Histamine Receptors in Guinea Pig Trachea and Their Relations to Cyclic Nucleotides

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University Moorilms International 300 N. ZEEB RD., ANN ARBOR, Ml 48106 8121805

Ja c k so n , G a y l e L a t r ic u M a r t in

THE EXISTENCE OF MULTIPLE HISTAMINE RECEPTORS IN GUINEA PIG TRACHEA AND THEIR RELATIONS TO CYCLIC NUCLEOTIDES

The Ohio Slate University PH.D. 1981

University Microfilms International 300 N. Z«b Road, Aon Arbor. MI 48106 THE EXISTENCE OF MULTIPLE HISTAMINE RECEPTORS IN GUINEA PIG

TRACHEA AND THEIR RELATIONS TO CYCLIC NUCLEOTIDES

DISSERTATION

Presented In Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in The Graduate School

of The Ohio State University

Gayle Latrlda Martin Jackson, B.S.

* * * * *

The Ohio State University

1981

Reading Committee: Approved By

Richard Fertel, Ph.D

Joseph Blanchlne, M.D., Ph.D

Jack Rail, Ph.D. Advisor Department of Pharmacology Gopl A. Tejwani, Ph.D ACKNOWLEDGEMENTS

I thank Dr. Richard Fertel, my adviser, for his support, guidance, criticism, patience and most of all his understanding.

1 wish to thank my Reading Committee: Drs. J. Blanchlne,

R. Fertel, J. Rail and G. Tejwanl for their critical evaluation of

this thesis.

There have been so many generous and kind people who have

helped me to reach this goal. Let me say a special "Thank You" to:

Dr. G. Tejwani for his assistance In biochemical techniques;

Carol Oravec, Novecia Custls and Christine Albrlghtson for

their technical assistance;

Janet Rice and Jill Williams for statistical assistance;

James Greenwald, photography;

Nancy Sally for graphic illustrations, and

Reece Smith for Xeroxing.

My friends, Ricardo Wright, Richard and Fannie Watson, Lee

Erby, Crosby and Florence Christian and the Kwaanza family, who in

their own special ways inspired me;

To my babysitters, Judy and Carol, who were always available

in my times of greatest need;

To Carol Jones for her patience in typing this manuscript and

her assistance in my years as a graduate student;

ii To my parentsr Abraham and Mattie who always encouraged me to

reach a little higher. To my brother, Abraham; my sisters Frances,

Mamie and Felicia; my sister-in-law, Theresa and my inlaws Hazel

and Charlie Allen, who were always supportive;

To my children, Nkenge and Atlba, who have shown the greatest

patience and understanding during this long ordeal;

Finally, to my husband Alvin, to whom I dedicate this work*

My love and thanks for all the encouragement and assistance you

have given me, and understanding the importance of this accomplishment

to me*

ill VITA

October 27 1950 Born - Baltimore, Maryland

1972 B.S., Morgan State University Baltimore, Maryland

1972-1973 University Fellow The Graduate School The Ohio State University Columbus, Ohio

1973-1974 Graduate Student Representative to Faculty Department of Pharmacology The Ohio State University Columbus, Ohio

1973-1974 Member of Council of Graduate Students The Ohio State University Columbus, Ohio

1973-1975 National Institute of Health Trainee Department of Pharmacology The Ohio State University Columbus, Ohio

1975-1976 University Fellow The Graduate School The Ohio State University Columbus, Ohio

1976-1979 Fellow, National Fellowship Fund (Ford Foundation)

1977-1978 Coordinated and participated in teaching Pharmacology7 400 Department of Pharmacology The Ohio State University Columbus, Ohio

iv 1977 Minority Recruiter Department of Pharmacology The Ohio State University Columbus, Ohio

1979 Participated In teaching Pharma* cology 600 Department of Pharmacology The Ohio State University Columbus, Ohio

HONORS

University Fellowship, The Ohio State University* 1972-1973, 1975-1976*

National Fellowship Fund Fellow (Ford Foundation)* 1976-1979*

Awarded Fellowship, American Association of University Women* 1976*

The ICSABER Society Award, The Ohio State University* 1978*

The Clayton S* Smith Memorial Award, The Ohio State University* 1979.

v PUBLICATIONS AND PRESENTATIONS

Martin, G.L., Lindower, J.O. and O'Neill, J.J. Barbiturate Induced . fine structure changes In guinea pig brain using an Improved terminal fixation procedure* Pharmacologist 16: 331, 1974 (abstract)

Martin, G*L. and Fertel, R. The effect of histamine on the cyclic AMP to cyclic GMP ratio In guinea pig lung* Pharmacologist 19: 284, 1977 (abstract)

Martin, G.L. and Fertel, R. Guinea pig lung may have both Hi and H2 receptors. Federation Proceedings 37: 393, 1978 (abstract)

Martin, G. Role of Histamine In Asthma. Research Seminar, October 1977. Morgan State University, Baltimore, MD and Coppln State College, Baltimore, MD

FIELDS OF STUDY

Major Field: Biochemical Pharmacology

Autonomic Pharmacology Drs. B.H. Marks and J.J. O'Neill

Biochemical Pharmacology Dr. J.J. O'Neill

Cardiovascular Pharmacology Dr. S. Dutta

Drug Metabolism Dr. D. Courl

Neuroanatomy Dr. A. Humbertsen

Neurochemistry Dr. L. Horrocks

Physiological Biochemistry Dr. A.J. Merola

Physiology Department of Physiology Staff

vi TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS ...... 11

VITA ...... iv

LIST OF‘FI G U R E S ...... ; ...... xlll

INTRODUCTION ...... 1

I. THE ROLE OF HISTAMINE IN THE CONTROL OF RESPIRATORY SMOOTH MUSCLE ...... 2

A. Historical Background ...... 1

Br Asthma and Anaphylaxis...... 2

C. The Normal Physiologic Control of 3 Airway Tone ......

II. THE MECHANISM OF ACTION OF H I S T A M I N E ...... 4

A. Evidence for the Existence of Two Histamine Receptors...... * ...... 4

B. Histamine Receptors In Respiratory Smooth Muscle ...... 5

1. Evidence for an H^ Receptor...... 5 2. Evidence for an H2 Receptor...... 7 3. Evidence for an H3 Receptor...... 9

III. CORRELATION BETWEEN AIRWAY TONE AND CYCLIC NUCLEOTIDES...... 10

A. Cyclic Nucleotides aa Second Messengers • . 10

1. Historical Background ...... 10

vil PAGE

2. Cyclic AMP- formation, metabolism and action...... 12 3* Cyclic GMP - formation, metabolism and action...... 13

B. The Mechanism of Action of Cyclic Nucleotides in Tracheal Smooth Muscle • • • ...... 14

1. Cyclic A M P ...... 14 2. Cyclic G M P ...... 15

C. The Role of Cyclic Nucleotides in the Actions of Histamine ...... 16

1. Cyclic A M P ...... 16 2. Cyclic G M P ...... 17

STATEMENT OF THE PROBLEM ...... 19

MATERIALS AND M E T H O D S ...... 21

I. ANIMALS...... 21

II. DRUGS AND SOLUTIONS ...... 21

A* Physiological Salt Solution (PSS)...... 22

III. PHARMACOLOGICAL AS S A Y ...... 23

A. Preparation of Tracheal Rings ...... 23

B. Passive Force VersuB Active Force...... 24

C. Equilibration of the Tissue...... 27

D. Monitoring of Tissue Sensitivity...... 30

E. Histamine Tachyphylaxis ...... 30

F. Assay of Tissue ...... 33

1. Dose Response ...... 33 2. Time Response...... 33 3. Evaluation of Histamine Antagonist Activity ...... 33 4. WaBhout of Drug •*..»...... 34

viii PAGE

G. Expression of Data • 34

IV. BIOCHEMICAL ASSAY ...... 34

A. Preparation of the Tracheal Smooth Muscle . 34

•B. Incubation of the T i s s u e ...... 35

C. Measurement of Cyclic AMP and Cyclic GMP . 35

1. Preparation of Tracheal Extracts • • 35

2. The Radioimmunoassay Procedure . . 38 a) Preparation of Assay Reagents . 39 1* Antiserum ...... 39 11. ^■’I-cydic nucleotide . . 39 111. Standard Cyclic Nucleotides 39

b) Radioimmunoassay Tracheal E x t r a c t s ...... 40

D. Protein Determination for Tracheal Smooth M u s c l e ...... 41

RESULTS ...... 44

I. THE PHYSIOLOGICAL EFFECTS OF HISTAMINE ON TRACHEAL SMOOTH MUSCLE ...... 44

A. The Effect of Histamine on Tracheal Smooth M u s c l e ...... - 44

B. A Comparison of the Effects of Acetylcholine, Carbachol and Histamine on Tracheal Contraction ...... 49

C. The Role of the Hi-Histamlne Receptor In the Blphaslc Response of Tracheal Smooth Muscle to Histamine...... 54

1. The Response of Tracheal Rings to 2-(2-aminoethyl)-pyridlne (2-AEP) . . 54 2. The Effect of Hi Receptor Blockers on the Blphaslc Response ...... 58

lx PAGE

D. the Role of Che H2 Receptor In the Blphaslc Response of Tracheal Smooth Muscle to Histamine 61

1. A comparison of the effects of histamine and dlmaprlt on tracheal smooth muscle • 61 2. The relaxation of tracheal smooth muscle by histamine and dlmaprlt . . . 66 3. The effect of metiamlde and cimetldine on the histamine response ...... 69 4. The effect of H2 antagonists on histamine Induced relaxation ...... 73 5. The enhancement of the histamine response by metiamlde and cimetldine . • 77

II. THE CORRELATION BETWEEN CYCLIC NUCLEOTIDES AND TRACHEAL CONTRACTION ...... 82

A. The Effect of Cyclic Nucleotides on Tracheal Smooth Muscle ...... 85 j III. THE EFFECT OF HISTAMINE ON TRACHEAL CYCLIC NUCLEOTIDE CONCENTRATIONS ...... 88

A. Concentrations of Cyclic Nucleotides In Guinea Pig Trachea ...... '. 88

B* The Effect of Histamine on Tracheal Cyclic Nucleotides ...... 89 | 1. Time course of the effect of histamine 2. Histamine dose response...... 89

C. The Effect of H^ and H2 Antagonists and Agonists on the Cyclic Nucleotide Response . . 92

1. The effect of the H], agonist 2-AEP on the concentration of cyclic AMP and cyclic GMP in tracheal smooth muscle. . 92 2. The effect of the H2 agonist dlmaprlt j on the concentration of cyclic nucleotide ! in tracheal smooth muscle...... 93 3. The role of the Hi and H2 receptor In the effect of histamine on tracheal cyclic nucleotides ...... 93

x PAGE

4. The effect of other bronchoactive agents on tracheal cyclic nucleotide concentrations . . • ...... 105

DISCUSSION

I. THE EXISTENCE OF MULTIPLE HISTAMINE RECEPTORS IN TRACHEAL SMOOTH MUSCLE AND THEIR RELATIONS TO CYCLIC NUCLEOTIDES ...... Ill

A. The Action of Histamine on Tracheal Smooth Muscle ...... 112

B. Role of H], Receptor in the Actions of Histamine on Tracheal Smooth Muscle ..... 113

C. The Role of the H2 Receptor In the Actions of Histamine ...... 114

D. Atypical Histamine Receptor ...... 116

E. Evidence that ■Histamine Receptors are Correlated with the Cyclic Nucleotide System...... 116

1. Cyclic A M P ...... 116 2. Cyclic GMP '...... 116

II. MECHANISM OF SMOOTH MUSCLE CONTRACTION ...... 121

A. Contractile Proteins ...... 121

B. Role of Calcium ...... 123

C. Drug Effects - Mechanism of Hormonal Induced Contraction ...... • 123

h i . c y cl i c N u c le o t i d es a n d tr a c h e a l sm oo t h muscle CONTRACTION ...... 125

A. Cyclic AMP ...... 125

1. Mediator of smooth muscle relaxation • 125 2. Cyclic AMP and smooth muscle contraction...... 126

xl PAGE

3. Cyclic AMP-dependent protein kineses 127 4. Role of calcium • •*••*••»•* 128

B. Cyclic GMP ...... 129

1. Evidence as mediator of smooth muscle contraction 129 2* Evidence as mediator of smooth muscle relaxation ...... 129 3* Cyclic CMP-dependent protein kinase 4. Role of calcium . • . • ...... 130

C. The Role of Cyclic Nucleotides In Tracheal Smooth Muscle 132

IV. A PROPOSED MECHANISM FOR THE ACTION OF HISTAMINE ON TRACHEAL SMOOTH MUSCLE ...... 133

A. Evidence for an Hi R e c e p t o r ...... 133

1. Evidence that histamine's activation of the Hi receptor produces tracheal contraction...... 133 2. Evidence that contraction results In Increased concentrations of cyclic AMP and cyclic GMP In tracheal smooth m u s c l e ...... 135 3. Evidence that cyclic GMP promotes the relaxation of tracheal smooth muscle 135

B. Evidence for an H2 R e c e p t o r ...... 136

1. Evidence that the activation of the H2 receptor Induces relaxation of the tracheal smooth muscle ...... 136 2* Evidence that activation of the H2 receptor leads to increased cyclic AMP ...... 136 3. Evidence that cyclic AMP mediates relaxation of tracheal smooth muscle. 137

C. Evidence of an Atypical Receptor...... 137

D. Future W o r k ...... 138

SUMMARY ...... 140

BIBLIOGRAPHY ...... 141

xll LIST OF FIGURES

Figure Page

1 Resting Force Versus Active Force ...... 26

2 Resting Force Versus Percent Active Developed Force 29

3 Desensitization of Tracheal Rings by Histamine . * 32

4 Time Course of the Decrease in Tracheal Cyclic

Nucleotide Concentration in Guinea Pig Trachea

With Incubation in Physiological Salt Solution • • 37

5 Cyclic AMP Standard Curve ...... 43

6 Cyclic GMP Standard Curve ...... 44

7 Polygraph Record of Hlatamlne Dose Response . • . 46

8 Histamine Dose Response ...... 48

9 Comparative Response of Tracheal Rings to Histamine,

Acetylcholine and Carbachol ...... 51

10 Time Course of the Effects of Carbachol and Histamine

on Tracheal Smooth Muscle Contraction ...... 53

11 A Comparison of the Dose Response Curves

of Histamine and 2-(2-Aminoethyl)-Pyridine • • • • • 56

12 The Antagonism of Histamine Response by Pyrilamine . 60

13 The Response of Guinea Pig Tracheal Rings to

Histamine and Diphenhydramine ...... 63

xili Figure Page

14 The Antagonism of Histamine Response by

Diphenhydramine . . • ...... 65

15 Comparative Response of Guinea Pig Tracheal Rings

to Histamine and Dlmaprlt ...... 66

16 The Relaxation of Tracheal Smooth Muscle by

Histamine ...... 71

17 The Relaxation of Tracheal Smooth Muscle by

D l m a p r l t ...... 72

18 Comparative Responses of Guinea Pig Tracheal

Rings to Histamine and Cimetldine ...... 75

19 The Time Course of the Effect of Cimetldine on the

Histamine Response of Tracheal Smooth Muscle . . 79

20 Comparative Responses of Guinea Pig Tracheal Rings

to Histamine and Metiamlde ...... 81

21 The Enhancement of the Histamine Response by

Metiamlde and Cimetldine ...... 84

22 The Effect of Dlbutyryl Cyclic Nucleotides on

Tracheal Smooth Muscle ...... •* 87

23 The Effect of Histamine on Cyclic AMP and Cyclic GMP

Concentrations in Guinea Pig Tracheal Smooth

Muscle ...... 91

24 The Effect of 2-AEP on the Concentrations of Cyclic

AMP and Cyclic GMP in Tracheal Smooth Muscle . . . 95

xiv Figure Page

25 The Effect of Dlmaprlt on the Concentrations of

Cyclic AMP and Cyclic GMP in Guinea Pig Trachea • 97

26 The Effect of Cimetldine on the Concentrations

of Cyclic AMP and Cyclic GMP in Guinea Pig Trachea 100

27 The Effect of Metiamlde on the Concentrations of

Cyclic AMP and Cyclic GMP in Guinea Pig Trachea • 101

28 The Effect of Hj. and H2 Antihistamines on the

Histamine Induced Alteration of Cyclic Nucleotides in

Guinea Pig Trachea ...... 10A

29 The Effect of Carbachol on the Concentrations of

Cyclic AMP and Cyclic GMP in Guinea Pig Trachea • 107

30 The Effect of Isoproterenol on the Concentrations

of Cyclic AMP and Cyclic GMP in Guinea Pig Trachea 109

31 The Effect of Histamine on the Contraction and

Cyclic Nucleotide Concentrations of Guinea Pig

Trachea ...... 118

32 A Proposed Mechanism for the Action of Histamine 134

xv INTRODUCTION

Histamine (4[5]-(2-amlnoethyl)lmldazole) is a biogenic amine which is found In lung mast cell granules, la releasable from the maBt cell by antlgen-antlbody action, and la able to act on respir atory smooth muscle to cause bronchoconstrlctlon. Histamine la not found exclusively In the lung, nor solely In the mast cell.

It has been Identified In all tissues studied. However, the fact that lung histamine may be Involved In respiratory diseases such as asthma has stimulated Interest In the role of histamine in lung function and the biochemical mechanisms of Its actions.

Although the ability of histamine to cause bronchoconstrlctlon in man is well known, the function of histamine in the control of respiratory smooth muscle in normal physiology has not been determined. Thus, It was the purpose of this dissertation to further investigate the physiological effects of histamine on respiratory smooth muscle. Since cyclic nucleotides are thought to mediate a variety of hormonal Induced responses, the effects of histamine on the concentrations of cyclic AMP and cyclic GMP in tracheal smooth muscle were investigated. In particular, the correlation between the actions of histamine on smooth muscle tone and its ability to alter the cyclic nucleotide concentrations of tracheal smooth muscle were Investigated.

1 2

I> The Role of Hlscamlne In the Control of Respiratory Smooth Muscle*

A. Historical Background

Histamine was first Identified as a constituent of ergot In the early 1900's* The subsequent synthesis of histamine by Wlndaus and

Vogt (1907) opened the door to research on the physiological effects of this substance.

The intravenous administration of histamine produced hypotension, an increase in capillary permeability, and a decrease in body temperature in cats and dogs (Dale and Laidlaw 1910, 1919); convulsions and bronchoconstrlctlon In guinea pigs (Barger and Dale 1911; Dale 1913); and dilation of arterioles and capillaries of the skin in man (Weiss et al., 1929). Along with the ability of histamine to produce symptoms

similar to anaphylaxis, Dale and Laidlaw (1919) observed that the local application of histamine produced an inflammatory reaction character

ized by redness, swelling, and edema of the skin. These observations

suggested that histamine may be involved in both local and systemic

allergic reactions. The possibility that histamine was involved in

anaphylaxis was enhanced by the discovery that histamine was contained

in lung tissue (Best et al.. 1927), the injection of histamine

isolated from cat lung into guinea pig produced death as a result of

asphyxia.

B. Asthma and Anaphylaxis

The respiratory disease, asthma, is characterized by bronchial

muscle spasms and bronchial obstruction. It is an allergic disease Involving the anaphylactic release of histamine and other mediators

(i.e., slow-reacting substance of anaphylaxis and klnins) from lung mast cells and blood basophils (Schlld 1936, 1939; Feldberg 1941;

Anggard et al., 1963; Kallner et al., 1972; Lichtenstein, 1973;

Kallner and Austen, 1975)* These substances then act on bronchial and tracheal smooth muscle to cause contraction (Schlld et al., 1951;

Hand and Buckner 1979).

The contraction of bronchi and trachea by histamine has been

demonstrated in several mammalian species including man and guinea

pig, In vivo (James 1969) and In vitro (Katsukl and Murad, 1977;

Dunlop and Smith 1977).

Whether or not histamine Is Important in the normal regulation of

airway tone has not been determined. However, histamine could play a

role In asthma. Several reports Indicate that histamine causes more

severe bronchoconstrlctlon in asthmatic patients than in healthy

subjects (Curry 1947; Benson 1975, 1978). Furthermore, asthmatics

have been reported to have increased plasma levels of histamine and

enhanced excretion of histamine metabolites during spontaneous and

allergen-induced attacks (Bhat£t al., 1976; Bruce et al., 1976;

Simon et al.t 1977; Lowhagen et al., 1980).

C. The Normal Physiological Control of Airway Tone

Histamine, serotonin, slow reacting substance of anaphylaxis, the

kinlns, and prostaglandins are present in the lung and are capable of

causing bronchoconstrlctlon (Brocklehurst, 1960; Berry and Collier,

1964). Most of the evidence suggests that airway tone is 4 modulated by adrenergic and cholinergic nervous activity, the neuro- transmitters epinephrine and acetylcholine mediating bronchodilatlon and bronchoconstrlctlon, respectively (Ahlqulst 1948; Foster 1964;

Furchgott 1967; Everett and Cairncross 1969; Ralanl et al., 1977; Ham-* marstrom and Sjostrand 1978)* There Is no evidence that histamine or the other bronchoconatrlctors are involved in the normal regulation of respiratory smooth muscle activity*

IX. The Mechanism of Action of Histamine

A* Evidence for the Existence of Two Histamine Receptors

Fournea an* Bovet reported in 1933 that 2-(l-plperindlnomethyl)

-1,4-benzodioxan prevented histamine Induced bronchospasms in animals*

Within the next thirty years a number of compounds were synthesized which were able to block the actions of histamine on smooth muscle

(Sherrod et al., 1947). These actions included the contraction of

arteries, veins, bronchi, trachea, gut, guinea pig ileum, and the

relaxation of capillaries. However, the ability of histamine to

induce gastric acid secretion, increase heart rate, and inhibit the

contraction of the rat uterus were not blocked by the antihistamines.

These observations led Ash and Schlld (1966) to postulate that the

actions of histamine resulted from the of activation of at least two

different receptors. Those actions of histamine which could be

blocked by low concentrations of antihistamines were said to be due

to the activation of Hj-hlstamlne receptors. Those actions not

antagonized by the antihistamines were said to be the result of

histamine's activation of a second receptor. This hypothesis was confirmed a few years later when In 1972,

Black and coworkers reported the synthesis of a new antihistamine, burlmamlde, which could specifically antagonize hlstamlne-lnduced gastric a d d secretion In rats, an action of histamine which was In sensitive to the "classical” antihistamines* The hlstamlne-lnduced In crease In heart rate and Inhibition of uterine contraction were also blocked by burlmamlde* These responses as well as hlstamlne-lnduced gastric a d d secretion were classified as H2 receptor responses*

Since that time, even more specific H2 antagodsts and agonists have been developed* This has led to the Identification of an H2 receptor

In several tissues Including guinea pig Ileum (Barelca and

Rocha e Silva, 1976), rat stomach (Ercan and Turker, 1977; Harkanson et al.. 1978), mouse vas deferens (Marshall, 1978), airway smooth muscle (Yen and Kreutner, 1979), heart (Verma and McNeil, 1977), and brain (Kanof and Greengard, 1979a).

B* Histamine Receptors In Respiratory Smooth Muscle

1* Evidence for an Hj receptor

The characterization of histamine receptors In smooth muscle has primarily been through the use of specific histamine agonists and antagonists. In 1948, Castillo demonstrated that hlstamlne-lnduced contractions of a guinea pig tracheal chain preparation could be blocked by the H^-antihlstamlnes, diphenhydramine, pyrlbenzamlne, and neo-antergan*

In a similar study using human bronchial smooth muscle, Hawkins and Schlld (1951) demonstrated that the Hpantagonlst mepyramine, at concentrations as low as 10”® m, inhibited hlstamlne-lnduced contractions■ More recently, H]^-antagonists have been shown to i antagonize hlstamlne-lnduced contractions of bovine, guinea pig, sheep, and dog tracheas (James, 1969; Byre, 1969; Katsukl and Murad,

1977; Ulmorl and Taira, 1978) and human and monkey bronchi (Dunlop and Smith, 1977; Krell, 1979).

Whether histamine acting on respiratory smooth muscle Hi receptors

Is Involved In bronchospasms which occur In allergic diseases such i as asthma Is widely debated. Both In vitro and ^n vivo experiments have produced conflicting results. Although Hi-antlhlstamlnes blocked hlstamlne-lnduced contraction of guinea pig trachea, the same antlhlstamlnes failed to block antigen-induced contraction of trachea from sensitized guinea pigs (Castillo, 1948). Schlld nt al. (1951) found that the concentration of antihistamines necessary to antagonize antigen-induced contraction of bronchial smooth muscle from asthmatics was 10,000 times that required to Inhibit contractions Induced by his

tamine. In contrast to these findings, other workers (Antonlssen et al., 1980; Smith and Dunlop, 1980) have reported that low concen- —8 tratlons of the blocker pyrllamlne (10 M) blocked antigen-induced contractions of sensitized dog tracheal smooth muscle and sensitized human bronchus, respectively.

In vivo attempts at blocking bronctioconstrlction have resulted in * i conflicting reports of the usefulness qf Hi type antihistamines In

the treatment of asthma. Oral admlnistjration of Hi-antlhistamlnes to asthmatics has been reported to Inhibit bronchospasms from inhaled ! histamine (Casterline and Evans, 1977, Nogrady and Bevan, 1978, Nathan et al; 1979) and Fopa (1977) reported that chlorpheniramine improved respiration in asthmatics through its bronchodilatlng activity.

However, Leopold et al.. (1979) found antihistamines Ineffective in reducing the severity of the asthma attack.

2. Evidence for an H2 receptor.

Although contraction mediated by an receptor is the major effect of histamine on the airways of man and guinea pig, a relaxation of respiratory smooth muscle by histamine has been demonstrated in some animal species. The relaxation of tracheal smooth muscle by histamine was first reported approximately 25 years ago. Hawkins

(1955) observed that although the general response of guinea pig

tracheal rings to histamine was a dose-dependent contraction, histamine

concentrations greater than 2 x 10“^ M produced relaxation. This re laxation was not antagonized by the nclasslcalM antihistamines.

Hawkins suggested that this inhibitory action of histamine on the

tracheal smooth muscle was related to the action of histamine on the

rat uterus, which was also insensitive to Hi-antihlstamlnes. This

inhibition of rat uterine contraction is now known to be mediated by

an H2“hiatamlne receptor.

In 1969, Eyre described a histamine-induced relaxation of sheep

bronchus which was also insensitive to blockade by the "classical"

antihistamines. Maengwyn-Davis' (1968) experiments with cat trachea

suggested that histamine-induced catecholamine release may be partially

responsible for the relaxant activity of histamine. After the intro

duction of H2-histamine antagonists, Eyre (1973) demonstrated that the relaxation of sheep bronchus could be blocked by the H2 antagonist burlmamlde and the relaxation of the cat trachea should be blocked by a combination of and Hj antihistamines. Thus Eyre postulated that an H2 receptor may be involved In the relaxant action of histamine on bronchial and tracheal smooth muscle. # Dunlop and Smith (1977) were the first to show evidence of an H2 receptor In human bronchus. Contractile responses to histamine were enhanced in the presence of the H2~antagonlst metiamlde. In the pres ence of the Hi receptor blocker mepyramine, histamine produced concentration-dependent relaxation. Prelncubatlon of the bronchus with the H2 blocker metiamlde blocked the hlstamlne-lnduced relaxation.

Okpako et al (1978) suggested that guinea pig airways may contain in hibitory H2 receptors which modulate histamine's contractile activity.

This theory is based on the observation that guinea pig trachea and

bronchus preincubated with an H 2 blocker produced a greater

contractile response to histamine. However, preincubation with the

H2 blocker failed to enhance contractile responses to the Hi

receptor agonist, 2-methylhlstamine, or to the cholinergic agonist,

carbachol. This theory Is supported by the finding that histamine,

as well as the H 2 receptor agonists 4-methylhistamlne and dlmaprlt,

produce dose-dependent relaxations of rhesus monkey bronchial smooth

muscle (Krell, 1979; Chand et al., 1980) which are blocked by H2-

antagonists. Evidence for the existence of airway inhibitory H2

receptors has since been presented for a variety of animal species (Drazen et al., 1978, 1980; Chand and Eyre; 1980; Hayashl and Toda,

1980; Snapper et al., 1980; Eyre and Deline, 1980).

In vivo experiments support the idea that inhibitory H2 histamine receptors are present In the airways. The Intravenous administration of the H2 blocker clmetidlne to dogs potentiated the constrictor response to histamine (Snapper et al.. 1980). Nathan and coworkers

(1979) reported that the oral administration of clmetidlne to asthma tics enhanced the bronchoconstrictive effect of inhaled histamine.

3* Evidence for an H3 receptor

The inability of specific drug receptor antagonists to block

the biochemical or physiological action of the drug on a tissue often leads investigators to postulate that the drug acts on a different

receptor in that particular tissue. Upon the advent of new antlhist- » amines in 1972, the existence of two histamine receptors, Hi and H2 was verified. However, there appear to be physiological actions of

histamine which are not blocked by either Hi or H2 receptor agonists.

For example, histamine-induced dose dependent relaxation of carbachol-

contracted rabbit trachea is not blocked by the Hi antihistamine py-

rilamine or the H2 antihistamines burimamide and metlamide (Fleisch and

Galkins, 1976). Since the beta-adrenergic blocker propanolol does

not inhibit the relaxation, this suggests that histamine-induced

relaxation is not a result of catecholamine release. Fleisch and

Calkins suggested that either the rabbit trachea contained an H2

receptor Insensitive to metlamide and burimamide or possibly an H3

receptor. Chand and Eyre (1977) demonstrated that histamine-induced 10 relaxation of carbachol contracted bronchi of cat, dog, and rabbit could not be blocked by metlamide, propranolol, or the prostaglandin synthesis inhibitor, lndomethacln. They suggested that these airways contained an atypical H2 receptor* In the rat, the H2 agonist dlmaprit, like histamine, relaxes the carbachol-contracted trachea

(Eyre and Besner, 1979)* Relaxation induced by either agent was not blocked by H2 antagonists.

Ill* Correlation Between Airway Tone and Cyclic Nucleotides

A. Cyclic Nucleotides As Second Messengers

1. Historical Background

Evidence for cyclic 3'5*-adenosine monophosphate (cyclic

AMP) as a hormonal mediator was first suggested by Rail and coworkers in their studies on the mechanism of epinephrine-, and glucagon- induced activation of liver phoBphorylase, the enzyme responsible for the conversion of glycogen to glucose: 1) Rail et al. (1956) observed that the activation of liver phosphorylase was enhanced in the presence of glucagon and epinephrine; 2) In cell free liver homogenates, the two hormones induced the formation of a heat stable factor (Rail £t al., 1957); 3) This factor was able to stimulate the formation of active liver phosphorylase in the supernatant fraction of liver homogenates, a cellular fraction In which the hormones themselveB were inactive; A) The purified factor contained adenine, ribose, and phosphate in a ration of 1:1:1; 5) In 1958, Sutherland and Rail identified the compound cyclic 3*,5t adenosine monophosphate

(cyclic AMP) in particulate fractions of liver, brain, heart, and skeletal muscle which was stimulated by epinephrine or glucagon In the presence of ATP and Mg^+ . These results suggested that cyclic

AMP activated liver phosphorylase, and thus mediated the glycogeno** lytic effect of epinephrine and glucagon in the liver. Consequently,

Sutherland and Rail (1960) proposed cyclic AMP as a "second" messenger*

The hormone which Initiated the response through its receptor activity was termed the first messenger* The receptor activation produced intracellular changes resulting in the production of a second messenger, cyclic AMP* This second messenger converted the biochemical signal into a physiological response, glycogenolysis* Other hormones and drugs were found to increase cyclic AMP in a variety of tissues, suggesting that cyclic AMP also mediated the effects of these agents*

This exciting discovery stimulated research attempts to find similar messenger nucleotides* Of the cyclic nucleotides since identified, cyclic 3'(S'-guanosine monophosphate (cyclic CMP) has

received the most support as a possible messenger* The discovery of cyclic GMP, unlike cyclic AMP, was not the result of the observation

of an effect resulting in the identification of the effector but

rather from the observation that cyclic GMP was present in urine

(Ashman et al., 1963)* Later it was shown by Goldberg et al. * 1973

that a variety of hormones could stimulate increases in cyclic GMP.

However the functions of these increases were not readily apparent.

However in many instances, the agents or hormone which induced

increases in intracellular cyclic GMP were antagonistic toward

agents which induced increases in intracellular cyclic AMP in the

same tissue or cell* For example, the neurotransmitter acetylcholine. 12 which suppressed cardiac contractility, stimulated Increases In cyclic GMP In a variety of tissues including the heart, whereas epinephrine, which stimulated heart function, stimulated an increase

In the cyclic AMP levels of heart (George et al., 1970)* Goldberg et al. (1975) described the possible role of these apparently antagonistic cyclic nucleotides In his Yln**Yang hypothesis. He suggested that cyclic AMP and cyclic GMP were like forces In nature which may act in opposition or In concert to maintain equilibrium.

Thus, in tlje cells these cyclic nucleotides may function as homeo static regulators. Therefore, an alteration in one of the cyclic nucleotides could possibly lead to an alteration In cell function or activity.

2. Cyclic AMP formation, metabolism, and action

Adenosine 3',5' cyclic monophosphate (cyclic AMP) is formed

intracellularly from adenosine triphosphate (ATP) in the presence of magnesium or manganese ions.

adenylate cyclase ATP ------> Cyclic AMP + PP1 Mg2+, Mn2+ Mg2+ cyclic AMP phosphodiesterase v 5'AMP

Adenylate cyclase, which converts ATP to cyclic AMP and pyrophosphate,

has been Identified In a variety of tissues (for reviews see Suther

land and Rail, 1960; Sutherland et al., 1962, 1968; Daly 1977; Weiss

and Fertel, 1977). This enzyme Is located on the cell membrane and

can be stimulated by drugs, hormones, and neurotransmltters. In

* some cases it Is suggested that the receptor site for these agents is actually a part of the adenylate cyclase.

Phosphodiesterase, which converts cyclic AMP to 5’AMP, is activated by magnesium ions and Inhibited by methylxanthines such as caffeine.

Thus, tissue levels of cyclic AMP are controlled not only by the rate of its synthesis by activation of adenylate cyclase, but also by the rate of its degradation by phosphodiesterase.

Cyclic AMP is proposed to produce its effects in tissues as a result of activation of a protein which acts both as a receptor for cyclic AMP and as an enzyme capable of phosphorylatlng tissue proteins

(for review see, Swlllens and Dumont, 1977). Activation of this protein kinase (cyclic AMP-dependent protein kinase) results in phosphorylation reactions which are able to affect the rate by which cellular processes occur. In the case of glycogenolysls, cyclic AMP activates a protein kinase, phosphorylase kinase, which In turn phosphorylates glycogen phosphorylase, the enzyme which promotes the breakdown of glycogen to glucose.

3. Cyclic GMP formation, metabolism, and action

The formation of cyclic 3',5'-guanosine monophosphate

(cyclic GMP) is similar to that of cyclic AMP.

guanylate cyclase GTP ♦ cyclic GMP + PPi

cyclic GMP phosphodiesterase

5 'GMP 14

The conversion of guanoslne triphosphate (GTP) to cyclic GMP by the action of guanylate cyclase has been described in. many tissues (Hardman and Sutherland, 1969; White and Aurbach, 1969). Guanylate cyclase apparently exists In both a soluble and particulate form and both forms require manganese Ions for activation* Although a number of hormones and neurotransmitter substances such as acetylcholine have been reported to Increase tissue levels of cyclic GMP, the activation of guanylate cyclase by these agents has not been observed* Cyclic

GMP phosphodiesterase, which catalyzes the hydrolysis of cyclic GMP, can be Inhibited by methylxanthlnes (for reviews see Goldberg and

Haddox, 1977). It has been suggested that cyclic GMP may, like cyclic AMP, act to promote phosphorylation of• cellular proteins through the activation of a cyclic GMP dependent protein kinase

(Coldberg and Haddox, 1977).

B* The Mechanism of Action of Cyclic Nucleotides In Tracheal Smooth Muscle

1. Cyclic AMP ,

Since epinephrine, which stimulates the synthesis of cyclic

AMP, also relaxes a variety of smooth muscles, Sutherland and Rail

(1960) suggested that cyclic AMP might mediate this Inhibitory effect of epinephrine* This Idea stimulatefl a number of Investigators to determine whether cyclic nucleotides are involved in the regulation of smooth muscle tone* The airway smooth muscle relaxant activities of beta adrenerglcs, as well as phosphodiesterase Inhibitors, haB been attributed to the ability of these agents to elevate cyclic AMP IS concentrations In respiratory smooth muscles (Buckner and Abel,

1974; Murad and Kimura, 1974; Katsukl and Murad, 1977; Triner et al., 1977; Wong and Buckner, 1978). Newman et al. (1978) ‘ demonstrated that the ability of several agents to inhibit cyclic

AMP-phosphodlesterase from the tracheal smooth muscle correlated well with their ability to Induce tracheal relaxation. The possi bility that cyclic AMP mediates tracheal relaxation was further supported by the observation that the application of cyclic AMP derivatives to .in vitro preparations of tracheal smooth muscle

results in relaxation (Moore jit al., 1968; Szaduykls-SzadurBki et al., 1972; Szaduykls-Szadurski and Bertl, 1972; Newman, 1978).

The effect of cyclic AMP on smooch muscle tone is suggested to

result from activation of cyclic AMP dependent protein kinase which catalyzes a protein phosphorylation leading to a reduction

in free calcium.

2. Cyclic GMP

The observation that agents which constricted smooth muscle

also Increased tissue levels of cyclic GMP led to the postulate

that cyclic GMP mediates smooth muscle constriction (Schultz and

Hardman, 1975). However, the evidence supporting this assumption

is not conclusive. There have been several reports that agents

which increase cyclic GMP levels in smooth muscle by activating

guanylate cyclase also cause smooth muscle relaxation (Schultz

et al., 1977; Axelsson et al., 1979; Kukovetz et al., 1979). Also,

exogenous cyclic GMP and its derivatives have been reported to

cause relaxation of aorta and vas deferens (Schultz et al., 1979), 16 vascular smooth muscle (Kukovetz et al., 1979; Napoli et al.t

1979) and tracheal smooth muscle (SzaduykisSzadurski andBerti,

1972; Katuaki and Murad, 1977). The role of cyclic GMP In smooth muscle tone remains to be determined.

C. The Role of Cyclic Nucleotides In the Actions of Ulatamlne

-1. Cyclic AMP

Histamine elevates cyclic AMP concentrations In a variety of tissues Including brain (Kakiuchl and Rail, 1968a,b; Palmer et al., 1972), lung (Palmer 1971, 1972, Mathe^t al., 1974; Ortez

1977), trachea (Murad and Kimura, 1974; Ohkubo et al., 1976;

Katsukl and Murad, 1977, Creese and Denborough, 1979) and heart

(Johnson et al., 1979; Kanof and Greengard, 1979b). Since histamine produces Its effects by interacting with a receptor, many attempts were made to determine whether the observed increases in cyclic

AMP resulted from the activation of a specific histamine receptor.

Histamine-sensitive adenylate cyclases have been demonstrated In cell-free preparations of guinea pig heart (Kanof and Greengard,

1979b) and mammalian brain (Kanof and Greengard, 1979a). Activation of these adenylate cyclases could be specifically blocked by H2 antagonists and mimicked by H2 agonists. Thus it was suggested that the Increases in cyclic AMP resulted from the activation of a adenylate cyclase coupled to the H2 receptor.

Although histamine-sensitive adenylate cyclases have not been demonstrated in smooth muscle, there is evidence that cyclic

AMP mediates some of the actions of histamine produced by the activation of the Hg receptor* The H2 antagonist metiamlde can

block the hlstamine~lnduced relaxations of K+ contracted guinea

pig ileum and rabbit mesenteric artery (Reinhardt et al., 1979).

This inhibitory effect of metiamlde correlates well with its ability to block the histamine Induced increases in cyclic AMP

in the respective tissues. The H} antagonist mepyramine was

ineffective in antagonizing the actions of histamine. These

results suggest that an adenylate cyclase associated with the H2

receptor may exist in the smooth muscles of the Ileum and mesenteric artery.

The role of cyclic AMP In the actions of histamine on tracheal

smooth muscle Is unclear. In the absence of studies on the effect

of histamine on adenylate cyclase prepared from tracheal smooth

muscle it 1 b not possible to know whether histamine stimulates the

synthesis of cyclic AMP by acting on a specific histamine

receptor.

2. Cyclic GMP

Just as cyclic AMP has been suggested as a second messenger

mediating some of the actions of histamine, there is evidence which

supports a similar role for cyclic GMP. Histamine elevates

cyclic GMP levels in human umbilical artery (Clyman et al.. 1975),

and bovine superior cervical ganglion (Study and Greengard, 1978).

The stimulatory action of histamine on the tissues as well as

the elevation cyclic GMP can be blocked by H} receptor antagonists.

Thus, it has been postulated that the actions of histamine on 18 these tissues is mediated by an increase in intracellular cyclic

GMP.

9 Histamine has also been shown to increase the level of cyclic GMP in mouse and guinea pig lung (Mathe et al., 1974;

Poison e£ al., 1976) and guinea pig and bovine trachea (Murad and Kimura, 1974; Ohkubo et al., 1976; Katsuki and Murad, 1977).

Since the H^-histamine antagonists can prevent histamine induced bronchoconstrlctlon as well as Increases in cyclic

GMP, it has been thought that the bronchoconstrlctlon is mediated by cyclic GMP.

The mechanism by which histamine elevates cyclic GMP in lung

* or trachea is not known. A histamine sensitive guanylate cyclase in lung has never been demonstrated, suggesting that the Increased cyclic GMP may not result from direct activation of the enzyme.

Lung contains cyclic GMP phosphodiesterase (Green et al.. 1977) which is not affected by histamine. Furthermore, the application I of exogenous cyclic GMP derivatives to Isolated tracheal preparations have been reported to produce both relaxation and contraction

(Szaduykls-Szadurski and Bertl, 1972; Szaduykla-Szadurskl et al.,

1972).

4 STATEMENT OF THE PROBLEM

At physiological concentrations histamine causes contraction of bronchial and tracheal smooth muscle In man and in certain animal species such as. the guinea pig> However, the function of histamine in the control of respiratory smooth muscle In normal physiology has not been determined.

The understanding of histamine's actions is based on the use of specific histamine antagonists* Bronchoconstrlctlon as well as the contraction of other smooth muscle can be blocked by the "classic" antihistamines referred to as blockers* Thus, bronchoconstrlctlon Is thought to result from histamine's activation of an Hi receptor on the smooth muscle cell* Whether an H2 receptor is Involved In the actions of histamine on tracheal * smooch muscle Is not known. How histamine contracts tracheal smooth muscle 1b also not clear. Histamine may have a direct effect on the tissue or it may cause contraction indirectly by stimulating some other system. One such system may consist of the cyclic nucleotides, cyclic AMP and cyclic GMP, which are thought

to mediate physiological processes In a variety of tissues*

Since mechanisms of histamine's actions In tracheal smooth muscle have not been established, It was the purpose of this

thesis to investigate this problem. This study was designed to

determine: 1 ) the effect of histamine on tracheal smooth muscle;

19 the presence of histamine receptors in the trachea and their relations to the actions of histamine; and 3) whether cyclic AMP and cyclic GMP mediate the actions of histamine on tracheal smooth muscle* MATERIALS AMD METHODS

I. Animals

Male albino (Hartley Strain) guinea pigs weighing between 250 and 450 grama were used In all experiments* The guinea pigs were housed In an environment maintained at 73a-74#F and 55% humidity with a 12 hour light-dark cycle* The animals were fed Purina

Lab Chow.

Choice of Animal. The guinea pig was chosen for this study because Its airway response to histamine Is similar to that of man (Dale and Laldlaw, 1910). Experiments using preparations of guinea pig trachea In vivo (James, 1969) add In vitro (Akcasu,

1952; Jamieson, 1962; Carlyle, 1963 and Constantine, 1965), have demonstrated that guinea pig airways are sensitive to histamine and other bronchoconstrlctors. In addition, Hanna and Roth (1978) have found no significant difference In the responses of guinea pig bronchi and trachea to bronchoactlve agents. Therefore, the

guinea pig trachea can be a useful tool for studying the effect of histamine and ocher drugs on the tracheobronchial tree.

II. Drugs and Solutions

Histamine dihydrochloride; diphenhydramine HC1; pyrilamlne maleate; acetylcholine chloride; carbamylcholine chloride;

DL-propranolol HC1; L-isoproterenol HC1; indomethacin; papaverine;

21 22 adenosine 3':5'-cyclic monophosphoric add; N6,02*-dibutyryl * adenosine 3':5t-cyellc monophosphoric acid; guanoslne 3*:S* — cyclic monophosphoric acid; and N6 , 02'-dibutyryl guanosine-3':5'- cydlc monophosphoric acid were obtained from Sigma Chemical

Company* 2-(2-Aminoethyl)-pyrldlne was obtained from the Aldrich

Chemical Company* Trlpelennamine HC1 was obtained from ICN Phar maceuticals, Inc* Clmetidlne was purchased from Smith Kline &

French Labs (Philadelphia).

Dlmaprit was a gift from Dr* Ganellin (Smith Kline & French,

U.K.). Metiamlde was a gift from Dr* T. Yellin (ICN United

States, Inc*). All other compounds used were reagent grade.

The drugs were solubilized In water, physiological salt solution, or other appropriate solvent. All dilutions were made with physiological salt solution (pH 7.4). The final pH of all test drugs was 7.0 to 7.4. Drug solutions were used the same day they were prepared.

A. Physiological Salt Solution (PSS)

The physiological salt solution had the following composition.

NaCl 114.0 mM KC1 4.9 mM MgCl2 6H20 1.2 oM NaHC03 24.8 mM Na2Ca EDTA 0.026 mM NaH2P0 4 1.8 mM CaCl2 2H20 1.6 mM Dextrose 10.0 mM Sucrose 50.0 mM

The pH of the solution was adjusted to 7.4 by the addition of 1 N HC1. III. Pharmacological Assay

A. Preparation of Tracheal Rings

For each experiment, four guinea pigs were killed by spinal dislocation and thoracotomies performed. Each trachea was quickly dissected out and placed in cold (4°C) physiological salt solution pH 7.4, which had been saturated with 95% O2 and 5% C0 2 *

The trachea remained In the cold salt solution until all four dissections had been completed. The tissue was washed several times with the cold salt solution to remove any blood, and the tracheal rings were prepared by a modification of the open ring method of Akcasu (1952), and allowed to come to room temperature during preparation. The trachea was opened by cutting longitudi nally through the cartilage, parallel to the tracheal smooth muscle. The opened trachea was pinned flat to beeswax in a petri dish containing physiological salt solution. Fasciae tissue was removed from the trachea and each trachea was cut into three- segment rings, according to the following procedure.

On each side of the pinned trachea, two segments of a single opened cartilage ring were isolated by cutting on both sides of the segment perpendicular to the tracheal smooth muscle.

Care was taken to avoid cutting the smooth muscle. A piece of

6 - 0 silk surgical suture was tied to each of the two ring segments.

The prepared ring was Isolated from the trachea by cutting completely through the tracheal smooth muscle parallel to the ring, one ring segment away from the ligatured segment on both sides. The prepared ring contained three cartilage segments with 24 ligatures tied to the middle segments* To prepare the second

ring from the same trachea, this procedure was repeated*

One of the sutures was tied to a plastic tissue holder which was equipped to support two tracheal rings* The other end of the

tracheal ring was connected by the second suture to the force

transducer (Grass Ft* .03 Servo displacement transducer). The

tracheal rings were suspended In an organ bath containing 10 ml of oxygenated (5J5 C02 and 95% CO2 ) physiological salt solution at room temperature. Two rings were suspended In each organ bath.

The tracheal rings were equilibrated under an initial tension of

500 to 600 mg for one to two hours, during which time the temper-* ature of the organ baths was gradually increased to 37°C with

the use of a circulating water bath. Isometric recordings of

the contractions of the tracheal rings were measured on a Grass

Model 5 or 7 polygraph.

B. Passive Force Versus Active Force.

The maximum contractile response which airway smooth muscle generates Is achieved at a specific muscle length (Stephens et al.,

1968 and 1969; Hahn and Nadel, 1979). An experiment was designed

to determine the amount of force to apply to the tracheal rings

In order to achieve a maximum contractile response to histamine.

Figure 1 shows the amount of force generated at three different

passive forces. The maximum contractile response was obtained at

550 mg wt force in three of the four tissues and appeared to be

independent of the initial length of the tracheal smooth muscle. 25

FIGURE I

Resting Force Versus Active Force

The length of the tracheal smooth muscle In each ring was measured prior to Its suspension In the organ bath* The amount of resting force applied to the tissue was varied by manipulating the force transducer. At each new resting forcet histamine (10~6 M) was added to the bath and Che amount of active force developed was measured. Each experiment was repeated twice to monitor any changes in tissue sensitivity upon reexposure of the tissue to histamine. Each test was followed by a complete washout of the drug. Each point represents the average of two tests. The values

In parenthesis indicate the initial length of the tracheal smooth muscle. ACTIVE FORCE (mg.wt. BOO 4 0 0 6 200

00 45

Resting Force Versus Active Force Active ForceVersus Resting 0 ETN FORCE RESTING m. w I . ) (mg. IUE I FIGURE 55 0 55

6 50 18mm I I 5mm 1 I7mm I 26 Averaging Che responses of Che four tracheal rings (Fig. 2) shows Chat Che percenc of accive developed force has reached a maximum at 550 mg wt force.

The cracheal rings were equlllbraced under an Initial force of 500*600 mg. Since Che tissue response can also be altered by

tissue manipulation (e.g. surgery, tying of sutures, etc.) the response of the tissue to a single concentration of histamine was routinely used to determine if the appropriate amount of force had been applied.

The application of resting force to the tracheal smooth muscle is important not only to obtain the maximum contractile

response but also to evaluate drugs which relax tracheal smooth muscle. In many cases, relaxation of the tissue cannot be observed

unless force has been applied to the tissue.

C. Equilibration of the Tissue

During the equilibration period, the rings were washed with

fresh oxygenated FSS every fifteen minutes. To determine

whether the rings were fully equilibrated, the response of the

tissue to a submaxlmal dose of histamine (5X10*7 M or 10*6 M)

was tested at the beginning of each experiment. This test was

necessary for two reasons. First, It was observed during the

equilibration period that the trachea spontaneously contracts

and relaxes. Exposure of the tissue to a low concentration of

histamine helps the tissue to stabilize. Secondly, It was

occasionally observed that the sensitivity of the trachea to FIGURE 2

Resting Force Versus Percent Active Developed Force

Each point represents the Mean + S.E.M. of determinations from A guinea pigs. kactive developed force

Ratting Force Versus Percent Active Developed Force Developed Active Percent Ratting Force Versus 100 4 60 80 20 0 450

ETN FORCE RESTING FIGURE 2 FIGURE ( w m g t . . ) 550

650 30 histamine increased after the first exposure. Therefore, the response of the tissue to 5X10-7 M or 10-6 M histamine was tested at least twice in order to evaluate changes in tissue sensitivity.

D. Monitoring of Tissue Sensitivity

Two tracheal rings were prepared from each animal. One ring served as a control for the other ring. Having a control tissue made it possible to determine if the responses produced were due to the test drug only, or to changes in tissue sensitivity.

E. Histamine Tachyphylaxis

When tracheal tissue is exposed to high concentrations (>10-4 M) of histamine it becomes desensitized to histamine. That is, the tissue is no longer responsive to histamine. In Fig. 3 the initial cumulative dose response to histamine (Test I) is compared to the tissue response to histamine after three hours of washing following exposure to 10“2 M histamine. Although a blphaslc tissue response is obtained, the maximum histamine response is reduced by

752. Since the high histamine concentrations Induced a prolonged reduction in the tissue response, the tracheal ring experiments were always discontinued once the tissue had been exposed to these higher concentrations. A paired tracheal ring preparation was used in these experiments. One tracheal ring was treated with histamine and the second tracheal ring was treated with the second drug. The responses of paired tracheal rings were usually within 203. of each other.

9 31

FIGURE 3

Desensitlzation of Tracheal Rings by Histamine

Cumulative doses of histamine (10-8 M to 10-2 M) were added to the organ bath> The contractile force was measured at each concentration after the response had reached a maximum* At the end of the first test the tissue was washed for 30 minutes and the tissue response to 10**6 M histamine was measured* Since the response to

1 0 - 6 M was reduced, the tissue was again washed and the test redone*

The washing and the testing were repeated several times. The test

(XI) represents the histamine response after three hours of washing.

All results are expressed as the percent maximum histamine response obtained in Test I. The values represents the Mean + S.E.M. of duplicate determinations from 4 animals* % M A XI M UM HISTAMINE RESPONSE 100 9 SO 40 0 8 20 60 80 Desensitization ofRings*by HistamineTracheal molar 8

on io t a r t n e c n o c HISTAMINE 6 FIGURE 3 FIGURE (-LOG) 4

of 2 32 33

F. Assay of Tissue

1. Dose Response

Two methods were used to measure the effect of drug concentration on the tracheal smooth muscle* There was little difference In the results of the two methods*

a* The drug was added to the bath and allowed to remain

In contact with the tissue until the drug response

had reached a maximum* The drug was then washed

out of the bath before the next higher concen

tration of the drug was tested*

b* The cumulative dose response method (Arlene and

deGroot, 1954) was also used* The concentration

of the drug In the bath was Increased after the D tissue response had plateaued*

In both cases, 100 ul of drug was added to the bath containing 10 ml of PSS. Therefore, approximately a 1:100 dilution of the original drug

concentration was made In the bath.

2* Time response

In these experiments, the response of the ring to one drug

concentration was monitored for 10-20 mlnuteB* At the end of

this period the drug was washed out completely*

3* Evaluation of histamine antagonist activity

The histamine antagonists were allowed to remain in

contact with the tissue for 1 0 - 2 0 minutes before histamine or a

histamine agonist was added to the bath* This procedure was

followed for both the time responses and dose responses* 4. Washout of drug

Drugs were washed out of the tissue by adding fresh oxygenated PSS to the organ bath and repeating this procedure until the tissue tension had again reached the initial baseline. The wash out tine depended on the dose of drug and the type of drug tested.

C. Expression of Data

In all experiments, the tissue response to 1 0 - 4 M histamine was tested. It was observed that the maximum contractile response to histamine occurred at 5 X 1 0 -5 M to 1 X 1 0 -4 M concentrations of histamine. Therefore, the tension developed with 1 0 -4 M histamine was considered'to be the maximum histamine response. In all experiments the drug responses were expressed as the percent of the maximum histamine response.

Data was analyzed using repeated measures analysis of variance.

Corrections for autocorrelation were made. Newman-Keuls post hoc tests were used when there was a significant F statistic.

IV. Biochemical Assay

A. Preparation of the Tracheal Smooth Muscle

The guinea pigs were killed by spinal dislocation. The trachea were quickly removed and placed in cold oxygenated PSS, pH 7.4. The tracheal tube was opened by cutting lengthwise through the cartilage parallel to the tracheal smooth muscle.

The trachea were pinned flat against the dissecting dish and

the connective tissue was dissected away from the tracheal smooth muscle. The smooth muscle was cut away from the tracheal rings and divided into segments*

B* Incubation of the Tissue

The dissected tissue was allowed to sit in oxygenated PSS at room temperature, thirty minutes before Incubation. This was done to avoid changing the temperature of tissues too abruptly.

It has been observed that handling of the animal and tissues alters cyclic nucleotide levels. Therefore, it was necessary to first preincubate the tissues in PSS before testing drugs on the tissue. This allowed the tissue to equilibrate and reduced variation in the tissue concentrations of the cyclic nucleotides. The tracheal segments required sixty minutes of incubation in oxygenated PSS at 37"C (Fig. 4). After the tissue was preincubated in 1.8 ml PSS, 200 jil of PSS or drug was added and the sample was incubated for the desired time. Incubations were terminated by the addition of 2 00 pi of 50% trichloracetic acid (TCA) to the incubation tubes. The tissues were then removed from the tubes and homogenised in 5X TCA.

C. Measurement of Cyclic AMP and Cyclic GMP

1. Preparation of tracheal extracts

The incubated tracheal segments were

homogenized in 0.8 ml of cold 5% TCA using a glass

tissue grinder with a ground glass pestle. The

homogenate was poured into 10 X 75 mm glass tubes.

The homogenizer was rinsed with 200 ul of 5X TCA 36

FIGURE 4

Time Course of the Decrease In Cyclic Nucleotide Concentration In Guinea Pig Trachea with Incubation In Physiological Salt Solution

Tracheal smooth muscle segments were Incubated In physiological salt solution at 37°C for 0, 30, 60 and 90 minutes. The cyclic AMP and cyclic GMP concentrations In the trachea were determined simul taneously* Each point represents the Mean + S.E.M. of determinations from 10 guinea pigs. The asterisk Indicates significant difference from control (P< 0.05).

i IU o o >•

p.moUs/mg protein INCUBATION INCUBATION Concentration Concentration in-Incubation with Pig Trachea Guinea Time Course of the Decrease in Cyclic Nucleotide Cyclic in Decrease the of Course Time in Physiological Salt Solution Salt in Physiological 30 Cci QMP Cyclic O FIGURE A FIGURE IE IMINI TIME 60 AMP * • 0 0

37 which was then added to the tubes containing the

homogenized sample* The samples were centrifuged

at 3000 x g for 20 minutes at 4°C. The tubes

were again placed in an ice water bath for

10 minutes to stabilize the precipitated protein*

The supernatant fraction was poured into

13 X 100 mm cubes. The precipitate fraction was

dissolved in 400 ul of 1 N NaOH and saved for

protein determination*

The TCA was removed from the supernatant

fraction by extracting three times with three volumes

of water saturated diethyl ether* The ether layer

was removed by suctionlng and any ether that re

mained after the last extraction was evaporated by

placing the sample tubes in 50°C water for ten

minutes* The pH of the extracts was adjusted to

6.2 by adding 0.1 volume of 1 M acetate buffer pH 6.5.

2* The radioimmunoassay procedure

In brief, 125i-labelled succinyl cyclic AMP tyrosyl methyl ester (THE) was added to the anti-cyclic AMP antiserum and known or unknown cyclic AMP samples were then acetylated to Increase the affinity for the antlserum and added to the mixture. The degree to which labelled cyclic AMP was displaced from the antibody was proportional to the concentration of unknown or standard in the sample* 39 a. Preparation of assay reagents

I. Antiserum* The antisera for cyclic AMP and

cyclic GMP were produced by Immunizing rabbits

with a mixture of Freund's adjuvant and either

succlnylated cyclic AMP or cyclic GMP coupled to

keyhole limpet hemocyanln* The animal was bled

periodically and the resulting antiserum was

shown to be highly specific for its appropriate

cyclic nucleotide*

II . 1251 cyclic nucleotide* Cyclic AMP TME

(02'-mono8uccinyl adenosine 3':5'-cycllc monophos-

phoric acid tyrosyl methyl ester) and cyclic GMP

TME (02'-aono8uccinyl guanoslne 3':5'-cyclic

monophosphorlc acid tyrosyl methyl ester) were

both obtained from Sigma Chemical Co. Cyclic

AMP TME and cyclic GMP TME were both iodlnated by

the lactoperoxldase method of Mlyachi et al., 1977).

The Iodlnated cyclic nucleotides were diluted In

0.05 M acetate buffer pH 6.2 containing 0.25Z bovine

gamma globulin.* Each sample contained about 10,000

counts per minute.

III. Standard cyclic nucleotides. Cyclic AMP

and cyclic GMP were purchased from Sigma Chemical

Co. The standards were prepared at a concentration 40

of 1 0 0 pmole/ml and scored at -2 0 *C until diluted

for use* Standard curves consisted of points

from 1 - 1 0 0 0 fmoles.

b. Radioimmunoassay of tracheal extracts

The tracheal extracts were diluted 1:1 for the cyclic

GMP and cyclic AMP determinations* All dilutions

were made with 0*05 M acetate buffer pH 6*2*

The assay procedure was as follows:

Standard cyclic nucleotide (1-1000 fmoles) or 2 0 0 pi Unknown sample

Acecylation reagent 10 pi (2 parts acetic anhydride + 5 parts triethylamlne)

1251-Succlnylated cyclic nucleotide** TME 50 pi

Sucdnylated cyclic nucleotide antiserum 50 pi (diluted 1:4000 for cGMP 1:8000 for cAMP

The standard or unknown sample wsb pipetted Into 12 X 75 mm glass tubes* The acetylation reagent was added to each tube and mixed Immediately* After acetylation, the samples remained at room temperature for 10 mlnuteB and then were put on

Ice* The 1251 cyclic nucleotide and the cyclic nucleotide antiserum were then added* The tubes were mixed and incubated overnight at 4°C* The next day 2*5 ml of 60% saturated ammonium sulfate (390 g (NH^^ SO4 + 1 liter H2 O) was added* The tubes were mixed and incubated at 4°C for 20 minutes. They were then centrifuged at 3000 g for 20 minutes at 4°C* The supernatant 41 fraction was poured off and the tubes were wiped inside without disturbing the precipitate. The precipitate fraction was counted by the gamma counter.

The binding of the cyclic nucleotide to the antiserum was expressed as a percentage of the binding which occurred in the absence of any cold cyclic nucleotide trace binding. The standard curve was plotted as percent of trace versus cyclic nucleotide concentration (1 fmole to 1000 fmole). Both the cyclic AMP and cyclic GMP assays were sensitive enough to measure 1 femtomole of cyclic nucleotides in 200 pi (Figs. 5 and 6 ). The unknown concentrations were determined from standard curves and expressed as pmoles per mg of protein.

D. Protein Determination For Tracheal Smooth Muscle

The tracheal protein was determined by the method of Bradford

(1976). The samples were diluted with 1 N NaOH to yield a concentration of 10 to 30 mg of protein per 100 ml solution.

The standard curve was done with BSA, 0 to 60 pg protein per 100 pi

To the 100 pi sample« 5 ml of the Coomassie Brilliant Blue

G-250 reagent was added. The color was allowed to develop for two minutes at room temperature. All samples were read at 595 run on the spectrophotometer within one hour. The colors were not scable beyond one hour. 42

FIGURES 5 and 6

Cyclic AMP and Cyclic GMP Standard Curves *

The binding of the cyclic nucleotide to the antiserum was expressed as a percentage of the binding which occurred in the absence of any cold cyclic nucleotide trace binding* The standard curve was plotted as percent of trace versus cyclic' nucleotide concentration (1 fmole to 1000 fmoles). PERCENT TRACE BINDING •99 90 50 10 1 YLC M (IMOLES) AMP CYCLIC 5 10 Cyclic AMP StandardCyclicAMP Curve FIGURE 5 FIGURE 0 100 50

0 1000 500 43 PERCENT TRACE BINDING 9 9 90 0 5 10 YLC M ( MOLES) (I GMP CYCLIC 10 Cyclic GMP Standard CurveStandard GMP Cyclic 4 FIGURE FIGURE 0 100 50 6 6 500 1000 RESULTS

I. The Physiological Effects of Histamine on Guinea Pig Tracheal Smooth Muscle*

A* The Effect of Histamine on Tracheal Smooth Muscle

The objective of these experiments was to determine the physiologic response of isolated tracheal rings to histamine* The polygraph recording of the responses to cumulative dose of histamine is shown in Figure 7. Slowly developing dose-related increases in force which reached a maximum at 10-4 M histamine were observed.

Concentrations of histamine above 10-4 M produced decreases in contractile force* In Figure 8 the same response is shown in the form of a concentration response curve* The response of the tracheal rings to either individual or cumulative doses of histamine was biphaslc* Concentrations of histamine from 10-8 M to 10**4 M caused contraction of the tracheal rings in a doBe related manner.

However, concentrations of histamine above 10-4 m induced a dose related decrease in tension. The maximum contractile effect occurred at 10**4 h histamine and at 10-2 M the tissue was 70% relaxed.

It has been established that the constrictor activity of histamine on tracheal smooth muscle is the result of activation of an % histamine receptor (Hawkins and Schild, 1951). However, the mechanism of the inhibitory action observed at high histamine concentrations is not known (Hawkins, 1955; Krell, 1979).

44 -0 45

FIGURE 7

Polygraph Record of Histamine Dose Response

Concentration activity recording for histamine obtained on a single tracheal ring* Cumulative doses of histamine were added in 100 1 quantities and changes in contractile force were measured* mg OF FORCE 1000 1800-1 0 Polygraph Record of Histamine Dose Response 020 2 10 IUE 7 FIGURE IE (MINI) TIME t 30 0 60 50 0 4 47

FIGURE 8

Histamine Dose Response

Individual or cumulative doses of histamine were added to the organ bath in 100 1 quantities* The maximum contractile response of the the trachea was obtained at each concentration* All results are expressed as the X maximum response obtained at (10**4 M Hist** amine)* Each point represents the Mean + S.E.M. of determinations from 15-23 guinea pigs* % MAXIMUM HISTAMINE RESPONSE 0 0 1 60 40 80 0 2 n - - - - —0 Cmltv Ds Responses Dose Cumulative 0 0— 8 ige oe Responses Dose Single OA DU CONCENTRATION DRUG MOLAR HistamineDoseResponse 6 -OG) G (-LO FIGURE 8 8 4

2 48 49

B. A Comparison of the effects of Acetylcholine* Carbachol and Histamine on Tracheal Contraction

Agents which contract tracheal smooth muscle independently of the histamine receptor were tested to determine If this blphaslc tissue response was unique to histamine* Accordingly, pairs of tracheal rings were exposed to increasing concentration of carbamylcholine (carbachol) and acetylcholine (Fig* 9)* Both of these agents increased tissue contraction In a dose-dependent manner with maximum effects which were, respectively, 80% and 40% greater than the maximum histamine response* In spite of this greater efficacy neither agent produced a decrease in force even at the highest concentration used (10-2 M). As had been seen previously, histamine produced a

50% decrease In force at 10-2 M. From this experiment, it is apparent that the histamine-induced decrease in force occurB well before the tissue reaches its maximum contractile force*

The differences between histamine and carbachol were further accentuated when the time course of their effects were examined* The response of tracheal rings to low (10-6 ), medium (10-4), and high

(10-2 ) concentrations of histamine and carbachol was monitored for

20 minutes (Fig* 10). Several differences in response were observed.

As was seen previously, the tissue contraction at each concentration of drug was greater with carbachol than with histamine. At 10-4 K drug concentration the contractile force obtained with carbachol was approximately double that obtained with histamine. The highest dose.of histamine tested (10-2 M) induced an initial increase in force followed by a rapid decrease in contractile force which was 34% of 50

FIGURE 9

Comparative Response of Tracheal Rings to Histamine, Acetylcholine and Carbachol

One tracheal ring was exposed to histamine and the second of the pair of tracheal rings was exposed to carbachol or acetylcholine.

After the response had plateaued the drug was washed out and the next higher concentration was tested. The histamine dose response is the result of 2 experiments and each value represents the Mean + S.E.M. of determination from eight guinea pigB. The data of carbachol and acetylcholine represent the Mean + S.E.M. of determinations from

4 guinea pigs. The responses are plotted as the percent of the maximum histamine response in the Individual tracheal ring. % % M A X IM UM HISTAMINE RESPONSE 200 160 120 80 40 R A L O M Comparative Response of Tracheal Rings toHistamine Rings Tracheal ofResponse Comparative Acetylcholine and Carbachol and Acetylcholine *# •* (-L 0 (-L G) FIGURE 9 FIGURE CARBACHOL ACETYLCHOLINE HiSTAMIN E HiSTAMIN

51 52

FIGURE 10

Tine Course of the Effects of Carbachol and Histamine on Tracheal Smooth Muscle Contraction

The tracheal rings were exposed to 10-6 M, 10-4 M and 10-2 M concentrations of histamine or carbachol* The control tissue received histamine and the test tissue carbachol. The change in tissue response was monitored for 20 minutes* After each concentration the drug was completely washed out before the next higher concentration was tested*

All responses are expressed as the percent maximum histamine response for the individual tracheal ring* All values represent the Mean +

S.E.M. of determinations from 6 guinea pigs. PERCENT MAXIMUM HISTAMINE RESPONSE 200 100 120 40 60 20 Time Course of the Effects of Carbachol and Histamine and Carbachol of theofEffects Course Time * IE F NUAIN (MIN) INCUBATION OF TIME on Tracheal Smooth Muscle Contraction Smooth Muscle Tracheal on 5 FIGURE 10 FIGURE 10 Carb achol Hlatamlna

20 53 *• 53 0 1 '* 54 maximum at 20 minutes. With carbachol, the contractile response reached a maximum in 10 minutes and remained stable over the 20 minute period. Thus, high histamine concentrations Induce a biphaslc response: a contraction followed by slowly developing relaxation which was not obtained with cholinergic agonists.

C. The Role of H^-Hlstamine Receptor in the Biphaslc Response, of Tracheal Smooth Muscle.

Experiments were designed to determine if the biphaslc response of histamine resulted from the action of histamine at the H} receptor.

The Hj receptor agonist 2-(2-aminoethyl)-pyridine (2-AEP) and the receptor antagonists, diphenhydramine and pyrllamine were used in the study.

1. The Response of Tracheal Rings to 2-AEP

The histamine agonist, 2-AEP, was tested on the guinea pig tracheal rings and the response to histamine was tested on matched control tracheal rings (Fig. 11). Two differences in response were observed. FirBt, the characteristic biphaslc dose response was obtained with histamine but not with the agonist. Secondly, the tracheal smooth muscle was more sensitive to the effects of histamine.

The 2-(2-amlnoethyl)-pyrldlne curve lies to the right of the histamine curve separated by approximately one log unit. Thus, histamine is the more potent contractile agent, since the dose of histamine which causes 502 of the maximum response (EC^q ) Is 2X10~6 m as compared with the EC5Q of 3X10~5 m for 2-AEP. The maximum histamine 55

FIGURE 11

A Comparison of Che Dose Response Curves of Hiscaolne and 2-(2-Aminoethyl)-Pyridine

Using Che paired Cracheal ring mechod, one cracheal ring was exposed Co cuaulacive doses of hiscaoine and Che companion cracheal ring was exposed Co cuaulacive doses of 2-(2-aminoeChyl)-pyridine.

All resulcs are expressed as Che percenc maximum hiseaaine response

(10-4 M Hiscaoine) for each ring. Each value represenCs Che

Mean + S.E.M. of decerminaCions from 6 animals. XMAXIMUM HISTAMINE RESPONSE 0 0 1 120 40 80 60 20 0 —o o— A Comparison of the Dose Response Curves of Histamine of Curves Response theDose ComparisonofA 2*• (2- AR Amlnoothyl )-Pyr I dine and and G U R 2 -( 2 -aminoefhyl)-pyridine (-L OG(-L ) FIGURE 11 FIGURE ONCENTRATION

56 57

response was obtained at 10”4 M and the maximum 2-AEP response was

reached at 5X10-2 M. It is also apparent that 2-AEP is more efficacious

than histamine, since its maximal contractile effect exceeds that of histamine. There is no decrease in contraction with even the highest

concentration of 2-AEP* On the basis of this and the experiments

shown in Figures 9 and 10, the decrease in contraction Induced by

high histamine concentrations is not due to a tissue fatigue

resulting from strong contraction, since both carbachol and 2-AEP are more efficacious than histamine* In addition, the data suggests that

the effect is not due to an receptor activation by histamine. 58

2. The effect of Hi receptor blockers on the blphasic response.

To further Investigate the role of the Hi receptor In the

blphasic response of the tracheal rings, the Hi blockers pyrilamine

and diphenhydramine were tested for their ability to antagonize the

histamine response* Figure 12 shows the effect of pyrilamine on the

tracheal response to histamine* Pyrilamine caused contraction of the

tracheal smooth muscle at concentrations as low as 10-9 M (not shown)

and at a concentration of 10-4 m , the contractile force is 14% of

maxlnum histamine response. At a concentration of 10-3 m , pyrilamine

induced relaxation In the tissue. A similar action of pyrilamine

and other H^-blockers has been observed In human bronchi (Hawkins

and Schlld, 1951) and guinea pig trachea (Hawkins, 1955). It has

been postulated that the blphasic response observed with H^-antagonists

is due to their ability to cause histamine release from tissue

(Arunlakshana 1952; Feldberg and Smith 1954; Hawkins 1955). Thus,

the blphasic effect observed is possibly a direct effect of histamine

on the smooth muscle. The ability of pyrilamine to contract tracheal

smooth muscle when used alone does not interfere with its ability to

inhibit histamine contraction. At a concentration of 5X10-9 M,

pyrilamine significantly decreased (p< .05) the contractile effect

of histamine at each concentration. However, the relaxation effect

was not blocked. The decrease in the contractile effect of histamine

induced by pyrilamine led to an enhancement of the relaxation response

seen with with higher concentrations of histamine. Although pyrila mine is a competitive antagonist of histamine at the H} receptor, I

FIGURE 12

The Antagonism of Histamine Response by Pyrilamine

In paired tracheal ring experiments, the control ring received

histamine and the second ring was Incubated with pyrilamine for

10 minutes prior to the addition of histamine* Cumulative histamine

dose responses were done on both tissues* The results are expressed

as the percent maximum histamine response* Each point represents

the Mean + S*E*M* of determinations from A animals*

The asterisk Indicates significant difference from the control

histamine response (p<0*05)> XMAXIMUM HISTAMINE RESPONSE 0 0 1 . 20 0 8 20 40 60 MOLAR CONCENT RATION CONCENT MOLAR The Antagonism of Histamine Response by by Pyrilamine Response Histamine of Antagonism The i FIGURE 12 FIGURE (—L O G ) Ml

Ml OF DRUG 60 /

It Is practically Irreversible. Thus* In the presence of pyrilamine! even high concentrations of histamine are not able to produce the maximum contractile force In the tracheal ring.

A further demonstration of the antagonism of histamine contractile response by the blockers is shown in Figures 13 and 14* Diphen hydramine resembles pyrilamine In Its effect on the tracheal rings, causing a contraction at 10"4 m followed by a decrease In force at higher concentrations (Fig. 13). In the presence of hlstamlnet diphenhydramine shifted the dose response curve to the right and significantly decreased the maximum response in a dose related manner

(Fig. 14). Thus diphenhydramine! like pyrilamine! is a competitive antagonist of histamine at the receptor.

It is clear that the blockers inhibit contraction of the tracheal ring. The results with pyrilamine provide evidence that the receptor is not responsible for the histamine-induced relax ation of the tracheal smooth muscle.

D. The Role of the Hj-Histamine Receptor in the Blphasic Response of Tracheal Smooth Muscle to Histamine.

1. A comparison between the effects of histamine and dlmaprlt on tracheal smooth muscle.

Studies with the receptor antagonists and agonists indicated that the contractile phase of the blphasic curve was due to the activation of an receptor. The relaxant phase however! was not blocked by the antagonist! but enhanced. The next set of experiments were designed to determine if the histamine induced relaxation of tracheal smooth muscle was mediated by another receptor, specifically an Hj-histamine receptor. 62

FIGURE 13

The Response of Guinea Fig Tracheal Rings to Histamine and Diphenhydramine

Using paired tracheal rings, the control tracheal rings received cumulative doses of diphenhydramine* The results are expressed as

the percent maximum histamine response for the individual tracheal ring* Each value represents the Mean + S*E.M* of determination from

A guinea pigs* XMAXIMUM HISTAMINE RESPONSE 100 .20 -80 .60 .40 60 40 80 20 0 OA CNETAIN OF CONCENTRATION MOLAR 6 2 4 6 8 Hlstamlna o lhnyr ini m Dlphanhydra • The Response of Guinea Pig Tracheal Rings Tracheal Pig Guinea Responseof The to Histamine and Diphenhydramine toand Histamine DRUGS FIGURE 13FIGURE I** LOGI

63 4 64

FIGURE 14

The Antagonism of Histamine Response by Diphenhydramine

Using paired tracheal rings, the control trachea was tested with histamine and the second tracheal ring was incubated with diphenhydramine (10~8 M to 10-6 M) for 10 minutes followed by the addition of cumulative doses of histamine* At the end of a test the drugs were washed out completely* The tissue was then Incubated with the next higher concentration of diphenhydramine* The control tissue, received histamine at each test in order to monitor the changes in histamine response* All results are expressed as the percent maximum histamine response for the Individual rings. Each point represents the Mean + S.E.M. of determinations from 6 guinea pigs* MAXIMUM HISTAMINE RESPONSE 100 40 80 60 20 The Antagonism of Histamine Response by by Diphenhydramine ofResponse Histamine Antagonism The OA CNETAIN OF CONCENTRATION MOLAR 7 5 6 7 8 HISTAM INE INE HISTAM FIGURE 14 FIGURE l-LOG Dlphanhydraml Dlphanhydraml i I

t 0 M 10 10”®M 10"*M a a 65 66

Dlmaprlc, (S-{3-(N,N-dimethylamino)propylJ IsoChloucea) is a very specific H2-hlstamlne receptor agonist as evaluated in H2 receptor containing tissues (Parsons e£ al., 1977; Durant et al.t

1977). It does not interact with the Hjl receptor, or cholinergic and adrenergic receptors.

To determine if the tracheal smooth muscle contained an H2 receptor, the effect of dimaprlt on the tracheal ring preparation was compared to the histamine effect (Fig. 15). Dimaprit relaxed the tracheal smooth muscle at 10-7 M. At 10-3 M a 20% decrease in force was measured. This response is similar to that seen with histamine.

However, at concentrations below 10-4 M, dimaprlt induces relaxation and histamine induces contraction of the tracheal ring although the relaxation obtained with dimaprlt is not significant. These results indicate that the H2 receptor may be present, although it is not readily seen in an uncontracted trachea.

2. The relaxation of tracheal smooth muscle by histamine and dimaprlt.

Since the relaxation of tracheal smooth muscle by histamine and dimaprit was observed mainly at high histamine concentrations, experiments were designed to attempt to demonstrate a relaxation of tracheal smooth muscle at lower histamine concentration. The acti vation of an H2 receptor is likely to be associated with the relax ation. However, the relaxation response of drugs on tracheal smooth muscle is more readily seen on a contracted tissue (Stephens, 1968).

Carbachol was chosen as the contractile agent since it is a strong 67

FIGURE 15

Comparative Response of Guinea-Pig Tracheal Rings to Histamine and Dimaprlt

In this experiment, one group of tracheal rings uas treated with cumulative doses of histamine (10“8 M to 10-2 M) and the second group was treated with cumulative doses of dimaprit (10-8 M to 10~3 M). All results are expressed as percent maximum histamine response for the individual tissue. Each point represents the Mean

+ S.E.M. of determinations from 14 animals. XMAXIMUM HISTAMINE RESPONSE 0 0 1 -20 40 20 60 80 8 ■ Comparative Response of Guinea-Pig Tracheal Rings Tracheal Guinea-Pig of Response Comparative ■ O A DU CONCENTRATION DRUG MOLAR Dimaprlt • *Dimaprit and toHistamine (-LOG ) 6 FIGURE 15 FIGURE 4

2 69 contractile agent and maintains a stable contractile response

Figs. 9 and 10). The tracheal rings were first contracted maximally with carbachol 10-4 M (102Z of maximum histamine response) (Fig. 16).

The addition of histamine produced a dose dependent relaxation of the tissue, 2X at 10-6 M and 15Z at 10-3 M (Fig. 16). In the presence of the blocker pyrilamine (SX10“9 M) a slightly greater relaxation was obtained, although it was not significantly different from that obtained with histamine alone.

A similar experiment was performed with dimaprlt (Fig* 17). The carbachol contracted tissue relaxed with the addition of dimaprit to the bath. A 3% relaxation occurred at 10-4M dimaprlt and in the presence of pyrilamine a 9% decrease in force occurred at the same concentration. However, the results were not statistically signi ficant. Thus, with a maximally contracted tissue, relaxation of the tracheal tissue by histamine and dimaprit was demonstrated. On the basis of these experiments it appears that histamine activates an inhibitory H2 histamine receptor at all concentrations. The effect is more pronounced at low histamine concentrations. The H2 effect is revealed with a maximally contracted tissue and blockade of the receptor.

3. The effect of metiamlde and clmetidlne on the histamine response.

To further investigate whether an H2 receptor mediating the relaxation response is present in tracheal smooth muscle, experiments were designed to attempt to block the histamine-induced relaxation 70

FIGURES 16 AND 17

The Relaxation of Tracheal Smooth Muscle by Histamine and Dimaprlt

The tracheal rings were contracted with carbachol 10-4 M In the presence and absence of pyrilamine 5X10-9 M. Cumulative doses of histamine or dimaprit was added to the bath and force was measured after the response had plateaued* The results are expressed as the percent relaxation following carbachol* Each point represents the

Mean + S.E.M. of determinations from 8 animals* The asterisk indicates significant difference (p< 0*05) from the response at

10”7 M histamine* % REL A X A T ION FOLLOWING CARBACHOL 25 20 30 OA CNETAIN O HISTAMINE OF CONCENTRATION- MOLAR The Relaxation of Tracheal Smooth Muscle by Histamine by Smooth Muscle ofTracheal Relaxation The 7 Control ylalo 5x10' Pyrllamlno♦ IUE 16 FIGURE - L I- O G I 5 % 2 71 X RELAXATION FOLLOWING CARBACHOL 20 0 5 0 5 OA CNETAIN F DIMAPRIT OF CONCENTRATION MOLAR The Relaxation of Tracheal Smooth Huscle by Smooth Dimaprit by Huscle ofTracheal Relaxation The 7 + Pyrilam ine ine + Pyrilam onlroI C •# O I LOG - I 5x10' FIGURE 17 FIGURE 5 3 72 73 by using two specific competitive ^-receptor antagonists, cimetidine and metiamlde. Both cimetidine (N4-cyano-N-methyl-N'-[2”(5-methyl- imidazol-4-yl) methylthloethyl] guanidine) (Parsons 1977) and metiamlde (N-methyl N'-2-[5-methylimidazole-4-yl)-methylthioethyl] thiourea) (Blaclc et al.. 1973) are structural analogs of histamine which have very specific U2 agonist activity.

The effect of cimetidine on tracheal smooch muscle was determined

(Fig. 18). At concentrations below 10-4 M, a small dose dependent contraction of the tracheal rings was observed. However, at concen trations above 10-4 M significant (p< .05) relaxation of the tracheal ring occurred. Thus the H2 blockers themselves are able to contract and relax the tracheal smooth muscle.

4. The effect of H2 antagonists on histamine-induced relaxation

The next set of experiments were designed to determine whether the ^-antagonists cimetidine and metiamlde could block the relaxation of tracheal rings induced by high histamine concentration and potentiate the tracheal contractile response to histamine, possibly by blockade of an H2 inhibitory receptor.

Tracheal rings were maximally contracted with 10-4 m histamine or acetylcholine followed by relaxation with 10-3 M histamine. At maximum relaxation, which occurred after approximately 30 minutes, increasing concentrations of the H2 blocker were added to the organ bath. Neither cimetidine nor metiamlde, at concentrations from

10-8 m to 10-4 m, reversed the histamine-induced relaxation of the tracheal smooth muscle. Furthermore, preincubation of the tracheal 74

FIGURE 18

Comparative Responses of Guinea Pig Tracheal Rings to Histamine and Cimetidine

Using paired tracheal rings, one tracheal ring,was treated with cumulative doses of histamine (1 0 “8 m to 1 0 ~2m) and the second ring was treated with cumulative doses of cimetidine (10-8M to

10~2m). All results are expressed as percent maximum histamine response for the Individual tissue. Each point represents the m Mean + S.E.M. of determinations from 19 animals* Asterisk indicates significant difference from control (p< 0.05). %M A X I MUM HISTAMINE RESPONSE 100 -20 40 60 20 Comparative Responses of Guinea Fig Tracheal Rings Tracheal Fig ofGuinea Responses Comparative 6 8 OA DU CONCENTRATION DRUG MOLAR oHletemlnm to Histamine and Cimetidine and to Histamine FIGURE 18 FIGURE ( “ LOG (“ LOG ) 4

2 76

rlnga with the U2 blockers also failed to alter the tracheal response

to high histamine concentrations. Thus it appears that mechanisms

other than the metiamlde- and cimetidlne-sensitive H2 receptor are

Involved in the tracheal relaxations which occur in response to

1 0 ~ 3 m histamine*

The previous experiments with antagonists demonstrated that

histamine-induced relaxation of acetylcholine contracted

trachea was potentiated in the presence of the Hj antagonists*

The results suggested the presence of an inhibitory H2 receptor

in the tracheal smooch muscle which could be activated at low

histamine concentrations* Experiments were designed to further

investigate this inhibitory H2 receptor* First, attempts were made to determine the effect of cimetidine on the dOBe response of

tracheal rings to histamine* Cimetidine was added to the bath, allowed to equilibrate with the tracheal ring for 2 0 minutes, and

then increasing concentrations of histamine were added* Inconsistent

results were obtained. Some tissues showed an enhanced contractile • response in the presence of cimetidine and others showed no change*

The difficulty in achieving reproducible results were mainly due

to the instability of the contractile response to histamine* It was occasionally observed that even the lowest concentrations of histamine produced increases in tension followed by slight decreases in tension* Thus it was necessary to monitor the histamine response in the presence and absence of the H2 blockers in order to distin guish the effects of blocker from the normal tissue response. A specific protocol was routinely used to demonstrate the effect of ^-blockers on the histamine response.

a). The tissues were preincubated with cimetidine for at least

ten minutes;

b). Only one dose of histamine was tested at a time, and

response was monitored for at least five minutes;

c). Changes in tissue sensitivity to histamine were

carefully monitored using a control tissue from the test

animal;

d). The drugB were completely washed out prior to the next

test. « Next, experiments were designed to test the effect of a single concentration of cimetidine on the time-dependent response of the tracheal ring to a single concentration of histamine using the above protocol. Figure 19 shows the effect of 10-5 M cimetidine on the contractile response to 10-5 M histamine. The contractile response was enhanced significantly at 10 minutes (p< .05) and remained significantly enhanced at 20 minutes. These results support the findings of Okpako et al. (1978) that H2 antagonists enhance the contractile response to histamines.

5. The enhancement of the histamine response by metiamlde and cimetidine.

The ability of the ^-blockers metiamlde and cimetidine to enhance the contractile response to histamine were compared.

Metiamlde resembles cimetidine in its ability to cause both con traction and relaxation of the tracheal tissue (Fig. 20). However, 78

FIGURE 19

The Tine Course of the Effect of Cimetidine on the Histamine Response of Tracheal Smooth Muscle

Histamine 10-5M was added to the organ bath of the control and

test tissue and the response was monitored for 20 minutes* The drugs were then completely washed out* Cimetidine (10~5 M) was added

to the test tissue and Incubated for 15 minutes* Histamine (10-5 M) was added to the control and test tissues and allowed to remain in

the bath for 20 minutes* The histamine response is the average of the control and test tissues response to histamine 10-5 M. Each value represents the Mean + S*E.M* of determinations from six animals*

The asterisk indicates significant difference from control histamine response (p< 0*05)* % % MAXIMUM HISTAMINE RESPONSE h ieCus f theCourseofTime Cimetidine ofThe Effect the on Histamine 0 6 1 1

40 8 20

0 I N (mini C UB AT TIME I ON Response of Tracheal SmoothResponseofTracheal Muscle */ *» itmn ■ Histamine Clmatl din* HI 1 1 a ml Cimetidine n + • FIGURE *19 10 15

I — 20

79 80

FIGURE 20

Comparative Response of Guinea Fig Tracheal Rings to Histamine and Metiamlde

Using paired tracheal rings, one tracheal ring was treated with cumulative doses of histamine (10-7 M to 10-2 M) and the second ring was treated with cumulative doses of metiamlde (10-7 M to 10-2 M).

All results are expressed as percent maximum histamine response for the individual tissue* Each point represents the Mean + S.E.M. of determinations from 4 animals* Asterisk indicates significance

(p< 0.05). %M A XIM U M HISTAMINE

RESPONSE * 100 60 40 80 20 80 60 20 40 Comparative Response of Guinea Pig Tracheal Rings Tracheal Guinea ofPig Response Comparative O A CNETAIN OF CONCENTRATION MOLAR 8 • Mat! Mat! amide • o Hist amine Hist o to Histamine and Metiamlde and Histamine to S G U R D FIGURE 20 FIGURE

« 81 82 a higher concentration of metiamlde (10-2 M) is necessary to achieve a significant (p. < 0 .0 S) relaxation of the smooth muscle*

Figure 21 compares the effects of metiamlde (10-7 M, 10-5 M and

10-3 M) and cimetidine (10-11 M, 10-9 M, 10-7 M and 10-5 M) on the response of the tracheal rings to 5X10-7 M histamine* Histamine

(5X10-7 M) produced a 12% Increase in force* In the presence of metiamlde, the contractile response was increased* At 10-5 M concentration of metiamlde the contractile response was enhanced significantly (p< .05)* However, higher concentrations of metiamlde failed to show any further increase* Enhancement of the histamine contraction was observed even with very low concentrations of cimetidine* The preincubation of the tissue with cimetidine (10-1 M to 10-7 m ) produced a dose related Increase in force. However, no f significant increase in force was seen at higher histamine concen trations. The enhancement of the contractile response to histamine by cimetidine and metiamlde may possibly involve inhibition of an inhibitory H2 receptor.

II. The Correlation Between Cyclic Nucleotides and Tracheal Contraction.

Cyclic AMP and cyclic CMP were originally proposed to be involved in the hormonally Induced relaxation and contraction respectively of smooth muscle (BAr, 1974; Andersson et al. 1975). Recently the role of cyclic CMP in smooth muscle contraction has been re-evaluated.

Many agents which Increase cyclic CMP levels in smooth muscle also cause smooth muscle relaxation (Schulte et al., 1977; Axelsson et al.,

1979; Kukovetz et al., 1979). 83

FIGURE 21

r The Enhancement of the Histamine Response of Metlamlde and Clmetldlne

The procedure as described for Figure 19 was used. At the end of a test, all drugs were washed out and the next higher concentration of metlamlde or clmetldlne was used. The control tissue received only histamine. The values represent the responses after five minutes of drug Incubations. All responses are expressed as a percent of the maximum histamine response for the Individual tissue. Each point represents the Mean + S.E.M. of determinations from 4 to 8 animals.

The asterisk denotes significant difference from the control response

(p< 0.05). XINCREASE IN HISTAMINE RESPONSE 120 100 20 80 60 40 OA CNETAIN F DRUG OF CONCENTRATION MOLAR The Enhancement of the ofHistamineResponse by The Enhancement M * t l • m i d * C l m*t I din* Clmetldlne endClmetldlne Metlamlde FIGURE 21 FIGURE G O L - ( )

84 85

Histamine has been shown to increase, the levels of cyclic AMP

and cyclic CMP in tracheal smooth muscle (Murad and Klmura, 1974;

Ohkubo at al., 1976, Katsukl and Murad, 1977). Thus, the cyclic

nucleotides may be Involved in the physiological response of the

trachea to histamine. To investigate the role of cyclic nucleotides

in the histamine response, the effect of cyclic nucleotides on

tracheal smooth muscle was studied as well as the effect of

histamine on the concentrations of cyclic nucleotides in the tracheal

smooth muscle.

A. The Effect of Exogenous Cyclic Nucleotides on Tracheal Smooth Muscle Contraction

In order to determine the effect of exogenous cyclic AMP and

cyclic GMP on tracheal smooth tone, tracheal rings were incubated with

cyclic AMP or cyclic GMP or their dibutyryl derivatives. None of

the cyclic nucleotides altered the tracheal smooth muscle tone,

(results not shown). In the next experiment the cyclic nucleotides were tested on tracheal tissue submaxlmally contracted with 10-5 M histamine (60% of the maximum histamine response). Neither cyclic AMP nor cyclic GMP had any effect on the histamine contracted tissue. However, their dibutyryl derivatives, Ng, 2'-0-dibutyryl cyclic AMP and Ng,2'-Odibutyryl cyclic GMP, relaxed the tracheal tissue in a concentration-dependent manner (Fig. 22). Dibutyryl cyclic AMP, at a concentration of 10~6m, significantly (p< 0.05) relaxed the histamine contracted tissue. At concentrations of

10“5 m and 1 0 * 4 M, 15Z and 75% decreases in forces were observed, 86

FIGURE 22

The Effect of Dibutyryl Cyclic Nucleotides on Tracheal Smooth Muscle

The tracheal rings were contracted submaxlmally with 10-6 M histamine (a dose which produced 602 of the maximum histamine response).

Cumulative doses of dibutyryl cyclic AMP or dibutyryl cyclic GMP

(10~7 M to 10~4 M) were added to the organ bath. Decreases In muscle tension vote measured. The results are plotted as percent of relaxation (following histamine). Each point represents the

Mean + S.E.M. of determinations from 4 animals. The asterisk denotes significant difference from the control response (p< 0.05). xrelaxation following histamine . 100 40 6 80 20 0

MO LA R LA MO 5 7 L The Effect ofEffect DibutyrylThe Nucleotides Cyclic RG CONCENTRATION DRUG. l-LOG I on Tracheal Smooth on Tracheal Muscle •v- FIGURE22 ylc GMP Cyclic Dibutyryl 5 7 L

60 40 100 20 80 0 87 88

respectively. Dibutyryl cyclic CMP, at concentrations of 1 0 -6 -

1 0 “ 4 m also, significantly (p<0.05) relaxed the tissue although

to a lesser extent than dibutyryl cyclic AMP. At 1 0 -4 M dibutyryl

cyclic GMP, a 20% decrease in force was produced. The lack of

effect of the authentic cyclic nucleotides on tracheal smooth muscle is possibly due to their inability to penetrate the smooth muscle cell membrane (Miller, 1977).

These results suggest that cyclic AMP and cyclic GMP, which

are proposed mediators of smooth muscle relaxation and contraction,

respectively, [Murad and Klmura, 1974; Andersson et al., 1975] can

both cause tracheal smooch muscle relaxation. Consequently, if

these two cyclic nucleotides play a role in tracheal smooth muscle activity, they must subserve Che same function - relaxation, and

thus are not involved in drug-induced contractions.

III. The Effect of Histamine on Tracheal Cyclic Nucleotide Concentrations

Histamine contracts and relaxes guinea pig tracheal rings.

We examined this biphaslc effect of histamine in view of the demon strated ability of histamine to alter tracheal cyclic nucleotides*

The objective was to determine whether there was a correlation between the biochemical and physiological effects of histamine.

A. Concentrations of Cyclic Nucleotides in Guinea Pig Trachea

The basal concentration of cyclic AMP in guinea pig trachea was approximately double the concentration of cyclic GMP. The average 89

cyclic AMP concentration was 5.6 + 5*53 pmoles/tag protein and the

average cyclic GMP concentration was 3.81 + 0.52.

B. The Effect of Histamine on Tracheal Cyclic Nucleotides

1. Time course of the effect of histamine

To determine the best incubation time for measuring the

effect of histamine on tracheal cyclic nucleotide concentrations*

tracheal segments were Incubated with histamine at concentrations of

5X10-7 M and 5X10-5 M for 0* 1* 5 and 10 minutes. Histamine

caused a rise In cyclic AMP and cyclic GMP concentrations at one minute. The concentrations returned to control level by ten

minutes (results not shown). On the basis of these results a one minute Incubation with histamine was used to determine the effect

of histamine on tracheal cyclic nucleotide concentrations.

2. Histamine dose response

The effect of histamine on tracheal cyclic nucleotide con centrations was evaluated. Histamine elevated cyclic AMP and cyclic GMP levels in a dose dependent manner (Fig. 23). The two cyclic nucleotide response curves are very similar. The responses are biphasic. Concentrations of histamine from 10-6 m to 10-4 M produced a dose related increase in cyclic AMP and cyclic GMP and the responses were maximum a concentration of 10-4 m concentration.

Histamine concentrations of 10-3 M and 10-2 M produced a decrease in cyclic nucleotide levels relative to the level at 10-4 M histamine. At a concentration of 10-4 m , histamine produced a

4-fold increase in cyclic AMP levels and a doubling of the cyclic 90

FIGURE 23

The Effect of Histamine on Cyclic AMP and Cyclic CMP Concentrations In Guinea Pig Tracheal Smooth Muscle

Tracheal smooth muscle fragments were preincubated with buffer for 60 minutes before the addition of histamine* The segments were

Incubated with histamine for one minute* Each point represents the

Mean + S.E*M* of observations from 7 to 17 guinea pigs. The asterisk denotes significant difference from the control response (p< 0*05)* CYCLIC NUCLEOTIDES N. -N m E O 01 la E a e 30 25 35 The Effect of Histamine on CycliconEffect andofThe AMP Cyclic GMP Histamine Concentrations in Guinea Pig Tracheal SmoothConcentrations in Tracheal Guinea Pig Muscle 20 10 OA CNETAIN OF CONCENTRATION MOLAR • O O CYCLIC* AMP YLC QMP CYCLIC 1 •o FIGURE 23 FIGURE HISTAMINE I 10

I 92

CMP levels. The Increases in cyclic AMP were significant (p< *05) at concentrations above 10-5M whereas the increase in cyclic GMP is only significant (p< .05) at 10-4 M concentration of histamine.

The decrease in cyclic AMP, relative to 10-4M histamine was also significant (p< .05).

C. The Effect of and H£ Agonists and Antagonists on the Cyclic Nucleotide Response

Evidence has accumulated suggesting that the histamine Induces increases in cyclic AMP and cyclic GMP concentrations in a variety of tissues Including lung (Palmer, 1971; Palmer, 1972; Mathe et al.,

1974; Poison et al., 1976; Ortez, 1977) are mediated by an interaction with specific histamine receptors. According to these authors the H^-histamine antagonists decrease the accumulation of cyclic

GMP and the ^-histamine antagonists decrease the accumulation of cyclic AMP In lung tissue. It has also been reported that Hi- histamlne antagonists block the increases in cyclic GMP in tracheal smooth muscle (Murad and Kimura, 1974; Katsuki and Murad 1977).

To further evaluate the relationship between histamine receptors and the cyclic nucleotide response, the effect of Hi and H2 histamine agonists and antagonists were studied on tracheal smooth muscle.

1. The effect of the Hi agonist 2-AEP on the concentrations of cyclic AMP and cyclic GMP in tracheal smooth muscle

The highly selective Hi agonist 2-(2-aminoethyl)-pyridine

(Durant, 1973) was used to assess the involvement of the Hi-receptor in the histamine Induced accumulation of cyclic nucleotides. 93

Figure 24 shows the effect of increasing concentrations of 2-AEP

1 0 - 6 m, 10-4 M and 10-2 M) on tracheal cyclic nucleotide concentra

tions* Like histamine (Fig* 23), 2-AEP increased both cyclic AMP

and cyclic GMP concentrations in tracheal smooth muscle. At the

highest concentration, both cyclic nucleotides are doubled* The

results, although not statistically significant, do suggest that

activation of the Hi receptor may lead to Increases in both cyclic

AMP and cyclic GMP concentrations.

2* The effect of the H2 agonist, dimaprit, on the concentrations of cyclic nucleotides in tracheal smooth muscle

The H2 agonist, dimaprit, was tested on the tracheal

smooth muscle to determine if an ^-receptor was Involved In the histamine-induced cyclic nucleotide response (Fig. 25). Dimaprit at a concentration of 10-5 M significantly (p< *05) increased cyclic

AMP concentrations, almost doubling the cyclic AMP levels of guinea pig trachea* Although changes were observed in cyclic GMP concentrations, the increases were not significant* According to

these findings, activation of the H2 receptor produces increases in the levels of cyclic AMP without significant alterations in cyclic

GMP levels*

3* The role of and H2 receptors in the effect of histamine on tracheal cyclic nucleotide

The previous experiments with the Hx and H2 histamine agonists suggest that histamine-induced increases in cyclic nucleo

tides may result from the activation of specific histamine receptors*

Most notably, the H2 agonist dimaprit significantly elevated cyclic 94

FIGURE 24

The Effecc of 2-AEP on Che Concentrations of Cyclic AMP and Cyclic GMP In the Tracheal Smooth Muscle

Guinea pig tracheal segments were Incubated with 2-AEP for one minute* Each point represents the Mean + S*E.M* of cyclic nucleotide determinations from 5 animals. CYCLIC NUCLEOTIDE The Effect of 2-AEP on the Concentrations of Cyclic AMP Cyclic of theConcentrations on 2-AEP of Effect The 16 1 5 a 12 20 24 0 and Cyclic GMP In the Tracheal Smooth Muscle theTracheal In Cyclic GMP and 2-12-AM I. NOETHYL I-PYRIDINE OA CNETAIN OF CONCENTRATION MOLAR 0 i i

* FIGURE 24 FIGURE 10"6 I

10"4 y lc AMP Cyclic m

10~2 I

95 96

FIGURE 25

The Effect of Dimaprit on the Concentration of Cyclic AMP and Cyclic GMP In Guinea Pig Trachea

Guinea pig tracheal segments were Incubated in Dimaprit

1 0 ~ 6 m to 1 0 ~ 2 m for one minute* Each point represents the

Mean + S.E.M. of cyclic nucleotide determinations from 5 guinea pigs* The asterisk denotes significant difference from the control (p< 0.05)* The Effect of Dimaprit on the Concentration of Cyclic AMP Cyclic of theConcentration on Dimapritof Effect The CYCLIC N UCLE OTIDES p.mol os/mg protoin 10 12 14 8 6 and Cyclic GMP in Guinea Pig Trachea Pig in Guinea Cyclic GMP and 0 OA CNETAIN OF CONCENTRATION MOLAR

ylc GMP Cyclic ylc AMP Cyclic FIGURE 25 FIGURE * * 10~7 maprit r p a im d

10 »-s ° " 10

' 97 98

AMP concentrations In tracheal smooth muscle without altering the

cyclic GMP concentrations*

to further evaluate the Involvement of histamine receptors In

the tracheal cyclic nucleotide response, specific histamine antag

onists were used* Clmetldlne and metlamlde (Black et al*, 1973;

Parsons, 1977) antagonists of the H2 mediated histamine response, and diphenhydramine, which antagonizes the activation of the Hi

receptor, (Ash and Schild, 1966) were used* Time studies done

with the antihistamines showed that maximum alterations in the cyclic nucleotides occurred after 1 0 minutes of incubation with

tracheal smooth muscle segments and the levels were decreased to control levels by 20 minutes (results not shown)* Thus, a 10 minute

incubation period was chosen to study the effects of the Hi and

H2 blockers on tracheal cyclic nucleotide concentrations.

Figures 26 and 27 show the effects of metlamlde and clmetidine on tracheal cyclic nucleotide concentrations* Although both agents altered cyclic nucleotide concentrations, none of the changes were significant* Thus It appears that H2 receptor blockade, in the absence of receptor stimulation, does not have a specific effect on tracheal cyclic nucleotide concentrations.

The objective of the next experiment was to attempt the separation of the histamine induced cyclic AMP and cyclic Q4P responses using specific Hi and H2 antagonists* Since It had been observed that the H2-blockers metlamlde and clmetldlne altered tracheal cyclic nucleotide concentrations after ten minutes 99

FIGURES 26 and 27

The Effect of H2 Antagonists on the Concentrations of Cyclic AMP and Cyclic GMP in Guinea Pig Trachea

Tracheal segments were incubated with clmetldlne or metlamlde for ten minutes* Each point represents + S.E.M. of cyclic nucleo tide determinations from 5 guinea pigs* CYCLIC NUCLEOTIDES p.moles/mg protein 20 24 16 12 8 The Effect of U of Effect The of Cyclic AMP and Cyclic GMP In Guinea Pig Trachea Pig Guinea In GMP Cyclic and AMP Cyclic of MOLAR 0 Cyclic • Cyclic O */>■ 2 Antagonists on the Concentrations the on Antagonists C l M E T l D I N E CONCENTR ATION CONCENTR FIGURE 26 10 ■8 GMP AMP 10 -4

OF 10"2 100 CYCLIC NUCLEOTIDES p.mol ••/mg protoln 20 24. 12 16 . 4 . oL 8 . . . of Cyclic AMP and Cyclic GMP in Guinea Pig Trachea Pig Guinea in GMP Cyclic and AMP Cyclic of h feto H of Effect The

0 I OA CNETAIN OF CONCENTRATION MOLAR

• • Cci GMP Cyclic O y lc AMP Cyclic 2 Antagonists on the Concentrations the on Antagonists 10-7 U M E T I AM I D E I FIGURE 27FIGURE

10 I * - 10 I * “

101 102

of Incubation It was assumed that this time period may not be

appropriate for evaluating the blocking effect, since the elevation

of Che cyclic nucleotides by the agonists may mask the blocking

effect* A twenty minute incubation of tracheal segments was chosen

for the blocking studies* this was similar to the time period for

studying the blocking effects of the antagonists on tracheal

contraction.

The tracheal segments were incubated with 10-5 M concentration

of the antagonist diphenhydramine or the H2 antagonist clmetldlne

for twenty minutes prior to the addition of 10-5 M concentration of

histamine (Fig. 28). The histamine response was measured after one * minute. Histamine increased cyclic AMP concentrations 80S and

cyclic GMP concentrations 120%.

Clmetldlne did not alter the histamine-induced Increase in

cyclic GMP* It did decrease the increase in cyclic AMP although

the results were not significant* These results do not discredit

the findings with the H2 agonist dimaprit which suggest that the

H2 receptor is involved in the histamine-induced increase in

cyclic AMP* Rather these data suggest that other mechanisms,

possible indirect, are also involved in the histamine-induced

increase*

Diphenhydramine did not alter the histamine-induced cyclic AMP response. However it did significantly (p< 0.05) block the hist amine induced Increase in cyclic GMP* These results, which suggest

that the H^ receptor is involved in the increase in cyclic GMP, are not consistent with the experiments with the H^ agonist 2-AEP 103

FIGURE 28

The Effect of and % Antihistamines on the Histamine Induced Alteration of Cyclic Nucleotides In Guinea Pig Trachea

Tracheal segments were incubated with 10-5M diphenhydramine or clmetldlne for 20 minutes prior to the addition of histamine* The histamine incubation was terminated after one minute* All columns represent the Mean + S.E.M* of determinations of 6 guinea pigB. The asterisk denotes significant difference from the control histamine response (p< 0.05). Induced TracheaAlteration of Cyclicin NucleotidesGuinea Fig

TheEffectthe of HiandHistamine Antihistamineson Hj XINCREASE ABOVE CONTROL 200 1 B 0 120 .40 40 80 0 Hltianln*. Q 1 Ml a* 01 0 D Cl * QD mall . m « cyclic

p m a 110~*I FIGURE 28FIGURE CYCLIC OHO

104 105 which failed Co demonstrate a direct association between receptor activation and cyclic GMP stimulation* Thus if the Hi receptor is Involved in this increase, then possibly activation of the receptor produces other responses which In turn lead to this Increase*

4. The effect of other bronchoactlve agents on tracheal cyclic nucleotide concentrations

The cyclic nucleotide changes observed with the histamine agonists and antagonists are quite complex and thus the exact mechanism of the elevation of tracheal cyclic nucleotides by histamine is difficult to explain. To aid in understanding this phenomenon, the effects of two bronchoactlve agents, carbachol and

Isoproterenol were studied for their effects on tracheal cyclic nucleotide concentrations* The cholinergic agonist carbachol contracts tracheal smooth muscle, and is reported to increase the concentrations of cyclic GMP in tracheal smooth muscle (Katsuki and

Murad, 1977). The beta-adrenergic agonist isoproterenol relaxes tra cheal smooth muscle and is reported to primarily increase cyclic AMP levels in tracheal smooth muscle (B&r, 1974). Carbachol (10-6 M) produces a slight decrease in the basal concentrations of cyclic

AMP and cyclic GMP (Fig. 29). At the 10-4 M concentration, the levels of cyclic AMP and cyclic GMP are nearly doubled and the maximum response is achieved. The results however were not statis tically significant. Isoproterenol (10-6 M) increased cyclic AMP concentrations significantly (p< .05). A 3-fold increase in cyclic

AMP level was obtained (Fig. 30). The levels of cyclic AMP remained high at 10~4 and 10-2 M concentrations. Isoproterenol had little 106

FIGURE 29

The Effect of Carbachol on the Concentration of Cyclic AMP and Cyclic GMP In Guinea Pig Trachea

Tracheal segments were Incubated with carbachol for one minute* Each point represents the Mean + S.E.M. of cyclic nucleotide determinations from 5 guinea pigs* The Effect of Carbachol on the Concentration of Cyclic AMP Cyclic of Concentration the on Carbachol of Effect The CYCLIC N UCLEOTI DES 10 1 £ to o a .20 24 12 and Cyclic GMP In Guinea Pig Trachea Pig Guinea In GMP Cyclic and OA CNETAIN OF CONCENTRATION MOLAR O ylc GMP Cyclic • ylc AMP Cyclic fh xf FIGURE 29 FIGURE CARBACHOL 10 -0

10 -4 10 -2

107 108

FIGURE 30

The Effect of Isoproterenol on the Concentrations of Cyclic AMP and Cyclic GMP In Guinea Fig Trachea

Tracheal segments were Incubated with Isoprotrenol for one minute.

Each point represents the Mean + S*E*M* of cyclic nucleotide deter minations from 5 guinea pigs* The asterisk denotes significant difference from the control response (p< 0*05). CYCLIC N UCLE OTIDES The Effect of Isoproterenol on the Concentration of Cyclic AMP Cyclic of Concentration the on Isoproterenol of Effect The p.mol ••/mg protoln 35 25 20 30 15 10 0 OA CNETAIN OF CONCENTRATION MOLAR and Cyclic GMP In Guinea Pig Trachea Pig Guinea In GMP Cyclic and • * ISOPROTERENOL 10 FIGURE 30 FIGURE * “ t 10 ylc GMP Cyclic ylc AMP Cyclic * “ 10“2 iiI 110 effect on tracheal cyclic GMP concentrations* A slight but insigni ficant increase was observed at the highest concentration*

The results obtained with isoproterenol are consistent with the idea that cyclic AMP may be involved in drug-induced smooth muscle relaxation* However! the data from the carbachol experiments fail to show a direct association between smooth muscle contraction and changes in tracheal cyclic nucleotide concentrations* DISCUSSION

Histamine, a primary amine which la found in mast cells of the lung and trachea, causes constriction of the bronchial and tracheal smooth muscles In a variety of mammals, Including man (Hawkins and

Schlld, 1951; Hawkins, 1955). This Is generally believed to be the primary function of histamine on airway smooth muscle of mammals*

However, there are numerous exceptions to this generalization* The cat trachea and sheep bronchus are completely relaxed by histamine

(Maengwyn-Davles, 1968; Gyre, 1969) and high histamine concentrations have been observed to cause relaxation of gulnea**plg trachea

(Hawkins, 1955).

The biochemical mechanism by which histamine produces its effect Is not known* One possibility is that the occupation of the receptor by histamine induces specific biochemical changes within the cell ‘such as an alteration in cyclic nucleotide concentration*

Histamine Increases the concentrations of cyclic AMP and cyclic

GMP In a variety of tissues Including tracheal smooth muscle (Murad and Kimura, 1974; Ohkubo et al*. 1976; Katsukl. and Murad, 1976)*

Both cyclic AMP and cyclic GMP are thought to be involved in the regulation of smooth muscle tone (B&r, 1974; Andersson et al,

1975).

Ill 112

It is the aim of this dissertation to clarify the mechanism by which histamine acts on tracheal smooth muscle. Although it is known that histamine can alter smooth muscle tone, the types of

receptors in the tracheal smooth muscle and their relationship to cyclic nucleotides has never been clearly defined.

I. The Existence of Multiple Histamine Receptors in Tracheal Smooth Muscle and Their Relations to Cyclic Nucleotides

A. The Action of Histamine on Tracheal Smooth Muscle

In the guinea pig tracheal ring preparation, histamine was found to not only cause a concentration-dependent contraction of the tracheal smooth muscle but also a concentration-dependent relaxation. The dose-response curve was bell shaped with the maximum'contractile force developed at a concentration of 10-4 M

(Fig. 31). The tracheal cyclic nucleotide dose response to histamine resembles the physiological response of the tracheal rings to histamine. Histamine induced a dose related Increase in cyclic AMP and cyclic GMP which reached a maximum at 10-4 M and decreased at concentrations above 10“4 m . A comparison of the effects of histamine on tracheal contraction and tracheal cyclic nucleotide concentrations suggests that the two phenomenon may be related.

In this work, the objective was to determine first if the physiological responses (contraction and relaxation) and the biochemical changes (alterations In cyclic AMP and cyclic GMP) were due to the activation of a specific histamine receptor, and second, if there was any relationship between the biphasic contractile 113

response and Che biphaslc cyclic nucleotide changes. Specific

histamine agonists and antagonists were used to determine if specific

receptors were Involved.

B. Role of the Hj Receptor In the actlonB of Histamine on Tracheal Smooth Muscle

The concentration dependent contractions of tracheal rings to histamine were significantly depressed by the receptor antagonists pyrllamlne and diphenhydramine. These results were not unexpected;

the bronchodllaclng activity of antagonists is well known.

However, a second effect of H^ antagonists was observed. Pyrllamlne at a concentration of 5X10-9 M significantly onhanced the Inhibitory effect of 10-2 M histamine on the tracheal smooth muscle. Approximately a 402 greater relaxation of the smooth muscle was obtained in the presence of pyrllamlne. These results strongly suggest the presence of an Inhibitory histamine receptor which is not associated with the Hi receptor. In fact, it suggests the presence of a receptor which directly opposes the Hj receptor effects.

The results obtained with the H^ agonist 2-AEP supports this hypothesis. Firstly, this agonist, which is less potent than histamine, Is also more efficacious than histamine, achieving a 202 greater contractile response of the tracheal smooth muscle. Thus the presence of an inhibitory histamine receptor apparently modulates the contractile response to histamine and maximum contraction is never obtained. Secondly, concentrations of histamine greater than

10**4 m produce a decrease in muscle tone; similar concentrations of 114

2-AEP produce contraction* These findings further suggest that

this Inhibitory effect Is Independent of the action of histamine at

the receptor* It appears that this Inhibitory effect may be a

protective mechanism, protecting the smooth muscle cell from both

the potent contractile effect of low concentrations of histamine as

well as from the immense airway constriction which could result

from high concentrations of histamine being released Into the

airways, during anaphylaxis*

In the experiments with the Hi antagonists paradoxical results were obtained. Pyrllamlne at a concentration of 10*3 M significantly

relaxed the tracheal smooth muscle, whereas concentrations less than

10*3 M had no effect on the muscle. Since high concentrations

(>10*5 M) of antihistamines have been shown to cause histamine

release from lung tissue (Dreyer, 1950; Feldberg and Smith, 1954),

the observed effect of pyrllamlne may be due to its release of histamine* Another possible explanation of the relaxant effect is

that if histamine Is Involved in maintaining the normal tone of

the tracheal smooth muscle, then complete blockade of the H^

receptor would reveal the inhibitory response.

C* The Role of the H2 Receptor In the Actions of Histamine

Recent reports that many inhibitory effects of histamine (i.e., inhibition of rat uterine contraction) were mediated by an H2 hist- 4 amine receptor^ suggested that this might be the case with the

Inhibitory action of histamine on tracheal smooth muscle. The appearance of this Inhibitory effect at the highest histamine 115 concentrations suggested that if both and H2 receptors existed in tracheal smooth muscle then possibly the affinity of histamine for the receptor is greater than the H2* Thus If the Hi receptor la fully occupied at 10-4 M histamine, the maximum contractile effect would be obtained* If the H2 receptor is not fully occupied at 10-4 M concentration, then higher histamine concentrations would be expected to produce a greater H2 effect leading to relaxation.

To further investigate the H2 effect, an H2 agonist was tested to determine if it mimicked the inhibitory action of histamine and

H2 antagonists were tested to determine if they would block the inhibitory effect*

The results obtained with H2 agonist, dimaprit, were unimpressive*

Although dimaprit had a general depressive action on the basal tone of tracheal smooth muscle the results were not significant. However, the fact that the higher dimaprit concentration (10-3 M), like histamine, showed the greatest decrease in tone, suggested that this phenomenon should be further investigated. The small relaxant effect of dimaprit, as compared to the histamine-induced effect, suggests that histamine is the more potent agoniBt of the hypothesized

H2 receptor*

The results with histamine obtained on carbachol-contracted tissue were more impressive. The theory that Ho receptors existed waB further supported by the observation that low concentrations of histamine significantly relaxed the carbachol-contracted tracheal rings in the presence of H^ receptor blockade* Furthermore, preincubation of the tracheal rings with the H2 antagonists cimetidine 116 and metiamlde significantly enhanced the contractile response of the tracheal rings to histamine* The results obtained demonstrated that cimetldine was the more potent of the two H2 blockers*

D* Atypical Histamine Receptor

Although the results obtained with the H2 agonists and antagonists supported the theory of an H2 inhibitory receptor they did not explain the mechanism of relaxation induced by high histamine concentrations. The H2 antagonists were unable to Inhibit or reverse the relaxation Induced by 10"2 m histamine* In fact, —2 the H2 antagonists at 10 M concentrations significantly relaxed the tracheal tissues* Whether the antagonists act as agonists of the H2 receptor at this concentration Is not known. There are several possible explanations for this effect: 1) the relaxation at this concentration may be the result of activation of an H2- like receptor - a receptor which can be activated by an H2 agonist but not reversed by H2 antagonist; 2) it may result from activation of a third type of histamine receptor*; 3) the Inhibitory response may result from the release of other factors such as prostaglandins which In turn relax the tracheal smooth muscle*

£. Evidence that Histamine Receptors are Correlated with the Cyclic Nucleotide System.

1. Cyclic AMP

Histamine significantly increased cyclic AMP concentrations in guinea pig tracheal smooth muscle* At a concentration of lCT4 M histamine, at which maximum contraction was obtained, the 117 maximum increase in cyclic AMP was achieved* In Figure 31 it can be seen chat the changes in the cyclic nucleotides is associated with the increase and decrease in contractile tone*

At K T 4 M histamine a 4-fold increase in cyclic AMP was observed and at 10**^ M a significant (71Z) decrease (relative to 10~4 M) was observed.

The major problem in interpreting these results is that it is difficult to determine if the increase precedes, occurs simultaneously with, or follows the change in contractile tone. The second problem is

that the effect of histamine on the tracheal cyclic AMP and tracheal tone were measured on different tracheal tissue preparations. However, * attempts were made to handle the tissues similarly.

The studies with the histamine agonists and antagonists provides insight into-the possible mechanlsm(s) by which histamine stimulates an increase in cyclic AMP. The H2 agonist dimaprit significantly increased cyclic AMP concentrations without altering cyclic GMP concentrations (p> 0.05). This suggests that histamine may stimulate increases in cyclic AMP via activation of the H2 receptor. The -2 H^ agonist, 2AEP, also Increased cyclic AMP concentrations at 10 M, although the differences were not statistically significant. Further experiments are necessary In order to determine the Involvement of

Hi receptors in the cyclic AMP response. The results obtained with the Hi and H2 antagonists are perplexing. Although both clmetldlne (10”^ M) and diphenhydramine (10**^ M) reduced the increase in cyclic AMP produced by 10~5 m histamine, the decreases were not significant. P*mols _ CYCLIC NUCLEOTIDE ■■ ■ — It MAX RESPONSE mi/prol*;ii 100 40 30 80 20 20 80 10 The Effect of Histamine on the Contraction and Cyclic and theContraction on Histamine of Effect The Nucleotide Concentrations of Guinea Fig Trachea Guinea Fig of Concentrations Nucleotide « OA CNETAIN OF CONCENTRATION MOLAR 0 ylc GMP Cyclic ylc AMP Cyclic io-# HI S T A M I N E FIGURE 31 FIGURE «

10 * " 118 119

What we can conclude from these experiments is:

1. Cyclic AMP relaxes tracheal smooth muscle;

2. An H 2 receptor is involved in the increase in cyclic AMP but other

mechanisms must be also involved since U2 blockade does not

inhibit the Increase;

3* No direct relationship betwen Hj receptor activation and cyclic

AMP can be drawn;

4* The increase in cAMP by histamine is greatest when muscle

contraction is at a maximum* At higher concentrations of histamine

(>10**^ M) both muscle contraction and cAMP concentration are

reduced*

5. The results with Isoproterenol show an Increase in cyclic AMP

without an alteration of cyclic GMP, suggesting again that an

Increase in cyclic AMP is associated with inhibition of smooth

muscle tone*

2. Cyclic GMP

Histamine significantly increased cyclic GMP in a pattern similar to that observed with cyclic AMP* At the peak contractile tone induced by 10**4 m histamine, cyclic GMP concentrations increased

3-fold (3.17 + 0*46 to 10.68 + 2.54). The increases in cyclic GMP, like the contractile response, were concentration dependent. The reduction in tension which occurred at 10”2 k (741) corresponded to 631 less activation of cyclic GMP relative to that at 10"4 M.

Although the Hj_ agonist, 2-AEP, increased cyclic GMP from basal level of 1.91 + 0.37 to 10.15 + 2.86 at 10~4 M, the 120 results were not statistically significant* Therefore, no conclusion could be drawn frosi this experiment about Hi receptor involvement in cyclic GMP activation.

Experiments with histamine antagonists showed that whereas the

H2 antagonist, clmetldine, did not alter the increase in cyclic GMP

Induced by 10*5 m histamine, the Hi antagonist, diphenhydramine

(10*5 M) reduced cyclic GMP concentrations 50% (p< 0.05).

Again, as with cyclic AMP, the relationship between cyclic

GMP and contractile activity of histamine is not clear cut.

Dibutyryl cyclic GMP relaxed tracheal smooth muscle. However, increases in cyclic GMP are associated with Increases in contractile

« tension induced by histamine. In contrast to the results with cyclic

AMP, the reduction in contractile tone at 10*2 M is not associated with an activation of cGMP (p> 0.05).

Apparently, the increase In cyclic GMP is not associated with H2 receptor activation. H2 agonists did not significantly Increase cyclic GMP; and H2 antagonists did not inhibit the histamlne*induced

Increase in cyclic GMP. However, the Hj receptor may be involved in the cyclic GMP response, since the agonist increases cyclic GMP and the Hi antagonist decreases the increase in cyclic GMP induced by histamine.

Whether cyclic GMP mediates contraction or relaxation of the tracheal smooth muscle is not clear. It appears that histamine may activate the H^ receptor to contract the tracheal smooth muscle.

Contraction of the smooth muscle leads to Increases in cyclic GMP. 121

II* Mechanisms of Smooch Muscle Contraction

The mechanism by which smooth muscle contracts has been an area of extensive research* A current and very popular theory suggests that a calcium-dependent phosphorylation of smooth muscle contractile proteins is an essential component of this process.

It Is suggested that this process Is to modulated by cyclic nucleo tides* I will discuss this theory In regard to the poslble mechanism of histamine-induced contraction of tracheal smooth muscle*

A. Contractile Proteins

All smooth muscles, regardless of the source, contain 2 major proteins, actln and myosin (for review see Adelstein and Hathaway

1979; Adelstein 1980). Actln exists as a double helical polymer in muscle. Myosin is a hexamer consisting of 2 pairs of light chains and 1 pair of heavy chains. Contraction results from the Interaction between the 2 proteins. The energy required for contraction is generated by the hydrolysis of ATP. The active site of ATPase Is located on the globular head region of myosin, where the substrate is hydrolyzed releasing ADP and Inorganic phosphate. The energy generated Is used to slide myosin and actln filaments past each other, thereby producing muscle shortening or contraction.

The interaction between actln and myosin and thus contraction does not occur unless myosin is first phosphorylated. Current evidence suggests that phosphorylation of smooth muscle myosin is i essential for the activation of myosin ATPase activity by actln 122

(Gorecka et al., 1976)* Myosin light chain kinase (MLCK) catalyzes the phosphorylation of the 20,000 dalton light chain of myosin

(Small and Sobieszek, 1977; Barron et al*. 1979, 1980; Aksoy and

Murphy 1979) and*MLCK is regulated by the binding of calcium to the enzyme, (Chacko et al., 1977)* MLCK, first Isolated from chicken gizzard, is composed of 2 subunits (Dabrowska et al., 1977)*

One of the subunits has been Identified as calmodulin (Dabrowska e£a l . , 1978), the protein which regulates the binding of calcium to MLCK. The dephosphorylation of myosin is catalyzed by a second enzyme myosin light chain phosphatase (Sobieszek and Small, 1977;

Chacko et al., 1977; Gorecka et al., 1976; Sherry et al., 1978).

The dephosphorylation results in smooth muscle relaxation.

MLCK PiATP

Relaxation <- Myosin Phosphorylated Myosin

Phosphatase

This cyclic phosphorylatlon-dephosphorylation of myosin light chain has been demonstrated in intact porcine carotid arteries

(Aksoy and Murphy, 1979) and chicken gizzard myofibrils (Yamaguchi and Watanabe, 1980). There is evidence that phosphorylation- dephosphorylation of myosin occurs in tracheal smooth muscle.

De Lanerolle and Stull, (1980) using antibodies to purify myosin from tracheal smooth muscle were able to separate the phosphorylated and dephosphorylated myosin light chains by isoelectric focusing. 123

B. The Role of Calcium

Although the cyclic phosphorylatlon-dephosphorylatlon reactions determine the contractile state of the smooth muscle, there are other regulatory mechanisms In the control of smooth muscle tone* Specifically, MLCK requires calcium for activation

(Barron et al., 1979). Unactlvated MLCK is not able to phosphorylate the 20,000 dalton myosin light chain (Walsh et al., 1980, Adelstein

1980; Stull 1980; Adelstein and Hathaway, 1979). The mechanism by which calcium regulates MLCK-lnduced phosphorylation has been extensively reviewed (Walsh et al., 1980; Adelstein and Hathaway,

1979). Apparently in the resting muscle, the concentration of free Intracellular calcium is low and myosin light chain kinase is in the inactive form. Contraction is initiated by an Increase

in calcium concentrations. The calcium binds to the protein calmodulin, the regulatory subunit of myosin light chain kinase.

This complex is the active form of MLCK, capable of phosphorylating myosin light chain and thus contraction. Barron et al. (1979) demonstrated that treatment of contracted arterial smooth muscle with calcium chelatory EGTA results in relaxation accompanied by dephosphorylation of the light chain. Thus contraction but not relaxation is calcium dependent.

C. Drug Effects- Mechanisms of Hormonal Induced Contraction

There is abundant evidence that endogenous and exogenous contractile agents induce contraction through this Ca2+-dependent phosphorylation mechanism. Agents such as histamine may initiate contraction by binding to a receptor, causing an Increase in the permeability of calcium channels (Stull jet al., I960), thus allowing intracellular calcium levels to Increase to the levels necessary for myosin light chain kinase activation and subsequent myosin phosphorylation. Increases in Ca*2-dependent myosin phos phorylation ware correlated with norepinephrine and KC1 induced contraction of arterial smooth muscles (Barron et al., 1979, 1980) and dephosphorylation of myosin were correlated with inhibition of contraction. Myosin phosphorylation and dephosphorylation have been correlated with drug and hormonally-induced contraction and relaxation, respectively of a variety of smooth muscles (Barron et al., 1979, 1980; Gualtieri and Janls 1977; Aksoy and Murphy,

1979; Stull etal., 1980). The addition of methachollne (10~5 m ) to tracheal smooth muscle Incubated with labeled phosphate, produced contraction accompanied by a 12-fold increase in myosin phosphate content (Paietta and Sands, 1978). Atropine-induced relaxation of the contracted tracheal smooth muscle was accompanied by a SOX decrease in phosphate content. Removal of calcium from the incubation medium of the resting tracheal smooth muscle relaxed the tissue and reduced the phosphate content of myosin below basal levels. Readdltion of calcium produced an increase in tension, concomittant with phosphorylation of myosin.

Histamine may act through this calcium-dependent phosphory lation mechanism to induce tracheal smooth muscle contraction by 125 acting at Its receptor to promote calcium Influx* There la no evidence for this as yet, but neither has histamine or any other contratlle agent been shown to Interact directly with the contractile apparatus to produce contraction*

III* Cyclic Nucleotides and Tracheal Smooth Muscle Contraction

A* Cyclic AMP

1. Mediator of smooth muscle relaxation

Cyclic AMP Is hypothesized as the mediator of horaonaly-, neurotransmlttor-, and drug-induced smooth muscle relaxation*

Epinephrine, isoproterenol, and other beta-adrenergic receptor agonists, which activate adenylate cyclase to Induce cyclic AMP synthesis, are potent bronchodilators (Andersson and Mohme, 1970;

Kukovetz and Poch, 1970; Triner et al., 1977; Andersson, 1972, M r

1974), Theophylline and other methylxanthlnes which Inhibit cyclic

AMP phosphodiesterase, thereby maintaining high intracellular cyclic

AMP levels, are able to relax a variety of smooth muscles including airway smooth muscle; and most Importantly, cyclic AMP relaxes tracheal smooth muscle*

Triner at al., 1977 has demonstrated a good correlation between the potency of Isoproterenol and other catecholamines as activators of adenylate cyclase and as relaxants of bronchial smooth muscle. Similarly, the Inhibition of phosphodiesterase by theophylline and papaverine resulting in increases In cyclic

AMP also corresponded to their ability to relax the bronchial smooth muscle* 126

At present, there Is no evidence that histamine-induced

Increases in cyclic. AMP generated in guinea pig tracheal smooth

muscle mediates relaxation of the tissue*

2. Cyclic AMP and smooth muscle contraction < In this work, it was observed that the contractile agents,

histamine, 2-AEP, and carbachol Induced Increases in cyclic AMP of

tracheal smooth muscle* These findings are in agreement with

those of Katuski and Murad (1977) and Wong and Buckner (1978) who

demonstrated that acetylcholine, histamine, carbachol and KC1

Increase cyclic AMP concentrations in guinea pig tracheal smooth

muscle, in vitro. Although the mechanism or function of this

increase is not known, it has been observed that removal of

calcium from the Incubation medium of the tracheal smooth muscle

prevents the Increase in cyclic AMP as well as contraction (Wong

and Buckner, 1978). This suggests that calcium may be responsible

for the increase in cyclic AMP. Andersson et_al. (1972, 1973) has

demonstrated that the Increases in cyclic AMP which occur in

rabbit colon muscle in the presence of carbachol actually follow

the contractile response. The observation that indomethacin

blocked this increase led Andersson to postulate that the increase was due to the release of prostaglandins, a phenomenon which is apparently dependent on the release of calcium as a result of

contraction.

Rasmussen and Goodman (1977) postulate that cyclic AMP is part of the regulatory feedback loop which modulates the contractile

response. The drug-induced increase in calcium concentration 127 causes contraction and blocks cyclic AMP hydrolysis by Inhibiting phosphodiesterase* The Increase In cyclic AMP stimulates cyclic

AMP reuptake by the membrane and e££lux from the cell, thus reducing contractile response*

Based on these findings, It Is possible that the Increase In cyclic AMP Induced by histamine may result from the contractile activity and may function to reduce or attenuate the contractile response, thus acting as a feedback regulator*

3* Cyclic AMP-dependent protein kinase

Current evidence suggests that the actions of cyclic AMP in organs and tissue are mediated by a cycllc-AMP dependent protein kinase (Kuo and Greengard, 1970; Langen 1973)* In smooth muscle, the cyclic AMP-dependent protein kinase is suggested to promote relaxation by phosphorylatlng myosin light chain kinase, thereby preventing it from phosphorylatlng myosin (Adelstein £t al., 1978).

Cyclic AMP protein kinase is a tetramer composed of a regulatory and catalytic subunit (for review see Glass and Krebs,

1980; Corbin and Lincoln, 1978; Krebs 1972). Binding of cyclic

AMP to the regulatory subunit dissociates the enzyme, and the catalytic subunit is free to catalyze myosin phosphorylation*

cAMP Inactive Protein Kinase — .,, > Active Protein Kinase + R£ * cAMP (R2C2) Myosin Light - P Chain Kinase Chain Kinase i Myosin— > Myosin (P)__» Contraction 128

Cyclic AMP dependent protein kinases have been isolated from a % variety of smooth muscles (Anderson al*, 1980) and bovine

tracheal smooth muscle (Sands et al*, 1976)* The latter was able to phosphorylate contractile (tropomyosin and actomyosin) and noncontractlie proteins of the tracheal smooth muscle.

Whether this noncontractlle protein is myosin light chain kinase has not been determined, but it appeared to be the preferred substrate for the protein kinase* Thus in tracheal smooth muscle the actions of cyclic AMP appear to be mediated by a cyclic AMP- dependent protein kinase*

Support for the hypothesis is given by the discovery of a dephosphorylation mechanism in tracheal smooth muscle. Paletta and Sands (1978) Isolated a phosphoproteln phosphatase from tracheal smooth muscle which was active against a phosphorylated specific substrate for tracheal muscle protein kinase. Again, the function of this protein is unknown and possibly is myosin light chain kinase*

4. Role of Calcium

Cyclic AMP-induced relaxation of smooth muscle apparently involves an alteration in intracellular calcium, which is necessary for the contractile response. Studies suggest that cyclic AMP enhances calcium uptake and binding by membrane fractions, accelerates calcium efflux from smooth muscle cells, and prevents the transmembrane efflux of calcium (Andersson and

Nelsson, 1977; Rasmussen and Goodman, 1977). 129

Current evidence, however, suggests that the chief effect of cyclic AMP on Intracellular calcium Involves cyclic AMP-dependent protein kinase-induced phosphorylation of myosin light chain kinase* Apparently the phosphorylated enzyme is unable to bind the calcium regulatory protein, calmodulin, as tightly as the unphosphorylated enzyme, and thus calcium activation does not occur. In the absence of myosin phosphorylation by myosin light * chain kinase, the muscle is relaxed.

B. Cyclic GMP

1. Evidence as mediator of smooth muscle contraction.

The role of cyclic GMP in smooth muscle contraction Is controversial. Early observations that contractile agents promoted increases In smooth muscle cyclic GMP concentrations with minor effects on cyclic AMP (Schultz et al., 1973b; Goldberg et al.,

1975) led to the theory that contraction resulted from these increases in cyclic GMP. However, data in support of this theory are not strong. Guanylate cyclase, the enzyme which catalyzes the formation of cyclic GMP, is insensitive to known contractile agents (Diamond, 1978; Schultz al., 1973b). Phosphodiesterase inhibitors, which inhibit the breakdown of cyclic GMP, thereby promoting intracellular accumulation of cyclic GMP, are potent tracheal and smooth relaxants (Triner et al., 1977). Furthermore, hs demonstrated in this work, dibutyryl cyclic GMP relaxes histamine-contracted tracheal smooth muscle.

2. Evidence as mediator of smooth muscle relaxation

Guanylate cyclase activators, i.e. nitroprusslde, nitroglycerin, hydroxylamlnc, Increase smooth muscle concentration of cyclic GMP and also relax smooth muscles (Schultz et al., 1975;

1977). In the ductus deferens, the phosphodiesterase inhibitor l-methyl-3-lsobutylxanthlne doubled the levels of cyclic GMP and increased cyclic AMP levels by 502 while causing relaxation

(Schultz et al., 1973b). Good temporal relations between Increases

In cyclic GMP and smooth muscle relaxation by guanylate cyclase activators have been.demonstrated. Katuski and Murad (1977) suggested that the Increase In tracheal cyclic GMP Induced by the guanylate cyclase activators were associated with relaxation since both occurred rapidly. Kukovetz et al. (1979) demonstrated that the increase in cyclic GMP in smooth muscle preparations proceeded relaxation by guanylate cyclase activators. Furthermore, this Increase as well as relaxation were potentiated by phospho diesterase inhibitors. The fact that cyclic GMP derivatives relax tracheal smooth muscle as reported In this work and by

Katsuki and Murad (1977) strongly suggests that cyclic GMP, like cyclic AMP, promotes tracheal smooth muscle relaxation.

3. Cyclic GMP-dependent protein kinase

There la much speculation about the mechanism by which cyclic GMP modulates smooth muscle tone. It is believed that cyclic GMP, like cyclic AMP, activates a protein kinase resulting in phosphorylation of membrane proteins (Casnellle and Greengard,

1974). There is strong evidence for this. Cyclic GMP kinases have been isolated from vascular smooth muscle (Casnellle at al.,

1980) as well as bovine lung (Nakazava and Sano, 1975; Gill et al., 131

1976; Lincoln et al.. 1977). Cyclic CMP-dependent protein klnase-

Induced phosphorylation of smooth muscle membrane proteins has been demonstrated In vascular smooth muscle (Ives et al.. 1980) and nonvascular smooth muscles (Casnellle and Greengard, 1974;

Wallach £t al., 1978).

The function of these cyclic GMF-dependent phosphorylations . are not known. In terms of cyclic GMP as a mediator of smooth muscle contraction, there Is no evidence chat cyclic GMP has any effect on the contractile apparatus such as promoting myosin phosphorylation, which In essence would stimulate smooth muscle contraction. On the other hand, there Is some evidence that cyclic

GMP**dependent phosphorylation may promote smooth muscle relaxation.

The phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (MIX) relaxed the rabbit colon and induced increases in cyclic GMP

(Andersson et al., 1980). The Increases in cyclic GMP correlated well with Increases In protein kinase activity. These authors suggested that MIX causes smooth muscle relaxation by activating protein kinase resulting In protein phosphorylation and Increase

In calcium sequestration.

4. Role of Calcium

Increases In smooth muscle cyclic GMP In vitro in response to the smooth muscle contractile agents KC1 and norepinephrine are diminished in the absence of calcium (Schultz and Hardman,

1975); Katsukl and Murad, 1977). In contrast, Increases of cyclic GMP Induced by guanylate cyclase activators do not require 132 calcium (Schultz-et al., 1977). It Is suggested that the Increases

In cyclic GMP Induced by contractile agents In secondary to the

Increase In calcium promoted by receptor activation (Schultz and

Hardman, 1975). This Increase In cyclic GMP is postulated to act as a negative feedback regulator, whereby Increases In cyclic CMP promotes calcium removal (Schultz et al., 1973a, 1977; Berrldge,

1977) and thus modulates or decreases contraction*

C. The Role of Cyclic Nucleotides in Tracheal Smooth Muscle

Based on the presented discussion the following Is proposed for the action of cyclic nucleotides.

In brief, it Is suggested that both cyclic AMP and cyclic

GMP mediate tracheal smooth muscle relaxation. There are possibly

2 mechanisms of cyclic AMP increase by histamine: a direct increase resulting from activation of the H2 receptor and Indirect Increase resulting from Hi receptor-mediated contraction which acts as a feedback regulator to modulate contraction. The increases in cyclic GMP probably also are a result of H^-receptor induced contraction. Like cyclic AMP, cyclic GMP probably also acts as a negative feedback regulator of the contractile response. 133

IV. A Proposed Mechanism for the Action of Histamine on Tracheal Smooth Muscle

Based on the results' presented In this dissertation, histamine

appears to have three different effects on guinea pig tracheal

smooth muscle:

1. Histamine Induces a contraction of the tracheal smooth

muscle which Is mediated by an H^ receptor and Is

associated with an Increase in cyclic AMP and cyclic GMP;

2. Histamine has an inhibitory action which is mediated by

an H2 receptor and Is associated with an increase In cAMP;

3. Histamine Induces relaxation of the tracheal tissue at

high concentrations. This effect may be mediated by a

third histamine receptor or an H2 subtype.

Evidence for this proposed mechanism will be presented in this *

section as described In Figure 32.

A. Evidence for H^ Receptor

1. Evidence that histamine's activation of the receptor produces tracheal contraction

It has been well established that hlstamlne-lnduced tracheal

contraction is mediated by an H^ receptor (Hawkins and Schlld,

1951; Hawkins 1955; James 1969; Eyre 1969; Murad et al., 1974;

Katsukl et al., 1977; Dunlop and Smith 1977; Himorl and Taira 1978;

Krell 1979). The data In this study support these findings.

In the isolated tracheal rings, histamine-induced concentration-

dependent contractions were blocked by the H^ antagonists diphen

hydramine and pyrilamlne. The H^ agonist 2-AEP also, produced a A PROPOSED MECHANISM FOR THE ACTION OF HISTAMINE

LOW HISTAMINE DIMAPRIT HIGH HISTAMINE HIGH DIMAPRIT PYRILAMINE CIMETID1NE (-r)

CA6CG

BETA

ACETYLCHOLINE L- ISOPROTERENOL 135 concentration dependent contraction of tracheal smooth muscle*

The H2 agonist, dimaprit, did not contract the tracheal ring.

The mechanism by which histamine promotes smooth muscle contraction

Is not known. It Is possible that the activation of the Hi receptor by histamine may cause membrane changes resulting in an

Increase in the permeability of calcium channels. The increase in intracellular calcium concentraction would promote tracheal smooth muscle contraction.

• 2. Evidence that contraction results in increased concen trations of cyclic AMP and cyclic GMP in tracheal smooth muscle.

In this work it was shown that the concentration-dependent contraction of the tracheal smooth muscle by carbachol and by histamine was associated with Inceases in both cyclic AMP and cyclic GMP. Furthermore, both the histamine-induced contraction and Increases in cyclic GMP could be blocked by the Hi antagonist diphenhydramine. Although diphenhydramine reduced the elevation of cAMP, the differences were not significant. Ttiese observations support the findings of Ohkubo (1976) and Katsuki and Murad (1977) who demonstrated that histamine as well as acetylcholine Increased the concentrations of cyclic AMP and cyclic GMP in guinea pig and bovine tracheal smooth muscle.

3. Evidence that cyclic GMP promotes the relaxation of tracheal smooth muscle

Exogenous cyclic GMP and Its derivatives have been reported to cause relaxation of tracheal smooch muscle (Szaduykls-

Szadurski et al., 1972; Katsuki and Murad, 1977). The data reported 136

In this work confirm these results* Dlbutyryl cyclic GMP causes a dose-dependent relaxation of the histamine contracted tracheal rings*

B. Evidence for H2 Receptor

1. Evidence that the activation of the H2 receptor Induces relaxation of the tracheal smooth muscle

In this work It has been shown that the H2 receptor agonist, dimaprit, causes a dose-dependent relaxation of the tracheal smooth muscle* Indirect evidence for an H2 receptor mediation of this relaxation is that the H2 blocker cimetldlne enhances the contractile response to histamine* This confirms the report of Okpako et al. (1978) that guinea pig trachea contains an Inhibitory H2 receptor* He demonstrated that the H2 blocker metiamlde enhanced the contractile reponse to histamine and that the H2 agonist, 4-methylhistamine, relaxed the guinea pig trachea, an effect which was blocked by metiamlde* In tracheal tissues of different species In which a direct relaxation of the muscle by histamine has been observed, neither nor H2 antagon ists were able to block the response (Chand and Eyre, 1978).

This suggests that there may be a second mechanism by which histamine Induces the relaxation of tracheal smooth muscle*

2. Evidence that activation of the H2 receptor leads to Increase in cyclic AMP

There is no direct evidence that histamine activates adenyl cyclase leading to synthesis of cyclic AMP In tracheal smooth muscle.

However, In brain and guinea pig ventricle, the stimulation of adenylate cyclase by histamine or the H2 agonist, dimaprit, can 137 be blocked by ttj histamine antagonists (Johnson et al., 1979; Kanof and Greengard 1979a,b). In this work It was observed that low

concentrations o£ dimaprit and histamine increased the concentrations

of cyclic AMP In the trachea and the increase in cyclic AMP was

reduced by the H 2 antagonist cimetldine>

3. Evidence that cyclic AMP mediates relaxation o£ tracheal smooth muscle.

Dibutyryl cyclic AMP relaxed the guinea pig trachea In a dose-dependent fashion. This was also observed In other tracheal prep arations (Moore jit al., 1968; Szaduykls-Szadurski £t al., 1972;

Newman, 1978). It was also observed in this work that isoproterenol, a known activator of adenyl cyclase (BHr, 1974) has a dual effect.

It increased cyclic AMP concentration In tracheal smooth muscle in addition to causing relaxation of tracheal smooth muscle.

C. Evidence for an Atypical Receptor

In this work it was observed that high concentrations of histamine, the U2 agonist dimaprit, the Hj antagonists clmetldine and metiamlde, and the Hi antagonists diphenhydramine and pyrllamine relaxed the tracheal smooth muscle. The hlstamlne-lnduced relaxation was enhanced by Hi receptor blockade and unaffected by

H2 receptor blockade. Thus it appears that the relaxation is due

to either a third histamine receptor or is not a receptor mediated event. The tracheal relaxation response at high histamine and high Hi antagonist concentrations has been reported previously

(Hawkins 1955). It has been observed that Hi antagonists are able to cause histamine release from lung tissue (Arunlakshana 1952) 138 which may explain why Che antagonises produce a histamine-like response* The relaxation response observed with high concentrations of H2 antagonists may-be due to an H2 agonist activity*

The fact Chat dimaprit and not 2-AEP mimicked the relaxation response suggests that the relaxation may be due to an t^-like receptor.

The relationship between this response at high histamine con centrations and the cyclic nucleotides is less dear* The data with dimaprit shows that this relaxation is not accompanied by increases in cyclic AMP. With histamine the Increases in cyclic

AMP and cyclic GMP are less than observed with maximum histamine response.

D. Future Work

More investigations are needed in this area before firm conclusions can be drawn about the effect of histamine on tracheal smooth muscle tone via cyclic nudeotldeB, calcium and possible prostagllndin8* The difficulty is simultaneously measuring all these parameters will make this quite a challenging endeavor*

There are some interesting implications of this work.

Firstly, these results emphasize the need to be aware of potential side effects when clinically using antagonists to endogenous substances having a multitude of actions. In particular, the H2 agonist dmetldlne administered to an asthmatic in order to treat 139 gastric ulcers, could possibly worsen the asthmatic condition by potentiating the constriction of the airways, by blocking airway

H2 receptors.

To prevent this problem U2 agonists could possibly be prepared as Inhalants for hlgh*-rlsk patients having both ulcers and asthma, for administration before chronic clmetldlne therapy Is begun.

Secondly, H2 agonists In combination with antagonists may be useful In bronchodilator therapy. An additive relaxant effect would be expected since the agents would be acting at different receptors. SUMMARY

HlBtamine has multiple effects on tracheal smooth muscle.

Activation of an H^ receptor produces contraction leading to

Increases in cyclic AMP and cyclic GMP which modulate the degree of contraction. Blockade of the receptor reduces the contrac tile responses and the increase in cyclic GMP. Blockade of the Hi receptor also enhances the relaxant effect observed at high hist amine concentrations.

Activation of the Hi receptor relaxes the tracheal smooth muscle as well as Increases cyclic AMP. Blockade of the Hi receptor enhances the contractile response to histamine as well as reduce the increase in cyclic AMP.

At high histamine and dimaprit concentrations the relaxation of the tracheal smooth muscle may be mediated by a third histamine receptor or an atypical Hi receptor, the increase in cyclic AMP and cyclic GMP is minimal. The relaxation is not blocked by or H2 antagonists. BIBLIOGRAPHY

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