* Activity* Increase Activity• Increase 6 1.03 1.44 +39.8 1.00 -2.9

7 0.17 0.25 +48.5 0.65 +290.9

8 1.74 2.11 +21.6 1.89 +8.9

9 0.12 0.10 -16.7 0.13 +8.3

iSSame subjects as in Table XXXII

*PDE activity expressed as nanomoles cyclic AMP hydrolysed/15 minutes/4 x 106 cells 146:

of beta adrenoceptor function could occur in vivo in patients taking large amounts of beta adrenergic bronchodilators for their asthma.

It is unlikely that the phenomenon of desensitization is confined to isoprenaline. Franklin et al. (1975) showed that depending on their ability to stimulate cyclic AMP production, other beta adrenergic agonists such as salbutamol and adrenaline desensitized cultured human fibroblast beta adrenoceptor function. The newer beta2 adrenoceptor agonists could similarly cause desensitization, although they were not tested in these experiments.

The asthmatic subjects showed a degree of lymphocyte beta adrenoceptor desensitization similar to that of normals. However, cyclic AMP levels stimulatid in controlAcells were significantly lower in lymphocytes from asthmatics than in cells from normal subjects (p4c0.025). This may be because the asthmatic subjects studied here were all taking beta adrenergic bronchodilators. As shown in Chapter IV, this can affect beta adrenoceptor responsiveness and 'control' cells may have been partially desensitized in vivo. Despite the lower control levels, in vitro exposure to isoprenaline resulted in a similar degree of desensitization in both groups. These results,therefore, do not support the idea that beta adrenoceptors of asthmatics are more easily desensitized than those of normal subjects.

The desensitization of beta adrenoceptor function by isoprenaline is specific in that the response to PGE1 remains unimpaired. However, culture of lymphocytes with PGE1 for 24 hours produces diminished responsiveness to isoprenaline as well as to PGE1.

The role of PDE in the desensitization process remains uncertain. Examination of cytosol PDE activity and PDE activity in whole-cell homogenates showed clearly that PGE1 had a significant effect on overall activity while isoprenaline did not. However, the relatively modest increase in PDE, activity following stimulation with PGE1 and the inconsistent results in both cytosol and membrane-bound PDE, in cells treated with isoprenaline indicate that alteration in PDE activity could contribute only slightly to the desensitization process. : 147 :

Recently, other workers (Romero et al., 1975; Mickey et al., 1975; Mukherjee et al., 1975) using radioactive alprenolol as ligand have shown a decrease in binding sites presumed to be beta adrenoceptors in various cells and tissues desensitized to beta adrenergic agonists. It seems possible that a similar mechanism, presumably loss of receptors or deformation, occurs in lymphocytes though this has not yet been shown.

The results presented in this chapter show that lymphocytes exposed to isoprenaline in vitro become desensitized to this drug while maintaining their response to PGE1. This phenomenon occurs in cells from normal subjects as well as those from asthmatics. Thus these findings are in agreement with and confirm the studies described in Chapters IV and V: cells isolated from subjects on varying regimes of increased beta adrenergic stimulants showed poor cyclic AMP responsiveness. : 11+8 :

CHAPTER VII

CONCLUSIONS : 149:

7. CONCLUSIONS Evidence has been presented that the lymphocyte beta adrenoceptor has the adrenergic receptor. Therefore, it may be a characteristics of a beta2 valid model for the beta adrenoceptors in lung or bronchial smooth muscle.

Cyclic AMP response to stimulation of this lymphocyte beta adrenoceptor is modified by exposure to beta adrenergic stimulants both in vivo and in vitro. This phenomenon was demonstrated in vivo in a range of subjects - asthmatics, obstetric patients, normals and patients with phaeochromocytomata. Similar results were obtained in vitro when lymphocytes were incubated with isoprenaline for 24 hours.

Asthmatics taking large amounts of beta adrenergic bronchodilators over a prolonged period of time showed a seven-fold reduction (1340.001) in maximal lymphocyte cyclic AMP response after stimulation with isoprenaline compared to normals or asthmatics not taking these drugs. In some patients restudied after stopping this medication, lymphocyte response improved significantly (1)-(0.001) and returned to within the normal range.

Non-asthmatic obstetric patients given large amounts of isoxsuprine, a beta adrenoceptor agonist, to prevent labour also had a significant reduction (p-c0.001) in lymphocyte beta adrenoceptor response. Normal subjects taking four times the recommended dose of salbutamol by inhalation showed reduced (p<0.01) responsiveness as well. A small but significant (1340.05) reduction was also found in normals on a standard dose (16 mg daily) of the drug orally. Thus, treatment of the asthmatic or normal subject in vivo affects lymphocyte beta adrenoceptor function.

Four patients with phaeochromocytomata who had increased levels of circulating noradrenaline also had reduced lymphocyte beta adrenergic responsiveness (134(0.001) compared to normal subjects. One was restudied after removal of his tumor: cyclic AMP response had improved and was within the normal range.

Dose-related desensitization to isoprenaline was also demonstrated in vitro. Lymphocytes from normal subjects cultured for 24 hours with isoprenaline 150 :

-8 -1 - 1 10 moles. litre to 10-6 moles. litre showed a reduced cyclic AMP response to subsequent isoprenaline stimulation depending od the initial concentration of drug (p<0.001) as compared to cells cultured without the beta adrenoceptor agonist. Lymphocytes from asthmatic patients had 6 1 a similar degree (68% in cells cultured with 10 moles. litre isoprenaline) of drug-induced desensitization in this in vitro system.

The results presented in this thesis do not support Szentivanyi's theory of a partial defect in beta adrenoceptor function in asthmatics. Decreased lymphocyte beta adrenergic function has been demonstrated in these patients but the evidence strongly suggests that this is almost all due to drug treatment.

There is a small amount of evidence suggesting that a minor abnormality may exist in the lymphocyte beta adrenoceptor of asthmatics. The unstimulated cyclic AMP level in asthmatics on non-adrenergic drugs was significantly lower (p<0.05) than in normals. When the results in this group of patients were analysed in terms of the absolute amount of cyclic AMP produced, rather than proportional increase, there was a significant difference between the beta adrenoceptor response of cells from these asthmatics on non-adrenergic drugs and normal subjects (1)4.0.001). However, it should be noted that there was also a significant difference between the data from these patients and from asthmatics on large amounts of adrenergic bronchodilators (1)4:0.001).

Lymphocytes from asthmatic patients incubated for 24 hours with no drug had a significantly reduced cyclic AMP response (p

This could also explain the findings of Kariman and Lefkowitz (1977) who have reported that there is significantly reduced dihydroalprenolol binding (presumably beta adrenoceptor number) to lymphocytes from : 151 :

asthmatic subjects both on and off adrenergic drugs. However, no significant reduction in this receptor binding was found in a smAller group of subjects with inactive asthma.

In the great majority of asthmatics who are treated with small prescribed amounts of bronchodilators as required the phenomenon of reduced beta adrenergic responsiveness due to therapy is probably unimportant. These drugs appear to continue to benefit patients clinically. However, there is data from some workers suggesting that bronchial response to beta adrenergic stimulants may be altered by prior administration of these drugs in normal (Holgate et al., 1977) and asthmatic subjects (Nelson et al., 1977; Jenne et al., 1977).

Drug-induced desensitization of beta adrenoceptors in the lung would be far more crucial to the asthmatic with an acute attack. In this situation, the patient may take increasing amounts of drug by aerosol but respond less and less. A vicious circle could be set up; as more bronchodilator is taken, the beta adrenoceptor would become progressively less sensitive, cyclic AMP response fall and an alpha adrenergic effect might then come to predominate causing increased mediator release and bronchoconstriction (Orange et al., 1971). Receptor desensitization may contribute to the well-recognized clinical phenomenon of tolerance or non-response to beta adrenergic stimulants seen in some patients with a severe asthmatic attack. It may also have been an important factor in the increased death rate during the 1960s as mentioned in the introduction.

The mechanism of drug-induced desensitization remains to be elucidated. It could be due to change in the receptor population itself, a change in adenyl cyclase or its coupling to the receptor, in the amounts of precursor or co-factors, or to an increase in the phosphodiesterase that metabolizes cyclic AMP. Activity of the latter enzyme in the cytosol and membrane fraction was not increased in lymphocytes desensitized by 24-hour incubation with isoprenaline. PGE stimulation of cyclic AMP 1 gave normal results in these cells suggesting that precursor and co-factors were available, although specific pools of these substances were not analysed. 152:

There are several areas where future work could be directed. The question of the clinical importance of drug-induced desensitization during long term treatment is still unanswered. There is some evidence that bronchial smooth muscle from some individuals studied in vitro can be desensitized to beta adrenoceptor agonists more easily than that from others (Davis and Conolly, 1977). Some asthmatics, therefore, may be more at risk. If this is the case, it would be useful to identify them, as one would be more cautious about prolonged administration of this group of drugs to these patients.

Although lymphocytes are easily obtainable and thus a useful source of beta adrenoceptors, the response is too varied for clinically useful data to be obtained from one individual. However, the in vitro culture technique includes its own control and is suited to explore the mechanism of desensitization. The number of beta adrenoceptors could be studied using a ligand such as radioactive alprenolol as could the time course of desensitization and its reversal. Other cell functions, such as lymphocyte transformation or the production of lymphokines, could be investigated. Techniques to prevent or reverse receptor desensitization, in particular, culture with prednisone could be examined and the effect of alpha adrenoceptor blocking agents as well as of other drugs could be evaluated. : 153 :

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APPENDIX 1

COMPUTER PROGRAMME

C-3K FOCAL CD 197 4 01.03 T !"PACKAP.D COUNTER PROGRAM FOR C- AMP" ! !; E 01.05 D 7:* 01.10 A 5; C S) 1 • 1 • 01.15 I ( S- 1) 1.4;1 C S-OSTOPDATA) 1 • 2, 1.65 01.20 S Z.FNEW(J*L*K, 5) 21.30 S K=K+1;1 CK-2) 1.1;D •1:1) I •1:G 1.6 01.40 0 1.1;G 1.1 01.60 S H=0; S Ju...I+ 1; S K=0:G 1.1 01•65 S 1=1+1;1 C I+1-3*GR) 1.7; S I S NTC/)=J-3:S P.I*1;G 1.75 01.70 S MT( 1 )2..1:G 1 • 1 01.75 i:F 1-3*GR, -2; S NC -I )=NTC -I )-NTC - 1- 1) 01.00 I CNTC 1)-11) 1.85.1.8 1. 1-45 01 •8 1 S N2NTC 1)-1;1 CNT( 1 )-NC 2*GR) I .85, 1.12. •05 21.82 F 1=2,GP; I C1*NC I ) -N( 1*GFI* 1-I )-4C I - 1 41*GP.) )1.159 1.9. 1.85 71.05 T !!"ERSOR IN DATA !"!;G 1.06 01..06 T 72, !"GROUP 1."NTC 1); F 1.2, 3*GR- I; T ! "GROUP"! "*"N( ) ; R 01.90 C 01.95 S J=015 Ka0;1) 4.05: D 2.79

02 . es s xc 1).Ps; s xc s x( 3)=Ps. • 3: S X( 4)2PS• .5; S X( 5)=PS+ 1 22•06 5 XC 6)=PS+2; 5 X(7)=PS+3; X(8)=11545; S X(9)=145+7; S XC 10)ups4.10 02.10 0 6:S CI=C/T; NC P)*INTC 2*GR)- 1; D 6 02.12 S EiGnFI TR( /2+.5) ; T 23, !!! !"BACKGROUND "BG 02.15 S Nc P)•NTC 3*G71- 1); 0 6;5 FIE=FI TRC C/T+• 5) ; S P)=N(F)+1 22.16 T !"PECOVERY BACXGROUND "1113:1) 6;5 C1*C/T; S NC P)=Nc P)• 1; 0 6 32 • 17 S RS=FI TR( /2+ •5) ; T !"RECOVERY STANDARD "RS 08.20 S NC P)=NT( 2*GR) 1:17 J=1.NTc 1)- I:5 5%1.0; D 6; D 3 22.21 S N=I Y •=FEXPCY*L 1): D 1'0;T !.16.04. "REGRESS! ON LINES Y="A."*X+"B 22.22 T ! !"CCRRELATI ON COEFFI C1 ENT: "R. !!"LOwER L IMI T TR( Y**• 5) 22•24 I CI-10)2.8;F I./1. 10;5 X(/)=FECPCXCI)*L13 02.25 T !!" NO EXP. DATA COMP. DATA DI FF • " ! 02.30 D 2.70;F ,./..1•NT( 1)-1; S 514=0; D 6:0 3: T 172,1: 0 1. 4 02.35 D 2.24:6 2.65 02.40 T FEXP( YtL 1) 5 SaFEXPCLI*C0:5 SY*01 S X2*0;S Y2=0:5 sP.e 02.79 G 2.2 02.00 F 0=1.02-1;S N( P+ 1)=NIC 2*GR- 1.0)-NTC 0): RAC 0)=0; I) 4 02.05 T ! !: 03.10 I (X(J))3•5: S XCJ)=FLOGCXC,J) )/L 1; SX*SX•Xca); 5 X20(2+X(J) , P. 03.20 S 1=14 1; 5 Y*C/T; D 3• 5; I) 6; S Y=FLOGC FI TR*C Y•C/T) /2+ • 5*-FIG) /L I 03.30 S SY*SY•Y: Y2*Y2+Y12; S SP-.SP•X( LI)*Y: R 03.50 5 W=I•1-1 04.05 S H-0; 5 A1.0:5 A30; 5 AA-0; S RA=0 04.10 T SOPRENPLINIF DOSE RESPONSE CURVE OF /"!!! 24.20. T "ASSAY EC. C-AMP REC. REC. 04.21 T " TOTAL. ABS. AV. AV.1"! 04.25 T " CPM P.M. ML CFM S. 3.4.25 T " C-AMP INCR. INCR. ABS. INCR."! I 04.30 S NC 74)=NT( P*G R-0- 1 )-NTC 0) ; S 2*GR-0)+ I; P.1.1 04.40 F J=NI( 0), NTC 0-6 1)-1: 5 SI./*0: ID 6;D 5;D R 74.50 T ! I "NB INDICATES ASSAY CPM OUTSIDE LIMIT : 168 :

APPENDIX 1 (continued)

05.05 5 V=11-2; 5 Y=C/T;D 6; S Y=FI TRICY*C/T) 5›-SG; T Z21,Y 25.07 1 C Y-Y+)5.08;T " '•;G 5.1 05.03 T '•* •• 05.10 5 X*FEXPCL I.< FLOGCY) /L I-2) /A3; T 14.02,X-PS; S CM=CPC 0)*(X-PS) 05.20 T :5. 02, CM; S P=P+ I; 1=c•i; S 4=0;D 6;S W=tSS p=p- 05.30 S Y=C/T-R8; T 24.Y;5 Ta=100*Y1( RS-RH) 05.40 T 26.02, IC: S RzsCM*100/TC; T RZ

06.10 S G=L*K; I ( 5W) , 6. 2; 5 G=G+N( P)4•41 26.20 S Z=FNEVCG*J); t H- 1)6 . 3; S C=Z; S K=0; S SW= 1:R 06.30 S T=Z; S H=H+ I;G 6.1

07 .02 A "DATE OF ASSAY "S,L,Z; S L=200; S L I=FLOG( 10) 07.04 A !"HOW MANY GROUPS OF EXP • DATA ? "GR; S GR*GR• 1 005 A 1"H014 MANY PI CO-MOLES IN HOT STANDARD 7"PS 07.07 T 1"CORRECT1 ON FACTOR FOR PI CO-"POLES/ML " ! 27.01 F 1,GP.- 1; T Z1, "FOR GROUP "0, ": .CP( 0), ! 07 • 10 T !"SAMPLE AVERAGE FOR REFERENCE GROUP, 4 AVERAGE SEQUENCE: 27.15 D 2• 6A; F 0= I,GR- 1; T % I, !"FOR GROUP "0,'•: ";A ARC 0)• 1; D 7.2 07 . 16 G 7 • 9 07 • 20 A -SEGUFNICE: "5, 1;1 ( 5)7.3; 5 H=1-1* I: S ASCH+20*0)=5:G 7.2 27 • 30 5 H=0; C 07.90 A ! !'•DATA TAPE IN READER 7 ••5; t 5-0YES)7 .9

23.10 S AI=A1 4 1; S RA=RA‘R%; I CAI-ARC 0»4 .3,1 .2,1 .4 28.20 S RR=RA/ARC 0); T RR, 1:S H*H*1 03.30 T !;R 03 • 40 S P•=R%-RR; T P.J, 100*A i/F:P.; 5 A3= A3+ AA=AA*A4 0:3.50 I C A3-AS39 • 3;1 CAS

10.10 5 S=SP-SX*SY/N; S AuSicx2- Sx 2/N) ; S 13*SY/N-A*SX/N 10.20 S R=S/FSCITc C X2- SX 2/N)*(y2- SY: 2/N) )

12.10 F NT( I ' )4.2; T 113,J; F H=0, I; T 212. 04, FNE1•7t ...1+L*K)