A LABORATORY STUDY or some BASIC PRINEIPLES 0F DRUG ACTION - 'Eh'esis for the Degree of M. S. MICHIGAN STATE UNIVERSITY SUSAPTA DlRDJOSUDjoNO 1972 -A.__. _.____._._ s BINDING BY " HUM; & SflflS’ 800K 8114an me LIBRARY BINDERS ABSTRACT A LABORATORY STUDY OF SOME BASIC PRINCIPLES OF DRUG ACTION BY Susapta Dirdjosudjono Experiments demonstrating some basic principles of drug action, i.e., absorption, distribution, elimination, biotransformation and drug-receptor interactions were performed. Strychnine, a weak base, was used to demonstrate the influence of ionization on drug absorption. It was shown that strychnine in basic solution.was absorbed more rapidly than that in acid solution. To study the distribution and elimination of drugs, 5,5 diphenylhydantoin 4 14 C was injected intra- venously in kittens. Blood and tissue samples were collected at appropriate times and their radioactivity estimated. It was found that the plasma half—life of diphenylhydantoin in kittens was 41.5 hrs and there were differences in the levels of diphenylhydantoin found in different tissues. The biotransformation of drugs was demonstrated by using sleeping times due to hexobarbital and alcohol and the onset times of tremor due to tremorine as biological indicators of the amount of active drugs in the body. It was demonstrated that phenobarbital pretreatment reduces hexobarbital sleeping times and the time of onset of tremor due to tremorine, indicating that phenobarbital induces the biotrans- formation of these drugs. In contrast, SKF 525A and carbon tetrachloride Susapta Dirdjosudjono pretreatment increased hexobarbital sleeping times. In other experi- ments sleeping time due to alcohol was increased by pyrazole while disulfiram had no effect. Some principles of enzyme kinetics were demonstrated since this is the model on which drug-receptor interactions are based. Noncom- petitive inhibition by ouabain, competitive inhibition by alcohol and indirect inhibition by ADP of K+ activated p-nitrophenylphosphatase were demonstrated. Using guinea pig aortic strips the interaction of norepinephrine and its antagonists competing at receptor sites (adrenergic receptors) was demonstrated, while the properties of muscarinic and nicotinic cholinergic receptors were demonstrated by using guinea pig ileum and frog rectus abdominis muscles. A number of experiments in organ pharmacology were conducted with diuretics, analgesics and cardiac drugs. The diuretic action of chloromerodrin and actions of hydrochloric acid and BAL on chloro- merodrin diuresis were demonstrated in rabbits. Using the same animals the-diuretic effects of the sequential administration of mannitol, hydrochlorothiazide and ethacrynic acid were demonstrated. The mouse hot plate method was used to compare the analgesic effects of morphine, meperidine, codeine and salicylic acid. The effects of chlorpromazine and the antagonist, naloxone, on the analgesic actions of morphine were demonstrated. Some actions of cardiac drugs were demonstrated using the Langendorff heart preparation. The effects of norepinephrine and isoproterenol on contractile force and coronary flow in guinea pig hearts were observed. Using the same preparation, the ability of quinidine sulfate and procaine hydrochloride to delay ouabain-induced arrhythmias was demonstrated. A LABORATORY STUDY OF SOME BASIC PRINCIPLES OF DRUG ACTION By Susapta Dirdjosudjono A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1972 to ciciel and tommy ii ACKNOWLEDGEMENTS The author very sincerely wishes to express his gratitude to Dr. T. Tobin, his major advisor, for encouragement, patient guidance in his graduate study program and for his valuable suggestions and assist- ance in the preparation of this thesis. The same appreciation is extended to Dr. T. M. Brody, the chairman of the Department of Pharmacology, for his interest. Also to Dr. J. B. Hook and Dr. R. K. Ferguson, his sincere thanks for their generous willingness to sit in on his guidance committee. Special thanks are given to Dr. S. Baskin and Dr. F. Welsch for their advice and assistance in the use of laboratory instruments; also his appreciation is given to Dr. T. Akera, Dr. G. L. Gebber and Dr. S. Stolman for their helpful assistance in the preparation of this thesis. Finally, the author expresses his gratitude to the Indonesian Government, also Gadjah Mada University, his Alma Mater and Mid~West Universities Consortium International Activities for most of the financial support for this work. Because of the above mentioned this thesis was made possible. 111 TABLE OF CONTENTS Chapter Page GENERAL INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . l I. ABSORPTION AND DISTRIBUTION OF DRUGS. . . . . . . . . . . . 3 OBJECTIVES . . . . . . . . . . . . . . . . . . . . . 3 INTRODUCTION . . . . . . . . . . . . . . . . . . . . 3 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 6 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 8 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . 11 REFERENCES . . . . . . . . . . . . . . . . . . . . . 12 II 0 DRUG BIOTMNSFORMATION. O O O O O 0 O O I O O O O O O O O O 19 OBJECTIVES . . . . . . . . . . . . . . . . . . . . . 19 INTRODUCTION . . . . . . . . . . . . . . . . . . . . 19 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 21 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 23 CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . 27 REFERENCES . . . . . . . . . . . . . . . . . . . . . 27 III. DRUG RECEPTOR INTERACTIONS. . . . . . . . . . . . . . . . . 33 INTRODUCTION 0 O O O O O O O O O O O O O O C O O O O 33 TI'IEORY O O O O O O O O O O O O O O O O O O O 0 O O O 34 REFERENCES 0 O 0 O O O O O O O O O O O O O O O O O O 3 7 IV 0 mzm KINET IC S O O O O O O O O O O O O O O I O O O O O O O 4 O OBJECTIVES . . . . . . . . . . . . . . . . . . . . . 40 INTRODUCTION . . . . . . . . . . . . . . . . . ... . 40 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 41 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 42 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . 45 REFERENCES . . . . . . . . . . . . . . . . . . . . . 46 v 0 CHOLINERGIC RECEPTORS O O O O O O O O O O O O O O O O O O O 5 3 OBJECTIVES . . . . . . . . . . . . . . . . . . . . . 53 INTRODUCTION . . . . . . . . . . . . . . . . . . . . 53 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 56 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 58 CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . 61 REFERENCES . . . . . . . . . . . . . . . . . . . . . 61 iv Chapter VI. ADRENERGIC RECEPTORS. . . . . OBJECTIVES . . . . . . INTRODUCTION . . . . . MATERIALS AND METHODS. RESULTS AND DISCUSSION CONCLUSIONS. . . . . . REFERENCES . . . . . . VII. DIURETICS . . . . . . . . . . OBJECTIVES . . . . . . INTRODUCTION . . . . . MATERIALS AND METHODS. RESULTS AND DISCUSSION SUMMARY. . . . . . . . REFERENCES . . . . . . VIII. CENTRALLY ACTING AGENTS: THE OBJECTIVES . . . . . . INTRODUCTION . . . . . MATERIALS AND METHODS. RESULTS AND DISCUSSION CONCLUSIONS. . . . . . REFERENCES . . . . . . IX. CARDIAC DRUGS . . . . . . . . OBJECTIVES . . . . . . INTRODUCTION . . . . . MATERIALS AND METHOD . RESULTS AND DISCUSSION SUMMARY. . . . . . . . REFERENCES . . . . . . Page 72 72 72 74 74 76 77 83 83 83 85 87 9O 90 97 97 97 98 99 101 101 107 107 107 109 110 115 116 Table 1.1 2.1 2.2 2.3 2.4 2.5 LIST OF TABLES Page Time to convulsions of rats injected with strychnine in alkaline and acidic solution . . . . . . . . . . . . . . 15 Effect of phenobarbital pretreatment on the sleeping times of rats challenged with ethanol or hexobarbital . . . 30 Effect of SR? 525A and chloramphenicol pretreatment on hexobarbital sleeping times. . . . . . . . . . . . . . . 31 Effect of carbon tetrachloride pretreatment on hexo- barbital sleeping times . . . . . . . . . . . . . . . . . . 31 Effect of pyrazole and disulfiram on alcohol sleeping times 0 C O O O O O O O O O O O O O O O O O O O O O O O O O 32 Effect of pretreatment with phenobarbital or SKF 525A on tremors due to tremorine or oxotremorine . . . . . . . . 32 vi Figure 1.1 1.2 1.3 1.4 3.1 3.2 4.1 402 4.3 4.4 4.5 501 5.2 5.3 5.4 5.5 LIST OF FIGURES Blood levels of diphenylhydantoin (DPH) in kittens following single intravenous injection of 14C DPH . . . Tissue levels of DPH in kittens following single intra- venous injection of 1 C DPH . . . . . . . . . . . . . . Tissue levels of DPH in kittens following single intra- venous injection of 1 C DPH . . . . . . . . . . . . . . Tissue levels of DPH 2n kittens following single intra- venous injection of 1 C DPH . . . . . . . . . . . . . . Plots of drug action against the concentration of drug (A) or log concentration of drug (B) . . . . . . The double reciprocal or Lineweaver and Burke transformation. . . . . . . . . . . . . . . . . . . . . Effect of ethanol on K+ activated p-NPPase activity . . Effect of ethanol on K+ activated p-NPPase activity, plotted according to the method of Hofstee. . . . . . . Effect of ouabain on Rf activated p-NPPase activity . . Effect of ADP on R+ activated p-NPPase activity . . . . Effect of ethanol or ouabain on K+ activated p-NPPase, plotted according to the method of Lineweaver and Burke Organ bath preparation for the frog rectus abdominis. . Typical cumulative dose response curve of frog rectus abdominis to acetylcholline . . . . . . . . . . . . . . Variation in the dose response curves obtained with different frog rectus abdominis muscle preparations . . Response of frog rectus abdominis to acetylcholine, alone and in the presence of d—tubocurarine . . . . . . Response of frog rectus abdominis muscle to acetylcholine, alone and in the presence of d-tubocurarine or atropine . . vii Page 17 17 18 18 39 39 48 48 50 so_ 52 65 65 67 67 69 Figure Page 5.6 Response of guinea pig ileum to acetylcholine, alone and in the presence of atropine 10’s, 10’9 M and d-tubocurarine 10"5 M . . . . . . . . . . . . . . . . . . . 71 5.7 Response of guinea pig ileum to acetylcholine, alone and in the presence of atropine 10‘9, 10"8 and 10"7 M . . . 71 6.1 Organ bath preparation for guinea pig aortic strip. A schematic representation of the organ bath system . . . . 80 6.2 Response of guinea pig aorta to norepinephrine, alone and in the presence of dibenamine 10’6, 10'5 M and pentholamine 10'6 M . . . . . . . . . . . . . . . . . . . . 80 6.3 Response of guinea pig aorta to norepinephrine, alone and in the presence of pyrilamine 10"6 M or phentol- amine 10-6 M. O O O O O O O O O C O O O O O O O O O O O 0 O 82 6.4 Response of guinea pig aorta to histamine, alone and in the presence of pyrilamine 10"6 M or phentolamine 10-6 M. O O O O I O O O O O O O O O O 0 O O O O O O O O O O 82 7.1 Initial responses of blood pressure (B.P.)and urine flow (U.P.) to chloromerodrin . . . . . . . . . . . . . . . 94 7.2 Response of urine flow to intravenous injection of 20 m1 Of 10: glucose. 0 O O O O O O O O O O O O O O O O O O 94 7.3 The diuretic response to chloromerodrin and the effects of HCl and B.A.L. on this response. . . . . . . . . . . . . 96 7.4 Diuretic responses to mannitol (MANN-), hydrochloro- thiazide (H.T.Z.) and ethacrynic acid (E.A.). . . . . . . . 96 8.1 Percent increase in reaction time on a mouse hot plate with morphine, meperidine, codeine and salicylic acid . . . 104 8.2 Interactions among naloxone, morphine and meperidine on the mouse hot plate test . . . . . . . . . . . . . . . . 106 8.3 Interaction between morphine and chlorpromazine on the muse hot plate test 0 O O O O O O O O I O O O O O O O O O O 106 9.1 Comparative effects of epinephrine and isoproterenol on the contractile force of isolated perfused guinea pig hearts. 0 O O O O O O O O O O O O O O C O O O O O O O O 120 9.2 Comparative effects of epinephrine and isoproterenol on the coronary flow of isolated perfused guinea pig hearts. 0 O O O O O O O O O O O O O O O O O I O O O O O O O 122 9.3 Effects of quinidine sulfate (0.8.) and procaine hydro- chloride-(P.H.) to delay ouabaineinduced arrhtymias in isolated perfused guinea pig hearts . . . . . . . . . . . . 122 viii GENERAL INTRODUCTION Pharmacology is the study of drugs, their chemical constitution, their biological action and their therapeutic applications. This thesis consists primarily of experimental demonstrations of some of the important principles and factors involved in the biological actions of drugs. The biological activity of a drug depends on a number of different factors. To act,the drug must penetrate from its site of administra- tion to its point of action in the target organ, the processes of absorption and distribution. At the same time, however, the drug is being removed from its site of action and from the body by the processes of metabolism and elimination, which generally tend to limit drug action in the body. The body of knowledge which quantitatively describes these processes for a given drug is known as the pharmaco- kinetics of that drug. In the first two chapters of this thesis a number of pharmacokinetic principles are experimentally demonstrated. In their target organs drugs are considered-to produce their pharmacological effects by interacting with specific receptors. A receptor may be defined as the portions of a macromolecule with which a drug interacts to produce its specific pharmacological response. Drug receptor interactions are highly specific and in general follow the broad outlines of Michaelis-Menten kinetics. Chapters III, IV, V and VI deal with the theory of drug receptor interaction, its rela- tionship to enzyme kinetics and the pharmacological characteristics of 1 2 some of the major receptor types are demonstrated. This body of knowledge concerning drug-receptor interactions is spoken of as the pharmacodynamics of drug receptor interactions. Though the general principles of pharmacokinetics and pharmaco- dynamics apply to all drugs, drugs are usually classified on the basis of the organ systems on which they act. Thus the remaining chapters of this thesis deal with the actions of selected groups of drugs on various organ systems of the body. In Chapter VII the action of a number of diuretics on kidney function is outlined, in Chapter VIII the action of various narcotic analgesics on the central nervous system, and in Chapter IX the actions of a number of cardiac drugs is presented. CHAPTER I ABSORPTION AND DISTRIBUTION OF DRUGS OBJECTIVES The objectives of these experiments were to determine the effect of ionization on the rate of absorption of an ionizable drug, and to study the distribution and elimination kinetics of a lipid soluble drug. INTRODUCTION For optimal drug action, an adequate concentration of the drug in its biologically active form is required in the vicinity of its pharmacological receptors. Attainment of this concentration will depend on the rates of absorption, distribution and the rate of disap— pearance of the drug from the body. The absorption, distribution and elimination of drugs involves the passage of drugs across biological membranes. Therefore the physico-chemical properties of drugs and the structures that comprise cell membranes determine the rates of absorption, distribution and elimination of drugs. Only free drugs can penetrate the membrane, and drugs that are bound by plasma proteins are confined in plasma (Goldstein, 1949). Therefore protein binding is also an important factor. In this chapter a number of the factors that influence the absorption and distribution of drugs and the kinetics of these processes will be discussed. There are three principal ways for a drug to penetrate a bio- logical membrane, i.e., by simple diffusion, by filtration through 4 small pores or by specialized transport systems. Which of these mechanisms a drug uses to penetrate biological membranes depends largely on the physico-chemical properties of the drug (Brodie, 1964). Size of drug molecules is not a critical factor when drugs pene- trate biological membranes by a simple diffusion mechanism. Since this is the most common mechanism, in general, size is not a limiting factor in the distribution of drug molecules. The smallest drug molecule, nitrous oxide, has a molecular weight of 46 and most drug molecules have molecular weights in the order about 300. Thus most drug molecules are small in comparison with protein molecules, the main building blocks of the body, which have molecular weights of about 100,000 or more. Drug molecules are therefore usually small enough to diffuse across the body membranes if their lipid solubility is favorable. The lipid solubility of a drug is its most important property and it depends on the "ratio" of its lipophilic and hydrophilic moieties (Schanker, 1962). Drugs which consist only of carbon and hydrogen and have no hydroxyl or polar groups tend to be highly lipid soluble (Albert, 1968). On the other hand drug molecules or those portions of a molecule that are electrically charged attract water molecules and this favors water solubility. Since drugs must penetrate lipid membranes to gain access to their sites of action in most cases but usually act in an aqueous environment, it is not surprising that most drugs are to some extent soluble in both lipid and water. One factor which greatly affects the lipid solubility of many drugs is the presence of ionizable groups on them. These drugs, which usually are either weak acids or weak bases, exist at physio- logical pH values as a mixture of their ionized and unionized forms, 5 the exact ratios of these two forms depending on the pKa of the drug and the pH of its environment. Since the unionized form of the drug may cross biological membranes readily whereas the ionized form cannot, this factor leads to marked differences in the distribution of drugs wherever a biological membrane separates two body fluids of different pH. Thus the absorption, distribution and elimination of many drugs are markedly affected by the pH of the body fluids (Brodie and Hogben, 1957). Another important aspect of ionization is that due to its electric charge the ionized compound may be able to bind to a particular protein. This is usually a readily reversible binding, due to hydrogen, hydro- phobic, ionic and van der Waals bonds or some combination of these (Brodie, 1964). Thus there is always a dynamic equilibrium between the drug molecules which occupy the protein binding sites and the‘ drug molecules which are free in solution. Binding is a function of the free drug concentration and with increasing free drug concentration the plasma protein binding sites are gradually saturated. If avid protein binding occurs, the concentration of drug in compartments other than plasma may be very low. Protein-bound drugs also act as a reservoir of inactive.drug which can prolong plasma half-lives and the duration of action of drugs. Occasionally, however, another drug will act to displace a protein bound drug and may cause unusually high blood levels of the free drug, which leads to drug toxicity. Because of these factors, a knowledge of the type and extent of protein binding occurring in vivo is important (Davison, 1971). Relative blood flow through tissues is also of importance in the transient kinetics of distribution of highly lipid soluble drugs. 6 With such a drug, initial distribution occurs to highly perfused tissues, such as the brain, kidney, etc., and these tissues rapidly equilibrate with the plasma. Later equilibration occurs with less well perfused tissues such as muscle and finally with poorly perfused tissues such as body fat. In this way, highly lipid soluble drugs attain transient high drug concentrations in the CNS which is followed by a rapid decrease due to redistribution. This is the mechanism by which the action of some ultra-short acting barbiturates is terminated (Brodie et al., 1950, 1952; Goldstein and Aronow, 1960). In the following section a number of experiments are presented which demonstrate some of these principles. Strychnine, a weakly basic drug, was selected to demonstrate the effect of pH on the passage of drugs across biological membranes. To demonstrate the pharmaco- kinetics of distribution and elimination of a drug, 5,5 diphenylhydantoin (DPH) was chosen, since this is a fairly lipid soluble drug which binds relatively tightly to the tissues to which it distributes (Whodbury and Swinyard, 1972). Since Cl4 DPH was used to facilitate blood and tissue measurement of drug levels, the cat, an animal with limited biotransformation capacity, was chosen as the test species. MATERIALS AND METHODS Experiment 1: The influence of pH on the toxicity of intraperitoneally administered strychnine. Male Sprague-Dawley rats weighing 175-200 grams were used through- out. The rats were divided into four groups. Each rat received 2 ms/kg or 4 mg/kg of strychnine intraperitoneally. A solution of 0.02% strychnine in 0.15 N NaHCO3 (pH: 8.8) was used for one group of rats, while for the other group 0.02% strychnine in 0.1 N HCl (pH: 1.2) was 7 used. Onset time for convulsions was recorded in each case (Table 1.1). Experiment 2: The distribution of 14C diphenylhydantoin (DPH) in kittens after a single intravenous injection. American short hair kittens weighing 500 to 1500 grams were used throughout. The kittens were anesthetized with dialurethane at a dose of 0.6 mg/kg intraperitoneally and the left femoral vein exposed and cannulated. The experiment was started by the rapid intravenous injec- tion of 3 mg unlabelled plus 10 uCi (0.54 mg) radiolabelled diphenylhydantoin in 1 ml of normal saline per 600 grams body weight. The DPH solution was prepared by adding 50 uCi of 5,5 diphenylhydantoin 4 14C (2.7 mg) (New England Nuclear, Boston, Mass.) to 15 mg of unlabelled DPH in 5 m1 of normal saline. Five minutes after the injection and at appropriate time intervals thereafter, 50 ul samples of blood were withdrawn through the femoral cannula. After each withdrawal the cannula was flushed with heparinized saline. The experiments were terminated after an appropriate period of time had elapsed (1/2, 1, 2, 4, 8, 12 hours) by opening the kitten's thorax. About one gram of cardiac, lung, liver, spleen, kidney, voluntary muscle, adipose tissue, cerebral cortex and cerebellum were taken for the determination of radioactivity. The drug content of these tissues was determined by liquid scintil- lation counting. One hundred milligrams of each tissue was placed in the liquid scintillation vial and incubated with 1 ml of Soluene TM 100 (Packard Inc., Downers Grove, Ill.) at 55° C for 12 hours. Then each vial was filled with a toluene base scintillation medium contain- ing 100 mg dimethyl POPOP (1.4-bis(2-(4-Methyl-S-phenyloxazolyl))-benzene) and 4 grams of 2.5 diphenyloxazole in 1 liter of toluene, and counted 8 in a Packard Tri-Carb Liquid Scintillation Spectrometer with automatic correction for quench. Tissue drug content was calculated as ugrams per gram.of tissue or per ml of blood. All experimental points are the means of three determinations plus or minus the standard errors of the means. The plasma half-lives of the drug were estimated from a linear regression line fitted to a semi-logarithmic plot. Statistical significance was determined by means of Student's t-test and p < 0.01 was taken as the level of significance. RESULTS AND DISCUSSION The effect of pH of the solution on the action of intraperitoneally administered strychnine is shown in Table 1.1. The onset of convulsions in rats injected with a basic (pH 8.8) strychnine solution at a dose of 2 mg/kg was significantly shorter than in those injected with an acidic (pH 1.2) solution. With the dose of 4 mg/kg the onset of con- vulsions due to the strychnine in acidic solution was about three times faster than with strychnine in the basic solution. Since strychnine is equally stable in acidic or basic solution, it is likely that these effects reflect differences in the amount of ionized or unionized drug present in the peritoneal cavity in each experimental condition. Strychnine is a weak base with the pKa of 8 (Albert, 1968). Its «degree of ionization can be calculated from the following equation (the Henderson-Hasselbalch equation) (Albert, 1968): Percent ionized: 100 1 + antilog (pH - pKa) Ifinia. in an acidic environment of pH 1.2, strychnine would be ionized loo/1.00000691peroent, or'almost one hundred percent. On the contrary, 9 in a basic environment of pH 8.8, strychnine would be ionized 100/8.53 percent or 11.7 percent. Because only the unionized form can penetrate cell membranes, the absorption of strychnine from the acidic solution might be expected to be much slower than from the basic solution. However, this is not altogether true in the case of this experiment. The membrane through which the drug must pass in the peritoneal cavity is the capillary membrane. The penetration of substances across this membrane may be by a combination of two processes, diffusion and filtra- tion (Pappenheimer, 1953). All substances, lipid soluble or not, traverse the capillary membrane at rates which are extraordinarily rapid in comparison with their rates of passage across other body membranes (Schanker, 1962). This might be the reason why the differ- ences in time to convulsions with strychnine are not dramatically great in these experiments, but the differences are significant. The second experiment shows blood levels and tissue distribution of DPH following the intravenous injection of a single dose of the drug. In Figure 1.1 the logarithm of the blood levels of DPH in ug/ml are plotted against time in hours. The blood levels of the drug show a biphasic decay, the rapid initial decline in the blood levels followed by a much slower second phase. Both phases of the decline are approximately linear, the rapid initial phase has a half-life of about 24 minutes and extrapolates back to a zero time drug level of about 8 ug/ml, while the slower phase has a half-life of about 41.5 hours and extrapolates to a zero time drug concentration of 5.3 ug/ml. These observations on the kinetics of the disappearance of the DPH from the blood stream of the cat correlate well with data obtained by other investigators in other species (Glazko at al., 1969; Noach et al., 1958) and in the cat by Firemark et al. (1963). 10 The initial rapid decline of blood DPH level seems to be associated with the movement of the drug from the plasma to tissue binding sites. If an apparent volume of distribution (Vd) is calculated from the apparent initial plasma concentration, the Vd is about 0.74 L/kg. This is somewhat lower than the body water content of young animals, and suggests that DPH distributes initially to the total body water. This interpretation assumes that during this initial distribution period the amount of DPH bound to tissue proteins is approximately equivalent to the amount bound to the plasma proteins (Lunde et aZ., 1970). This rapid initial distribution to body water is then followed by a slower movement of the drug to tissue binding sites. This process appears to take about twenty minutes to complete and at the end of this period plasma levels of the drug have fallen to about 5.3 ug/ml. This corresponds to a volume of distribution of about 1.1 L/kg, pro- viding indirect evidence for tissue binding. These observations are further supported by the high drug concentrations observed in the brain, liver, heart and kidney at 30 minutes. Similar data, which have been interpreted as representing the movement of DPH to tissue binding sites, have been observed by other investigators (Suzuki et al., 1970). The reasons for the marked differences in tissue levels of DPH are not clear. The high liver levels could be due to biotransforma- tion of the drug, with the radiolabelled carbons being incorporated into liver constituents. However, this appears unlikely because the liver drug levels were unusually high at thirty minutes, at which stage drug metabolism would be minimal. It appears likely that some liver constituents have a high affinity for DPH, for a similar pattern is 11 observed in other species (Noach et al., 1958). Also other tissues show a tendency for drug levels to equilibrate early, with little subsequent change being observed over the time period of the experi- ments. This result was rather surprising in view of the lipid solu- bility of DPH, but is borne out by the results of other workers (Noach et aZ., 1958). The long plasma half—life of DPH in the kitten observed in these experiments agrees well with the earlier results of Firemark at al. (1963), who reported a plasma half-life for DPH in the cat of about 72 hours. This long plasma half-life is to be expected in view of the lipid solubility of DPH and the limited biotransformation ability of the cat (Brodie, 1964a). From these data a dosage schedule for DPH in the cat may be estimated. A loading dose of 10 mg/kg would give plasma levels in the order of 9.0 us/ml. Daily maintenance doses in the order of 3.3 mg/kg should serve to maintain the plasma levels of DPH between 9.0 and about 7 ug/ml, generally considered a safe thera- peutic range. SUMMARY Rats injected with a basic solution of strychnine convulsed sooner than rats injected with an acidic solution of strychnine. Since strychnine is a weak base and is highly ionized in acidic solution the experiments suggest that unionized strychnine is more rapidly absorbed than the ionized form. After a single intravenous injection of DPH, plasma levels of the drug declined in two linear phases with half-lives of 24 minutes and 41.5 hours, respectively. These data give calculated Vd's of 0174 L/kg and 1.1 L/kg. The data suggest that DPH distributes initially 12 to the body water and then more slowly to various tissue binding sites. High levels of DPH were observed in the liver which may be due to a high affinity of some liver constituents for DPH. REFERENCES Albert, A.: Selective toxicity. Methuen & Co Ltd., London, 1968, p. 250. Brodie, B. B.: Physico-chemical factors in drug absorption. In Absorption and distribution of drugs. Ed. by T. B. Binns. E & S Livingstone Ltd., London, 1964, p. 1. Brodie, B. B.: Distribution and fate of drugs; therapeutic implica- tions. In Absorption and distribution of drugs. Ed. by T. B. Binns. E & S Livingstone Ltd., London, 1964a, p. 199. Brodie, B. B., Mark, L. C., Papper, E. M., Lief, E. B., Bernstein, E. and Rovenstine, E. A.: The fate of thiopental in man and a method for its estimation in biological material. J. Pharm. Exp. Ther. 98:85, 1950. Brodie, B. B., Bernstein, E. and Mark, L. C.: The role of body fat in limiting the duration of action of thiopental. J. Pharm. Exp. Ther. 105:421, 1952. Brodie, B. B. and Hogben, C. A. M.: Some physico-chemical factors in drug action. J. Pharm. Pharmacol. 9:345, 1957. Davison, C.: Protein binding. In Fundamentals of drug metabolism and drug disposition. Ed. by B. N. La Du, H. G. Mandel and E. L. way. The Williams & Wilkins 00., Baltimore, 1971, p. 63. Firemark, B., Barlow, C. F. and Roth, L. J.: The entry, accumulation and binding of Diphenylhydantoin 2 14C in brain. Int. J. Neuropharmacol. 2:25, 1963. Glazko, A. J., Chang, T., Baukema, J., Dill, W. A., Goulet, J. R. and Buchanan, R. A.: Metabolic disposition of diphenylhydantoin in normal human subject following intravenous administration. Clin. Pharmacol. Ther. 10:498, 1969. Goldstein, A.: The interaction of drugs and plasma protein. Pharm. Rev. 1:102, 1949. Goldstein, A. and Aronow, L.: The duration of action of thiopental and pentobarbital. J. Pharm. Exp. Ther. 128:1, 1960. Lunde, P. K. M., Rane, A., Yaffe, S. J., Lund, L. and Sjoqvist, F.: Plasma protein binding of diphenylhydantoin in man. Clin. Pharm. Ther. 11:846, 1970. 13 Noach, E. L., woodbury, D. M., and Goodman, L. 8.: Studies on the absorption, distribution, fate and excretion of 4 14C labelled diphenylhydantoin. J. Pharm. Exp.T her. 122:301, 1958. Pappenheimer, J. R.: Passage of molecule through capillary walls. Physiol. Rev. 33:387. 1953. Schanker, L. S.: Passage of drugs across body membranes. Pharm. Rev. 14:501, 1962. Suzuki, T., Saitoh, Y. and Nishibara, R.: Kinetics of and diphenyl- hydantoin disposition in man. Chem. Pharm. Bull (Tokyo) 18:405. 1970. WOodbury, D. M. and Swinyard, E. A.: Diphenylhydantoin. Absorption, distribution and excretion. In Antiepileptic drugs. Ed. by D. M. WOodbury, J. K. Penry and S. P. Schmidt. Raven Press, Publishers, New York, 1972, p. 113. 14 TABLE 1.1. Time to convulsions of rats injected with strychnine in alkaline and acidic solution. Rats were injected intraperitoneally with a 0.02% solu- tion of strychnine in alkaline (pH 8.8) or acidic (pH 1.2) solution and the time of onset of convulsions noted. All values are the means of at least four determinations, j;the standard errors of the means. 15 TABLE 1.1. Time to convulsions of rats injected with strychnine in alkaline and acidic solution Doses 0.022 Strychnine 0.022 Strychnine in 0.15 N NaHC03_ in 0.1 N HCl P Mean _-i_-_ S.E. Mean 1 S.E. (min.) (min.) * 2 mg/kg 8.36 (4) __-l_-_ 0.575 11.52 (4) -_+_ 1.076 < 0.001 4 mg/kg 2.99 (4) i 0.279 7.64 (4) i 0.811 < 0.001 * Number of animals . 16 FIGURE 1.1. Blood levels of diphenylhydantoin (DPH) in kittens following single intravenous injection of 1 C DPH. Anesthetized kittens were injected intravenously with 6 mg/kg of 14C DPH and 50 ml blood samples withdrawn at the indicated intervals. Blood radio- activity levels were determined by liquid scintillation counting and blood levels of DPH calculated, all points being the means of at least three separate determinations i the standard errors of the means. All lines are least squares estimates and the extrapolation from CPM to blood levels of DPH assumed that biotransformation of the drug was insignificant. FIGURES 1.2, 1.3, and 1.4. Tissue levels of DPH in kittens fol- lowing single intravenous injection of 14C DPH. Kittens injected with 14C DPH as in Figure 1.1 were sacrificed at the indicated times and tissue samples taken. The symbols show the radioactivity and the estimated amounts of drug in each of the sampled tissues at the indicated times after the initial injection. All points are the means of at least three separate determinations 11the standard errors of the means. 17 FIG. I .I. Blood Levels of Diphenylhydantoin (DPH) in Kittens Following Single Intravenous Injection of MC DPH. 10 60 O T g 0 9 8-1 l- 3 1° 1 .40 '3 E 6" Q 1 35 -836 _ E Q Q I 0‘0”“35. Q l" 6 o ‘1‘ O m 41 U M. +20 LT" “r r 1 j I I l I I I T F ‘l 3 5 7 9 ‘ll HOURS FIG-1.2. Tissoe Levels of DPH in Kittens Following Single Intravenous Injection of MC DPH. I; fit O—LIVER 450 ._HEART .. o—FAT _ 20- e—MUSCLE __ m b «a 2 03 -I ,- g a: no 3.“ - 3 E l’ _-_-l 0 .. a L\ .50 (L A\"l l\~.e - ‘1’ ll I- I I r I fil I 1 2 4 8 12 18 FIG. 1.3 and l.4. Tissue Levels of DPH in Kittens Following Single Intravenous Injection of 14C DPH. 20 :1. O— KIDNEY 9— LUNG " 0— SPLEEN i-lOO 4 - “10« ‘l’\ é “1 5‘9 é fill fie p50 9, m V MN ' g g I I I I JUL r r3 0 l 2 4 E 8 HOURS 12 3 ° 4% r 9— o —CERE8ELLUM 400g 15" e —CORTEX .- m 10‘ '- l:<,> . i-SO I I I I _l% l O 'l 2 4 8 12 HOURS CHAPTER II DRUG BIOTRANSFORMATION OBJECTIVES The objectives of these experiments were to demonstrate some mechan- isms of drug biotransformation; the concept of a rate limiting step in a metabolic pathway; the induction and inhibition of the liver micro- somal drug metabolizing system and the importance of biotransformation as a mechanism of drug toxicity. INTRODUCTION The great majority of drugs undergo biotransformation in the body and this transformation has variable effects on their biological activity. Usually, biotransformation results in an increase in the water solubility and rate of excretion of the drug and thus in loss of biological activity. Therefore, the rate of biotransformation often determines the duration of action of a drug in the body (Brodie and Maickel, 1962). Occasionally, however, the biotransformation of a compound results in its conversion to a more active or toxic compound and such conversions are often involved in teratogenic actions of drugs (Brodie, 1972). Thus the biotransformation of drugs may either limit, enhance or radically alter the pharmacological actions of drugs and is therefore a very important area in pharmacology. The biotransformation of drugs proceeds by two distinct mechanisms. The simplest mechanism occurs where the drug resembles a normal 19 20 substrate of intermediary metabolism and is transformed by these enzymes. Examples of such mechanisms are the hydrolysis of local anesthetics by plasma cholinesterases (Goldstein et al., 1969) or the reduction of chloralhydrate to the active form, trichloroethanol, by alcohol dehydrogenase (Friedman and Cooper, 1960). This mechanism, however, requires that the drug be closely related to a normal body constituent and this is normally not the case for the great majority of synthetic chemicals of plant origin that mammalian systems (especially herbivores and omnivores) are exposed to during their life. To meet this requirement mammals have developed a specialized "drug" metabolizing system, the liver microsomal drug metabolizing system. This drug metabolizing system is located primarily in the smooth endoplasmic reticulum (ER) of liver cells from.which it may be iso- lated by differential centrifugation (Axelrod, 1955). In the presence of NADPH, Mg++, nicotinamide and molecular oxygen these membranes transform.many drugs by a series of intermediary steps. However, this system has a number of characteristics which determine the rate at which it metabolizes drugs and these give it its considerable pharmacological importance. One of these is its very broad substrate specificity, which enables this system to deal with many different types of chemicals (Rubin at al., 1964). This broad specificity may also be associated wdth the ability of many agents to inhibit drug metabolism by this pathway and thus prolong the pharmacological effects of various agents. .Another important characteristic is the fact that prolonged exposure to some drugs results in the induction of this system and in a con- siderable increase in the rate of drug metabolism (Conney and Burns, 1962). Finally, the ability of the liver microsomal system to metabolize 21 drugs is dependent on the functional integrity of the liver and agents or pathological conditions which produce liver damage are liable to greatly prolong drug action. In the following section a number of experiments are presented which demonstrate these characteristics of drug metabolizing systems. Alcohol and hexobarbital, both sleep inducing agents whose action is terminated by metabolism, were chosen as the test drugs, with sleeping time as the pharmacological response. Alcohol is an example of an agent metabolized by enzymes of intermediary metabolism, while hexo- barbital is an example of an agent which is degraded by the liver microsomal drug metabolizing system. In the experiments reported here the actions of a number of specific inhibitors and inducers on the rate of metabolism of these drugs is reported. MATERIALS AND METHODS Emperiment l: The effect of pretreatment with phenobarbital on ethanol and hexobarbital sleeping times. Four groups of four female Sprague—Dawley white rats were pre- treated with either phenobarbital, 50 mg/kg intraperitoneally (i.p.) or an equivalent volume of normal saline i.p. twice daily for four days. On the fifth day all rats were challenged with either hexo- barbital (100 mg/kg) or 2.5 ml of 33.31 ethanol i.p. Within one to four minutes these animals lost their righting reflex and didinot regain it for up to eight hours. Loss of the righting reflex was con- sidered to have occurred if an animal did not attain eternal recumbency within sixty seconds after being placed supine on a plane surface. The period for which the righting reflex was lost was taken as the "sleeping time." Table 2.1 shdws the effects of pretreatment with 22 phenobarbital and normal saline on sleeping times due to hexobarbital and ethanol. All sleeping times are estimated to the nearest minute. Experiment 2: Acute inhibition of hexobarbital metabolism by SKF 525A and chloramphenicol. White female Sprague-Dawley rats were used as previously. SKF 525A (B-diethylaminoethyl diphenylpropylacetate), 25 mg/kg in 0.6 m1 of normal saline or chloramphenicol 50 mg/kg in 0.6 ml of normal saline or 1.25 ml of normal saline, were administered i.p. to groups of four rats. Twenty minutes after pretreatment the animals were challenged with hexobarbital 100 mg/kg i.p. The time of onset and mean sleeping times are presented in Table 2.2. Experiment 3: Effect of carbon tetrachloride pretreatment on hexo- barbital sleeping times. Three groups of four Sprague-Dawley white rats were pretreated with either 2.5 ml carbon tetrachloride orally or an equivalent volume of normal saline (groups 2 and 3) 24 hours prior to challenge with 100 mg/kg hexobarbital i.p. Twenty-four hours later one of the saline treated groups was treated with 25 mg/kg SKF 525A and 100 mg/kg chloramphenicol and twenty minutes later all three groups were chal- lenged with hexobarbital and the sleeping times measured as before (see Table 2.3). Experiment.4: Effects of disulfiram and pyrazole on alcohol sleeping times. Three groups of four white female Sprague-Dawley rats were treated with 4 ml of 1.72 pyrazole, 6 ml normal saline i.p. or with 1.5 m1 of 2.52 disulfiram orally. Ten minutes after pretreatment all 23 rats were administered 2.5 ml of 33.32 alcohol orally and their sleeping times (Table 2.4) monitored as before. Experiment 5: Effect of pretreatment with phenobarbital or SKF 525A on tremors due to tremorine and oxotremorine. White Swiss Webster mice in groups of eight were injected with 25 mg/kg tremorine or 5 mg/kg oxotremorine subcutaneously and the time of onset of tremor was noted. Tremor was monitored by picking up mice by the tail, by which method the fine muscular tremors were readily detected. Similar experiments were carried out on mice which had been pretreated with 50 mg/kg phenobarbital twice daily for three days prior to the experiment. The effects of pretreatment with 10 mg/kg SKF 525A or 15 mg/kg atropine were also studied and the data are presented in Table 2.5. RESULTS AND DISCUSSION In these experiments sleeping times due to hexobarbital and alcohol or the onset times for tremor have been used as biological indicators of the amount of active drug in the body. The assumption is made that the various interferences of these experiments produce their effects on sleeping times (or time to tremor) by altering the rate of biotransformation of these drugs and these assumptions are well supported by the literature (Goldstein at al., 1969). The results show the importance of drug metabolism as a mechanism of both initiation and termination of drug action in the body. The anesthetic and sedative effects of short acting barbiturates such as hexobarbital are terminated largely by biotransformation in hepatic tissue, and to a lesser extent in other tissues (Cooper, 1955). Therefore the sleeping time due to hexobarbital may be taken 24 as an index of the rate of biotransformation of this drug, and varia- tions in this parameter are presumably due to alterations in the rate of drug metabolism. Experiments 1, 2 and 3 are interpreted on this basis. One of the characteristics of the liver microsomal drug metabolizing system is that it is induced by exposure to certain drugs. Under these conditions the amount of smooth endoplasmic reticulum in the liver and the rate of drug metabolism is increased (Remmer, 1962). Thus in Table 2.1, the sleeping time due to hexobarbital was reduced four fold by pretreatment with phenobarbital, consistent with the hypothesis that the ability of the liver to metabolize hexobarbital was increased. However, if these animals were challenged with ethanol an eight fold increase in sleeping time was observed. This increased sleeping time was presumably due to residual phenobarbital in these animals and demonstrates the difference between the pathways of alcohol and hexobarbital metabolism. The liver microsomal drug metabolizing system is inhibited spe- cifically by certain drugs and nonspecifically by liver damage and in both of these situations the rate of drug biotransformation is markedly reduced. Table 2.2 shows the effects of SKF 525A and chloramphenicol on sleeping times of animals challenged with hexobarbital. In each case an approximately four fold increase in sleeping time occurred. These data are consistent with in vitro observations that SKF 525A and chloramphenicol are noncompetitive inhibitors of the microsomal drug metabolizing system (Cooper et al., 1954; Dixon and Fonts, 1962). Such inhibition of drug metabolism is of clinical importance in barbiturate anesthesia, where treatment with chloramphenicol either prior to or during anesthesia can greatly prolong recovery time (Adams and Dixit, 1970). 25 Nonspecific liver damage also reduces the rate of drug metabolism, as shown by the data presented in Table 2.3. Here the animals pre- treated with carbon tetrachloride, an agent which produces acute liver damage (Brody, 1959), exhibited a ten fold increase in hexobarbital sleeping time. This increase was more than twice that observed with a combination of SKF 525A and chloramphenicol. The reason for this increased effectiveness of carbon tetrachloride is not clear, but may be due to an anesthetic like action of carbon tetrachloride itself. In the clinical situation, deficits in the drug metabolizing system may be seen in cases of liver damage (Brodie at al., 1959) or in newborn animals in which the drug metabolizing system is not fully developed (weiss et al., 1960). The metabolism of ethanol was selected as a model of drug metabolism.proceeding via a pathway of intermediary metabolism (Hawkins and Kalant, 1972). Ethanol first interacts with alcohol dehydrogenase to give rise to acetaldehyde, and this is the rate limiting step in the metabolism of ethanol (Lundquist and Wolthers, 1968). The acetaldehyde formed then interacts with aldehyde dehy- drogenase to give rise to acetic acid which is further metabolized in the TCA cycle (Hawkins and Kalant, 1972). The data of Table 2.4 show the effects of selective inhibition of alcohol dehydrogenase on sleeping times due to ethanol. Pyrazole is a potent inhibitor of alcohol dehydrogenase in vitro (Reynier, 1969) and its action in vivo is consistent with these observations. As shown in Table 2.4 the administration of pyrazole results in a four fold increase in sleeping times after the administra- tion of.ethanol. This marked prolongation of sleeping time is associ- ated with relatively stable blood alchol levels (Bustos et aZ., 1970), 26 and the effectiveness of pyrazole is presumably associated with the fact that oxidation by alcohol dehydrogenase is the rate limiting step in the metabolic degradation of ethanol. Unlike pyrazole, disulfiram, a potent inhibitor of aldehyde dehydrogenase (Graham, 1951), does not increase ethanol sleeping time (Table 2.4) or decrease the rate of disappearance of ethanol. This is despite the fact that disulfiram is a competitive inhibitor of aldehyde dehydrogenase and after its administration blood acetaldehyde levels are markedly increased (Bald and Jacobson, 1948). There is, however, no change in the rate of disappearance of ethanol. This is because the blood levels of acetaldehyde rise sufficiently to overcome the inhibitory action of disulfiram on aldehyde dehydrogenase but not enough to "reverse" the ethanol-acetaldehyde reaction. Thus the only action of disulfiram on ethanol metabolism is to increase the steady state levels of acetaldehyde, the basis of the "antabuse" reaction in human medicine. These observations show that inhibition of a non rate-limiting step in a metabolic pathway can occur without inhibition of the rate of the overall pathway. Only at the rate limiting step does inhibition necessarily mean inhibition of the overall pathway. The experiments with oxotremorine represent an attempt to demonstrate drug activation by biotransformation. Tremorine is pharmacologically inactive and only becomeswactive after undergoing metabolic transformation to oxotremorine, which occurs in the liver microsomal system. Thus pretreatment with phenobarbital, by inducing drug metabolism, produced a significant decrease in the time to con- vulsions. This decreased time to convulsions is presumably associated with:an increased rate of drug metabolism in the induced liver 27 microsomal enzyme system. In contrast to this action of phenobarbital pretreatment, SKF 525A, which acutely blocks drug metabolism, delayed the onset of tremors consistent with the hypothesis that the delayed action of oxotremorine is due to inhibition of drug metabolism. However, these data per as do not rule out the possibility of central effects of SKF 525A for atropine also delays the onset of tremorine dependent tremors, presumably by central action, since it does not affect drug metabolism. CONCLUSIONS The experiments reported above show that the duration of action of some drugs can be increased or decreased by certain drugs. Pheno- barbital, a drug which induces the microsomal drug metabolizing system, reduced hexobarbital sleeping times and time of onset of tremors due to tremorine. SKF 525A and chloramphenicol increased hexobarbital sleeping time apparently by inhibiting non-competitively the microsomal drug metabolizing system. Carbon tetrachloride increased hexobarbital sleeping time, presumably due to its ability to cause liver damage. Pyrazole increased alcohol sleeping times due to inhi- bition of the rate-limiting step of alcohol metabolism, i.e., it inhibited alcohol dehydrogenase. Disulfiram, a competitive inhibitor of aldehyde dehydrogenase, did not increase alcohol sleeping times. Thus inhibition of a non rate-limiting step in a metabolic pathway can occur without inhibition of the rate of the overall pathway. REFERENCES Adams, H. R. and Dixit, B. M.: Prolongation of pentobarbital anesthesia by chloramphenicol in dogs and cats. J. Am. Vet. Med. Ass. 156:902, 1970. 28 Axelrod, J.: The enzymatic deamination of amphetamine (Benzedrine). J.Biol. Chem. 214:253, 1955. Brodie, B. B.: Enzymatic activation of foreign compounds to more potent or more toxic derivatives. In Proceeding of the Fifth International Congress of Pharmacology, San Francisco, 1972, p. 5. Brodie, B. B., Burns, J. J. and Weiner, M.: Metabolism of drugs in subjects with Laennec's cirrhosis. Med. Expt. 1:290, 1959. Brodie, B. B. and Maickel, R. P.: Comparative biochemistry of drug metabolism. In Metabolic Factors Controlling Duration of Action, Proceeding of First International Pharmacological Meeting. Vol. 6 ed. by B. B. Brodie and E. G. Erdos, Macmillan Co., New York, 1962, p. 299. Bustos, G. 0., Kalant, H., Khana, J. M. and Loth, J.: Pyrazole and the induction of fatty liver by a single dose of ethanol. Science 168:1598, 1970. Brody, T. M.: Toxicology symposium. Mechanism of action of carbon- tetrachloride on liver cells. Fed. Proc. 18:1017, 1959. Conney, A. H. and Burns, J. J.: Factors influencing drug metabolism. Adv. Pharm. 1:31, 1962. Cooper, J. R., Axelrod, J. and Brodie, B. B.: Inhibitory effects of B diethylaminoethyl diphenylpropyl acetate on a variety of drug metabolic pathways in vitro. J. Pharm. Exp. Ther. 112:55, 1954. Dixon, R. L. and Fout, J. R.: Inhibition of microsomal drug metabolic pathways by chloramphenicol. Biochem. Pharm. 11:715, 1962. Friedman, J. P. and Cooper, J. R.: The role of alcohol dehydrogenase in the metabolism of chloralhydrate. J. Pharm. Exp. Ther. 129: 373, 1960. Goldstein, A., Aronow, L. and Kalman, S. M.: Principle of drug action. Harper Row Ltd., New York, 1969, p. 237. Graham, W. D.: In vitro inhibition of liver aldehyde dehydrogenase by tetraethylthiuram disulphide. J. Pharm. Pharm. 3:160, 1951. Held, H. and Jacobson, E.: The formation of acetaldehyde in the organism after ingestion of antabuse (tetraethylthiuram di- sulphide) and alcohol. Acta Pharm. Tox. 4:305, 1948. Hawkins, R. D. and Kalant, H.: The metabolism of ethanol and its metabolic effects. Pharm. Rev. 24:67, 1972. Lundquist, F. and Wblthers, H.: The kinetics of alcohol elimination in man. Acta Pharm. Tox. 14:265. 1968. 29 Remmer, B.: Drugs as activators of drug enzymes. In Metabolic factors controlling duration of drug action. Proceeding of First International Pharmacology Meeting. Ed. by B. B. Brodie and E. G. Erdos. Macmillan Co., New York, 1962, p. 235. Reynier, M.: Pyrazole inhibition and kinetic studies of ethanol and related oxydation catalysed by rat liver alcohol dehydrogenase. Acta Chem. Scand. 23:1119, 1964. Rubin, A., Tephley, T. R. and Mannering, G. J.: Kinetics of drug metabolism by hepatic microsomes. Biochem. Pharm. 13:1007, 1964. Weiss, C. F., Clark, A. J. and Werton, J. R.: Chloramphenicol in the new born infant. A physiologic explanation of its toxicity when given in excessive dose. New Eng. J. Med. 262:787, 1960. 30 TABLE 2.1. Effect of phenobarbital pretreatment on the sleeping times of rats challenged with ethanol or hexobarbital Onset of Sleeping Time Sleeping Time Pretreatment Treatment Mean : S.E. Mean i S.E. (min.) (min.) Saline 10 ml/kg Hexobarbital * B.I.D. for 4 days 100 mg/kg 2.5 (4) 10.28 64.0 (4)19.10 Phenobarbital Hexobarbital 1.6 (4) 19.33 16.3 (4):3.17 50 mg/kg B.I.D. 100 mg/kg for 4 days Saline 10 mllkg B.I.D. for 4 days Phenobarbital 50 mg/kg B.I.D. for 4 days Ethanol 33.32 2.5 m1 Ethanol 33.32 2.5 ml 2.7 (4) 10.47 3.3 (4) 330.30 19-25(4)i3-03 124.0 ((03.00 * Refers to the number of animals. 31 TABLE 2.2. Effect of SKF 525A and chloramphenicol pretreatment on hexobarbital sleeping times Sleeping Times Pretreatment Mean : S.E. 2 Control (min.) a Normal saline, 1.25 ml 135.5 (4) j;15.39 100 SKF 525A, 25 mg/kg 544.0 (4) 1:52.89 400 Chloramphenicol, 50 mg/kg 479.0 (4) i;20.27 3S4 * Refers to the number of animals. TABLE 2.3. Effect of carbon tetrachloride pretreatment on hexobarbital sleeping times Sleeping Times Pretreatment Mean : S.E. 2 Control (min.) a Normal saline, 1.25 ml 97.5 (4) i; 5.97 100 SKF 525A, 25 mg/kg plus 632.5 (4) i 46.72 650 chloramphenicol 100 mg/kg Carbon tetrachloride 2.5 ml 1,276.0 (4) $.93°49 1,308 * Refers to the number of animals. 32 TABLE 2.4. Effect of pyrazole and disulfiram on alcohol sleeping times Sleeping Times Pretreatment Mean i S.E. 2 Control (min.) Normal saline, 4 m1 i.p. 33.0 (4)f:_ 1.77 100 Pyrzole, 1.72, 4 ml i.p. 126.0 (4) : 31.70 381.8 Disulfiram, 2.5%, 1.5 m1 orally 30.0 (4).il 5.70 90.7 * Refers to the number of animals. TABLE 2.5. Effect of pretreatment with phenobarbital or SKF 525A on tremors due to tremorine or oxotremorine Drugs Tremorine 25 mg/kg S.C. Oxotremorine 5 mg/kg S.C. Onset of Tremor Onset of Tremor Pretreatment Mean 1 S.E. Mean 1 S.E. (min.) (min.) * Saline 10 mg/kg daily 19.0 (4) i 0.40 1.00 (4) _-*_-_ 0.00 for 3 days (control) Phenobarbital 50 mg/kg 16.5** (4) -_+_ 0.28 1.75 (4) i 0.25 B.I.D. for 3 days SKF 525A 10 mg/kg 30 27.5** (4) i 0.64 1.0 (4) i 0.0 minutes before treatment (dead after 4- 6m n.) Atropine 15 mg/kg 30 32.0** (4) i 0.57 1.5 (4) i 0.25 minutes before treatment (dead after 10-15 min.) * Refers to the number of animals. ** Refers to p 0.01, significantly different from control. CHAPTER III DRUG RECEPTOR INTERACTIONS INTRODUCTION Pharmacological theories of drug action are based on the concept of specific receptors for drugs. This concept is largely a consequence of the remarkable effectiveness and specificity of some drugs. Acetylcholine and atropine are both active in concentrations of less than 10..6 M (see Chapter V) and many substances are effective at even lower concentrations. The lethal oral dose of botulinum toxin is only 10-10 gm/kg (van Heyningen, 1950). This remarkable specificity has given rise to the concept that drug molecules act by binding at specific sites in the cell to produce their effects. This concept was first proposed by Ehrlich (1898), who suggested that specific sites on the surfaces of antigens and antibodies are essential for interaction between them. He further suggested that arsenical drugs killed trypanosomes by binding at specific receptors and that drug resistance was due to the loss of these specific receptors. The concept of drugs competing for specific receptor sites was first proposed by Langley (1905), who suggested that nicotine and curare competed for a receptive substance in the neuromuscular junction. More substantial evidence for a competitive interaction between binding sites was shown by Gaddum (1926), who demonstrated a classical parallel 33 34 shift in the dose response curve for adrenaline in uterine smooth muscle after exposure to ergotamine. The first significant quantitative treatment of the drug receptor interaction was that of Clark (1937). Clark applied the kinetics of enzyme action developed by Michaelis and Menten (1913) to the action of acetylcholine on frog heart. Subsequently, this basic work of Clark was developed by others, notably Ariene and co-workers. Ariene (1964) introduced the concept of intrinsic activity into drug receptor kinetics. Intrinsic activity is a measure of the ability of the drug to produce its pharmacological effect once bound. Drugs which are pharmacologically effective when bound are considered to have a high intrinsic activity and the intrinsic activity of the drug in a series which produces maximal response is defined as one. Drugs which produce a lesser response, though bound, are said to have fractional intrinsic activities and drugs which are bound and do not activate have zero intrinsic activity. THEORY Current theory of drug receptor interaction is based almost entirely on our understanding of enzyme-substrate interactions. The bonds formed between the drug and the receptor are those of the so- called "Michaelis Menten" enzyme-substrate complex, i.e., hydrogen, hydrophobic and van der Waals bonds. Though individually weak, these interactions summate to give a relatively tight drug-receptor inter- 6 to 10.7 Moles/L. action with dissociation constants in the order of 10- From this Michaelis Menten type drug-receptor complex the reaction can proceed in a number of ways. Binding without the induction of the specific conformational change required for receptor activation results 35 in reversible receptor blockade. Special subgroups on drugs may react chemically with the receptor after binding and this type of irreversible blockade has been referred to as competitive nonequilibrium blockade (Ariene, 1964). If binding results in a specific alteration of the receptor, receptor activation occurs and the pharmacological response occurs. These interactions may be schematically outlined as below: k1 k3 D + r--——————3Ih:-————-—) 1k v——-—--- k 2 Where k3 is greater than zero a pharmacological response occurs. D = drug r = receptor kl and k2 = rate constant for association and dissociation k3 = proportionality constant A = pharmacological response This reaction follows mass law and all the constituents are con- sidered to be in equilibrium. Thus the occupation of the drug by the receptor and the response (which is considered proportional to receptor occupation) may be treated in terms of the simple Michaelis-Menten treatment. At equilibrium the rate of formation is equal to the rate of dissociation, therefore . k2 (Dr) 3 k1 (D) (r) Rd is a dissociation constant of the drug receptor complex and is equal to k2/kl. __J; (D) (r) Kd k1 (Dr) ....................(1) r = free receptor = total receptor (R) - drug receptor complex (Dr). 36 r-(R)-(nr)-£d—0§1§£l (D) (R) - (Dr) (D) ' Kd (Dr) (Dr) . 3M)— ...(2) Rd + (D) ............ ..... ... The interaction between the drug and the receptor produces an action (A). It can be expressed as the fraction of the maximum activity which can be generated as A/Am. Am is activity when all the receptors are occupied by the drug (Dr is maximal), therefore k k D +-r'-—-;L--—3 Dr 3 )= Am ‘__________ k2 When Dr is maximal, Dr is equal to R (total receptor). Am - k3 (Dr) = k3 (R) A k3 (Dr) (Dr) Am k3 (R) (R) (Dr) - 52111151 (3) if equations (2) and (3) are matched, . (D) (R) _A(m (D) Kd+(D) Am A , (D) (R) Am Kd + (D) (R) Am SD) A = Kd + (D) ............. ..........(4) This relationship can be represented graphically by plotting the action (A) against concentration of drug (D). This will give a rectangular hyperbola as shown in Figure 3.1A. From this plot the dissociation constant of the drug receptor complex (Kd) can easily be determined 37 similar to the Michaelis-Menten constant (Km) in enzyme-substrate interaction. Equation (4) then can be rearranged: Am Rd: (D) (r-l) eoeoeeeoeoooooooeeoo(5) and from this relationship, Kd can be calculated since if A equals to 1/2 Am, Kd - (D). This can be determined graphically as shown in Figure 3.1A. For a wide range of drug concentrations a semilogarithmic plot is convenient as shown in Figure 3.18. For the determination of Kd linear transformations such as the method of Lineweaver and Burke (1934) can be used. This plot presents the data as points on a straight line. Taking the reciprocal of each side, equation (4) can be rearranged as follows: l.Kd-(D)- Kd + D A Am(D) Am(D) Am(D) 1 Rd 1 1 K‘E(B)+Xm- ooooooooo.oooooooooooo(6) As shown in Figure 3.2, this plot yields a straight line and Rd is easily determined. REFERENCES Ariene, E. J.: Molecular Pharmacology. Academic Press, New York, 1964. Clark, A. J. (1937), Cited by E. J. Ariene: Molecular Pharmacology. Academic Press, New York, 1964. Ehrlich, P. (1898), Cited by A. Albert: Selective Toxicity. Methuen & Co. Ltd., London, 1968. Gaddum, J. H.: The action of adrenalin and ergotamine on the uterus of the rabbit. J. Physiol. 61:141, 1926. van Heyningen, W.: Bacterial Toxins. Blackwell, Oxford, 1950. 38 Langley, J. N.: 0n the reaction of cells and of nerve endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and to curari. J. Physiol. 33:374, 1905. Michaelis, L. and Menten, M. L.: Die Kinetik der Invertinwirkung. Biochem. Z. 49:333. 1913. 39 1/2Am. , ;_A ‘3‘" R :1 K8 (D) LOG (0) FIG. 3.1. Plots of Drug Action against the Concentration of Drug (A ) or Log Concentration of Drug ( B ). 1 (0 “ F A +b\ // 2. ......... Amdp-m-no - _ 1 1 _1_ Kd Kd (0) FIG. 3.2. The Double Reciprocal or Lineweaver and Burke Transformation. CHAPTER IV ENZYME KINETICS OBJECTIVES The objective of these experiments was to demonstrate Michaelis- Menten type enzyme kinetics on which drug-receptor theory is based. INTRODUCTION The enzyme (Na+ - K+) ATPase (adenosine triphosphate phospho- hydrolase E C 3.6.1.3.) (Skou, 1965) was chosen for these experiments because of its ready availability in this laboratory. In the presence of Mg++ and appropriate monovalent cations, preparations of this enzyme catalyze the hydrolysis of p-nitrophenylphosphate to paranitrophenol and inorganic phosphate at a rate approximately one third of that of ATP hydrolysis (Judah et al., 1962). The hydrolysis of p-nitrophenyl- phosphate is markedly stimulated by K+ and it appears that, in producing this effect, K+ binds at specific activating sites on this enzyme, distinct from the sites at which p-nitrophenylphosphate is hydrolyzed (Robinson, 1969). Therefore K+ acts in much the same way as a drug, binding at a distinct site and producing a conformational change which results in an increase in a specific activity. This activation of the p-nitrophenylphosphatase (p-NPPase) by Rf is inhibited by a number of drugs and therefore makes a useful model for the investigation of enzyme activation and inhibition kinetics. 40 41 MATERIALS AND METHODS Rat brain (Na+ - Rf) ATPase was prepared by the method of Akera and Brody (1969). Rat brains were homogenized in five volumes of ice cold 0.25 M sucrose, 5 mM disodium ethylenediaminetetraacetic acid (NazEDTA) and 0.22 deoxycholate, pH 6.8. The homogenate was centri- fuged at 12,000 x g for 30 minutes and the supernatant taken and centri- fuged at 35,000 x g for 30 minutes. The resulting pellet was suspended in 2 M NaI, 2.5 mM Na EDTA, 3 mM MgATP and 5 mM histidine hydrochloride, 2 pH 7.3. This suspension was stirred gently for 30 minutes at 0° C, diluted with water to give 0.8 M NaI, and centrifuged for 30 minutes at 100,000 x g. The resulting pellet was washed twice by centrifuga- tion and resuspension in 10 mM Tris-HC1 buffer, pH 7.3. The final residue was suspended in a medium containing 0.25 M sucrose, 5 mM histidine HCl and 1.0 mM Tris EDTA. The protein content of prepara- tion was assayed by the method of Lowry et al. (1951). p-Nitrophenylphosphatase (p-NPPase) was measured by a modification of the method of Robinson (1969). Forty micrograms of enzyme were incubated in 1 m1 of a medium containing 5 mM MgCl 5 mM p-nitrophenyl- 2. phosphate, 50 mM Tris-HCl buffer, pH 7.4, with other additions as indicated. Incubation was for 15 minutes at 37° C. At the end of this period the reaction was stopped by the addition of 0.5 m1 of 151 TCA. The assay tubes were then centrifuged at 3000 x g for ten minutes and“ 2.5 ml of 1 M Tris base added. This addition developed the yellow color of paranitrophenol and the absorbence of the solution was read at 420 nm on a Shimadzu spectrophotometer. p-Nitrophenylphosphatase activity was expressed as umoles Pi/mg protein/hour. All experimental points are the means of three separate determinations with different 42 enzyme preparations and are expressed as a percentage of the activity observed in the presence of 16 mM K+. RESULTS AND DISCUSSION In the presence of Mg++, a low background level of p—nitrophenyl- phosphatase activity was observed. As this activity is probably related to the nonspecific Mg++ ATPase activity in these enzyme preparations (Robinson, 1969), it was subtracted from the activity observed in the presence of Mg++ and K+ as a blank. Only the K+ activated portion of the p-nitrophenylphosphatase activity is plotted. The kinetics of the K+ activation and the inhibition of this enzyme by three inhibitors were investigated. Figure 4.1 shows the activation of this enzyme by Rf and the inhibition of this activation by ethanol. The curve for activation of this enzyme by K+ is approximately hyperbolic and the apparent Km for the activation of this enzyme by K+ is about 2 mM. The addition of 12 (v/v) ethanol inhibited the K+ activation of the enzyme. High concentrations of K+ partly overcame the ethanol inhibition of this enzyme, suggesting that the inhibition of the enzyme activity by ethanol is reversible and the interaction between ethanol and K+ is competitive. Replotting the data by the method of Lineweaver and Burke (1934) also showed that this interaction is at least partly competitive (Figure 4.5). Replotting these data (Figure 4.2) and the data of Figure 4.3 (replot not shown) by the method of Hofstee (1956) indicated that the data deviate considerably from simple Michaelis- Menten kinetics, consistent with the results shown in Figure 4.4. Alcohol is a relatively weak inhibitor of the (Na+ - K+) ATPase activity associated with this enzyme. Therefore, the effect of ouabain, 43 a more potent inhibitor of this enzyme preparation, was tested. Figure 4.3 shows the inhibitory effect of 10..5 M ouabain on the K+ activated p-NPPase activity of this enzyme. At this concentration ouabain produced an approximately 70% inhibition of this enzyme, and when plotted by the method of Lineweaver and Burke both lines inter- cepted on the horizontal axis suggesting that the interaction was essentially noncompetitive (Figure 4.5). Adenosine diphosphate, which is a product of the (Na+ - Rf) ATPase reaction, binds tightly to this enzyme and is a potent inhibitor of this enzyme system under certain conditions (Nagai and Yoshida, 1966). Figure 4.4 shows the inhibitory effect of 10-4 M ADP on this enzyme. At low concentrations of K+, the enzyme is almost completely inhibited by ADP. Increasing the concentration of K+ produces a sigmoidal increase in enzyme activity but did not completely overcome the inhibitory effect of ADP. ADP shifted the potassium concentration for half maximal stimulation of enzyme activity to about 6 mM, a more than three fold reduction in the apparent affinity of the enzyme for K+. The experimental results presented here approximate the major kinetic patterns of enzyme inhibition. Ethanol was chosen as a proto- type competitive inhibitor since it has been suggested to inhibit the (Na+ - K?) ATPase competitively with respect to K+ (Israel at aZ., 1965). The results presented here support Israel's observations, since raising the concentration of H+ partially overcame the ethanol inhibi- tion of the enzyme. If the interaction was strictly competitive raising the concentration of Ki would be expected to completely over- come the inhibition by ethanol. When the data were plotted by the method of Lineweaver and Burke (Figure 4.5) the two lines intercept 44 at approximately the same point on the 1/V axis indicating that at infinite substrate levels the effect of the inhibition is overcome. Similarly, when plotted by the method of Hofstee the Vmax values observed in the presence and absence of ethanol were not significantly different (Figure 4.2). The pattern of inhibition produced by ethanol suggests that the interaction between Rf and ethanol on this enzyme is competitive. Ouabain was chosen as an inhibitor because it is a potent and specific inhibitor of the (Na+ - Rf) ATPase, giving rise to a rela- tively stable enzyme-ouabain complex (Akera and Brody, 1971). The ouabain inhibition of the p-NPPaee reported here has many of the characteristics of a noncompetitive inhibitor. In particular, there was no tendency for increasing concentrations of K+ to overcome the actions of ouabain. This action of ouabain is equivalent to the removal of a portion of the enzyme from the reaction mixture and thus its effect appears as a reduced Vmax with no change in the apparent Km for K+. Though ouabain produces this effect by binding very tightly to this enzyme, reversible inhibitors may produce the same effect if increasing the substrate concentration does not affect the affinity of the enzyme for the inhibitor. Adenosine diphosphate produces a completely different pattern of inhibition. It reduces the Vmax markedly, shifts the apparent Km for R+ from about 1.5 mM to more than 4 mM and changes the activation curve for K+ from an approximately rectangular hyperbola to a markedly sigmoidal activation curve. These data suggest that the action of ADP is to bind to the enzyme and stabilize a form which is not readily activated by K+. More K+ is therefore required to activate the enzyme and the concentration for half maximal activation of the enzyme is 45 increased. The sigmoidal shape of the activation curve in the presence of Rf suggests that at least two potassium ions must bind to activate the enzyme. The inhibition of this enzyme by ADP is an example of allosteric (indirect) inhibition of this enzyme (Monod et al., 1965; Akera and Brody, 1971). Similar patterns of inhibition are also seen in drug receptor interactions. Competitive inhibition which resembles the pattern of alcohol inhibition occurs when increasing the concentration of the drug overcomes the action of the inhibitor. Since drug receptor inter? actions are usually presented as log dose response curves, competitive inhibition appears as a parallel shift in the dose response curve (change in Km) with no change in Vmax (Figure 6.3). Noncompetitive inhibition appears as a reduction in Vmax with a reduced slope (Figure 6.2). An indirect drug inhibitor interaction such as the Kf - ADP interaction may appear as a series of parallel dose response curves such as the atropine-acetylcholine antagonism presented in Figure 5.7. SUMMARY The inhibition of K+ activated p-nitrophenylphosphatase by ethanol, ouabain and adenosine diphsophate were demonstrated. The curve for activation of the enzyme by K+ shows that 12 ethanol inhibited competitively whereas 10“5 ouabain inhibited essentially noncompeti- tively the K+ activation of the enzyme. Linear transformations of the data by the methods of Lineweaver and Burke and Hofstee allowed statistical analysis and showed similar results. Adenosinediphosphate inhibited the K+ activation of this enzyme markedly at low concentrations of K+, but less effectively at higher concentrations. The activation of this enzyme by K+ was sigmoidal in 46 the presence of ADP. These results suggest that the interaction between K+ and ADP on this enzyme is allosteric or indirect. REFERENCES Akera, T. and Brody, T. M.: The interaction between chlopromazine free radical and microsomal sodium— and potassium-activated adenosine triphosphatase from rat brain. Mol. Pharmacol. 5: 605, 1969. Akera, T. and Brody, T. M.: Membrane adenosine triphosphatase. The effect of potassium on the formation and dissociation of the ~ ouabain-enzyme complex. J. Pharm. Exp. Ther. 176:545, 1971. Hofstee, B. H. J.: Graphical analysis of single enzyme systems. Enzymologia 17:273, 1956. Israel, Y., Kalant, H. and Laufer, 1.: Effect of ethanol on Na+, K+, Mg++estimu1ated microsomal ATPase activity. Biochem. Pharmacol. 14:1803, 1965. Judah, J. D., Ahmed, K. and McLean, A. E. M.: Ion transport and phosphoproteins of human red cells. Kalant, H. and Israel, Y.: Effect of ethanol on active transport of cations. In Biochemical factors in alcoholism. Ed. by R. P. Maickel. Pergamon Press, New York, 1967, p. 25. Lineweaver, H. and Burke, D.: The determination of enzyme dissocia- tion constants. J. Am. Chem. Soc. 56:658, 1934. Lowry, G. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265, 1951. Monod, J., Wyman, S. and Changeaux, J. P.: On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12:88, 1965. Nagai, K. and Yoshida, H.: On potassium dependent phosphatase (guinea pig brain). Biochem. Biophysic Acta 128:410, 1966. Robinson, J. D.: Kinetic studies on brain microsomal adenosine tri- phosphatase. II Potassium dependent phosphatase activity. Biochemistry 8:3348, 1969. Skou, J. C.: Enzymatic basis for active Na+ and KI transport across cell membrane. Physiol. Rev. 45:546, 1965. 47 .Aeo.o A av ucoumooee maonmofiwaamam uoo one mean Hmuauuo> one so moeoououea woo woo moumnvm momma mo wonooa one an cause one: omega use .Hooonuo NH mo monomoua may a“ oowum>auum +M one soon moaouao madam one use moommom mo eczema one an wouuoao oommm2|a one mo coaum>auom +M onu 30am ooaouwo some one .H.¢ ouswfim mo omono one muse one .ooummox mo cosmos moo ou wnauuooom wouuoaa .mufi>auum omommzna wouo>wuom +¥ so Hoemnuo mo uuowwm .~.a mmauHm .ommmmZIm «so to coaum>auom +M use emoeneeae Aao.o v av maunmowmamwam Hoamnuo .umum one an voumoavsa .+M :8 N u< .mumn Hmoauuo> one an woumUHmfiW mum gowns momma ecu mo muouuo uumvamuo ago + nnoaumumaoua oshuoo uaouommav news mooaumnfiauoumv wounu mo some onu ma uaaoa zoom .auw>fiuom ommmmZIn woum>wuom +M no +M mo meoaumuucooaoo waammouooa mo uoommo one soon moaouao vHHom any .mow>wuom ommmmzua umum>auom +M so Hosonuo mo uoommm .H.q mmsuHm 48 ...x «(E >:>:U< .x. Ow ON O hi F“!- w! ..V. / .o.oAa I .1 x. .293 to 33:: 9 .0...coU l O ON AllAllDV 7. F7"? 100? LR .0330... *0 tofoi of 0. 85.83 ooze: 5.2.: .322 (a 3.3:: .0. .3 .295 .0 Unified: Or +0. 26 m .n N r — b p p p 7 .. Sovnlui e. WAG .. . 0:0 0 00 I . m ON oxo— _ fun . :m 0 MI ._o:coUl @ a \ . 100% V 3 H W a m .09 .>._>:o< oaomaZIQ po.0>:o<+x co _0co£w .0 .ootw ._.V.0E 49 FIGURE 4.3. Effect of ouabain on Ki activated p-NPPase activity. The solid circles show the KT activation of the p-NPPase and the star circles show the effect of 10‘5 M ouabain on this activation. FIGURE 4.4. Effect of ADP on Rf activated p-NPPase activity. The solid circles show the KT activation of the p-NPPase and the open circles show the effect of 10"4 M ADP on this activation. 50 FIG.4.3. EHect of Ouoboin on KIActivoted p—NPPose Activity, 100* /Q l ; é a a? 60_ / I H T / I 20. /§ § Q @_Control. “a G—Effect of Ouoboin IO-SM. -.. r f I I T fiF—I—J 1 2 4 8 mM K‘ 16 FIG.4.4. Effect oi ADP on K’Activoted p—NPPose Activity. 1 1 00+ /€§/H ('3 ( .2 / ... § 3 20.. fl /§ ® _ Control. / o :3 / O~ Effect of ADP 10‘ ‘M. 51 onofiuoasuamo.nowonoumou onu ca wowsaoea nos use :mouw was» Baum vouoaao we namomso mica mo ousowoua one ca +M 2e m.o um unwon one .moumsum umoma mo cosmos one an mmswaumuow mums muoan ona .aaonmoo z mica mo uncommon ecu ow hufi>wuom moo zoom moaouwu umum one .Hoamnuo NH «0 monomoua one a“ auH>Huum one 305m moaouao vfiaom emu .omomm2ta may no nowumbwuom +M onu Bonn moaouao ammo oeH .Aqmmav oxusm mam umbmosmsag mo monuoa onu he wouuoaaou .m.« use H.e muswwm mo mmoau mum some may .oxuom one uo>mosonHA mo wonuoa one On wnfivuouom mouuoae .ommmmZIa voum>auom +M so damnmso no Hoamnuo mo uommmm .m.¢ MMDUHm 52 .2 Op 503030 We .30th9 .ob— _oc0.tw *0 .ootmlfi. ..0:coUIO \. moo IOOO I All/\llDV‘); .05 o .0425 .25 occasion: .0 .0050: o... o. 95:00:. notoE .onoaaZIQ “0201.04. +0. :0 £03030 to .9553 .0 .00.; .n... .0: CHAPTER V CHOLINERGIC RECEPTORS OBJECTIVES The objectives of these experiments were to demonstrate some proper- ties of cholinergic receptors. INTRODUCTION Acetylcholine (ACh) is the transmitter substance at the neuro- muscular junction, in pre- and postganglionic parasympathetic nerve fibers and in preganglionic nerve fibers. It is stored in vesicles in the preganglionic terminal and it is released when an action potential reaches the nerve terminal (Feldberg and Gaddum, 1934; Dale at al., 1936). The released ACh diffuses across the synaptic cleft and inter- acts with specific receptors on the postsynaptic membrane or muscle end plate (Castillo and Katz, 1955). Its interaction with these receptors triggers an increase in membrane permeability which ultimately results in the tissue (pharmacological) response. Because it is experi- mentally convenient to monitor muscle contraction most of the early studies and the work reported here deals with the cholinergic responses of skeletal and smooth muscle. As well as interacting with ACh these receptors will also inter- act with structural analogs of ACh to varying degrees. Early work with two such analogs, nicotine and muscarine, showed that the cholinergic receptors in skeletal muscle reacted preferentially with nicotine and 53 54 poorly with muscarine, whereas the reverse was the case with smooth muscle. This different reactivity points to differences between cholinergic receptors in different tissues and cholinergic receptors are now classified as either nicotinic or muscarinic (Korolkovas, 1970). Besides interacting with these receptors, ACh combines with acetylcholinesterase during hydrolysis. Ehrenpreis (1967) noted that there is a close similarity between cholinergic receptors and acetyl- cholinesterase. It is assumed, however, that the active sites of the ACh receptor cannot be identical to that of the acetylcholinesterase, because the former does not hydrolyze ACh (Korolkovas, 1970). Drugs that inhibit acetylcholinesterase potentiate the action of ACh whether the ACh is exogenous or released by nerve stimulation. The nicotinic receptors of autonomic ganglia and skeletal muscle are not completely identical; they respond differently to certain stimulating agents. Volle and Koelle (1970) noted that there are at least three cholinoceptive sites in sympathetic ganglia. The primary site is an excitatory receptor site that can be activated by endogenous ACh and by ganglion stimulating drugs, exogenous acetylcholine, tetra- methylammonium, nicotine and dimethylphenylpiperazine. The activation of the receptors results in ganglion depolarization and postganglionic firing and is sensitive to blockade by hexamethonium, d-tubocurarine and tetraethylammonium. Volle and Koelle further noted that the second excitatory. receptor site is activated by acetylcholine,. methacholine or muscarine. The result of activation of this second receptor is the same as the activation of the first receptor except that the depolarization starts 4-5 seconds later. This second receptor can be blocked by atropine. The third receptor is an inhibitory one which can be activated 55 by ACh and methacholine and is blocked by atropine. The activation of this receptor produces ganglionic hyperpolarization. The nicotinic receptor on the postsynaptic membrane of the neuro- muscular junction is activated by ACh and nicotine. High doses of nicotine cause blockade of the receptor. Koelle (1970) noted that neuromuscular blocking agents are classified into two groups. The first group are the competitive or nondepolarizing agents (Lewis and Muir, 1967). These agents combine with the receptor on the postsynaptic membrane leading to an inactive complex and a rise in the threshold for stimulation of the skeletal muscle; the classic example is d-tubocurarine (Dale at al., 1936). In consequence, the end plate potential caused by liberated ACh undergoes a progressive reduction in amplitude, duration and rate of development, terminating in its complete disappearance (Thesleff, 1955). Impulse transmission then ceases because the amplitude of the end plate potential has fallen below the threshold necessary to trigger an action potential in the membrane of the muscle fibers (Eccles at al., 1941). Paton (1953) and Taylor (1959) stated that the mechanism of the d-tubocurarine antagonism of ACh is competitive in nature. The second group of neuromuscular blocking agents are the depolar- izing agents which mimic the action of the transmitter and whose combina- tion with the receptor leads to the formation of an active complex and to depolarization of the postsynaptic membrane; the classic example is decamethonium. In high doses this depolarizing agent produces a blockade that has two distinct aspects (Zaimis, 1951). The phase one block is a short duration block due to depolarization of the motor end plate (Jenden et al., 1951) which is followed by membrane repolarization. The phase two block, called desensitization (Axelsson and Thesleff, 56 1958) is more prolonged and is terminated only by the removal of the drug. A drug that selectively produces cholinomimetic effects at the muscarinic receptor is muscarine. The localization of the muscarinic receptors is on the postganglionic parasympathetic neuroeffectors, primarily in smooth muscle and secretory glands. Such sites probably exist also in certain autonomic ganglia such as in the inferior mesen- teric ganglia of the cat (Gyermek at al., 1963). Some authors consider that muscarinic receptors also exist in the central nervous system. Drugs that antagonize the muscarinic actions of ACh are defined as muscarinic blocking agents. The mechanism of action of such agents is by competitive antagonism of ACh and other muscarinic agents. The antagonism can therefore be overcome by increasing the concentration of ACh at the receptor sites in the effector organ. Atropine is a highly selective antagonist of muscarinic agents in smooth muscle and exocrine gland cells (Innes and Nickerson, 1970). MATERIALS AND METHODS The rectus abdominis of the frog was used to demonstrate the action of ACh on nicotinic receptors and the guinea pig ileum was used to demonstrate the muscarinic receptors (Long and Chiou, 1970). The rectus abdominis muscles were obtained from 20 gm Rana pipiens. The frogs were decapitated and their spinal cords destroyed with a long needle. The muscles were then isolated from the pelvic girdle to their insertion in the cartilage of the pectoral girdle. Threads were attached to both ends of the muscle and they were dissected from the body. A These muscle preparations were stored at 4° C in frog Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.08 mM CaCl 2.4 mM NaHCOa) for up to 24 2’ 57 hours (Welsch, 1972). For the experiments, the muscle strips were suspended in an organ bath as indicated in Figure 5.1. The long thread was connected to a Grass force displacement transducer and the contrac- tion was recorded on a Grass polygraph. The medium in the muscle bath was frog Ringer's solution containing 10-4 M eserine sulfate to inactivate any cholinesterase present. A 1 mM stock solution of ACh in eserinated frog Ringer‘s was pre- pared fresh each day and appropriate dilutions were made for each experiment. Because the cumulative dose response curve was the method used, very small volumes of the ACh solutions were added to the organ bath so that minimal changes in the bath volume were obtained. Dose response curves were obtained for ACh alone and in the presence of atropine and d-tubocurarine. The guinea pig ileum preparation was used to demonstrate the muscarinic receptors. Guinea pigs were sacrificed by a blow on the head and the terminal portion of the ileum was excised and placed in a dish of modified Tyrode's solution (NaCl 0.8%, KCl 0.02%, CaCl2 0.02%, NaHCO 0.12, NaHPO 0.0052, glucose 0.1:, morphine sulfate 40 mg/l, 3 3 eserine and pyrilamine maleate 10-4 M) at 23° C. The mesentery was trimmed away and the ileum was cut into 2 cm long strips suitable for mounting. The guinea pig ileum was mounted, as with the frog rectus muscle, in the modified Tyrode's solution, and the muscle contractions were recorded as previously. Dose response curves to acetylcholine alone and in the presence of d-tubocurarine or atropine were prepared as previously. All experiments were performed at room temperature and the preparations were oxygenated by bubbling air slowly through the muscle bath (Figure 5.1). 58 RESULTS AND DISCUSSION The cumulative dose-response method (Figure 5.2) was chosen because of its rapidity and the fact that it eliminates errors due to repeated washing of the muscle preparations. Because this method depends on the cumulative increase in ACh concentrations in the bath, 10"4 M eserine was present in the bathing medium in all experiments to inhibit muscle acetylcholinesterase. In addition morphine and pyrilamine were added to the guinea pig ileum preparation to reduce spontaneous activity (morphine) (Paton, 1957; Schaumann, 1957) and to prevent the action of endogenous histamine (pyrilamine) and 5-hydroxy— tryptamine (morphine). There was considerable variation in the dose-response curves obtained with the different muscle preparations, as shown in Figure 5.3. Therefore all the experiments reported here were performed on single muscle preparations. Figures 5.4, 5.5, 5.6 and 5.7 show typical log dose-response curves of frog rectus and guinea pig ileum to ACh. In each case the muscle responds to increasing concentrations of ACh with a sigmoidal dose-response curve, half maximal activation being obtained at about 10.6 M ACh. The data indicate that ACh has roughly equal affinity for the cholinergic receptors in each type of muscle preparation, with an apparent affinity constant in the order of 10.-6 M. The data obtained using ACh give no indication of the differences between the cholinergic receptors in these two systems. Figures 5.4 and 5.5 show the differential sensitivity of the frog rectus to d-tubocurarine and atropine. In the presence of 10”5 M d-tubocurarine the log dose response curve to ACh is shifted about two log units to the right and the slope of the dose response curve is 59 markedly decreased. If additions were made beyond 5 x 10.4 M ACh, as in other experiments, these higher concentrations of ACh completely overcame the action of d-tubocurarine, suggesting that the interaction is competitive in nature. Atropine at 10-5 M concentration did not reduce the reactivity of the frog rectus muscle to ACh, in marked contrast to its effects on the guinea pig ileum preparation (Figure 5.6). This differential sensitivity of the skeletal muscle receptor to d-tubocurarine and atropine is characteristic of the nicotinic cholinergic receptor. d-Tubocurarine is considered a simple competitive inhibitor of the cholinergic receptor since increasing concentrations of ACh can over- come its action. In keeping with this its action in vivo is antagonized by agents which release ACh, such as tetraethylammonium (Koketsu, 1958) and by anticholinesterases and depolarizing agents. However, in vitro the antagonism does not follow simple Michaelis-Menten kinetics, which predict parallel shifts in the dose-response curves, for in the presence of d-tubocurarine the dose-response curve to ACh was markedly flattened. These observations suggest that the inter- action between d-tubocurarine and the nicotinic receptor is more complex than simple competitive inhibition. Electrophysiologically, d-tubocurarine is considered a non- depolarizing agent (Lewis and Muir, 1967) due to its effect on the end plate. It does not affect the electrical properties of the end plate or those of the muscle membrane but it reversibly inhibits the depolariz- ing effect of ACh applied to the end plate. The presence of d-tubocurarine inhibits the end plate potential triggered by the depolarizing effect of ACh, thereby preventing the firing of action potentials. With increasing d-tubocurarine concentrations the end plate potential becomes less and less and finally disappears. 60 Figures 5.6 and 5.7 show the differential response of the guinea pig ileum preparation to d-tubocurarine and atropine. At 1 x 10..9 M, atropine produced a marked shift to the right of the dose-response curve to ACh and 1 x 10.5 M atropine completely inhibited the response. In contrast d-tubocurarine, which was an effective inhibitor in the frog rectus preparation had no effect in the guinea pig ileum. These results demonstrate the high affinity of the cholinergic receptors in guinea pig ileum for atropine and the relative ineffectiveness of d-tubocurarine in this preparation. If the concentration of atropine in the system was increased, classical parallel shifts in the dose-response curves were obtained (Figure 5.7). However, despite these data it appears unlikely that the interaction between ACh and atropine may be explained on the basis of simple competitive inhibition. Theoretically, a simple competitive inhibitor binds reversibly to receptors and thus is readily removed by washing. With atropine, however, it was early observed that the inhibition did not wash out (Clark, 1926) and recent experiments with radio-labelled atropine (Paton and Rang, 1965) have shown that atropine dissociates from the muscarinic receptors of guinea pig ileum with a half-life of about 20 minutes. Further, the rate of dissociation of atropine from the receptors as measured by the rate of recovery from atropine inhibition was not accelerated by large amounts of ACh, ruling out an indirect drug receptor interaction. These observations suggest that atropine forms a stable complex with its receptors and since a pharmacological response can still be obtained (Figure 5.7), more receptors are present in the tissue than are required for a maximal response. This and other data obtained by Nickerson (1956) have given rise to the concept of spare receptors, i.e., only a fraction of the 61 receptors present in a tissue are required for a full pharmacological response (Nickerson, 1956). Thus, ACh and atropine initially compete for the same receptors but the slow rate of dissociation of atropine receptor complex allows additional ACh to act only on the unoccupied receptors. CONCLUSIONS In the frog rectus preparation d-tubocurarine inhibited muscle contraction produced by ACh, but under similar conditions no inhibition was observed in the presence of atropine. On the other hand, atropine markedly inhibited the response of the guinea pig ileum to ACh, whereas d-tubocurarine was ineffective. In both cases inhibition (where observed) was qualitatively competitive but there are reasons for thinking that these interactions are in fact more complex. The differ- ences between the cholinergic receptor in skeletal muscle and smooth muscle have led to the classification of cholinergic receptors as either muscarinic (smooth muscle) or nicotinic (skeletal muscle). REFERENCES Axelsson, J. and Thesleff, S.: The desensitizing effects of acetyl- choline on the mammalian motor and plate. Acta Phys. Scand. 43:15, 1958. delCastillo, J. and Katz, B.: On the localization of acetylcholine receptors. J. Physiol. 128:157, 1955. Clark, A. J.: The reaction between acetylcholine and muscle cells. J. Physiol. 61:530, 1926. Dale, H. H., Feldberg, W. and Vogt, M.: Release of acetylcholine at voluntary motor nerve endings. J. Physiol. 86:353, 1936. Eccles, J. C., Katz, B. and Kuffler, S. W.: Nature of the end plate potential in curarirized muscle. J. Neurophysiol. 4:362, 1941. Ehrenpreis, 8.: Molecular aspects of cholinergic mechanisms. In Drugs affecting the peripheral nervous system, Vol. I. Ed. by A. Burger, Marcel Dekker, New York, 1967, p. l. 62 Feldberg, W. and Gaddum, J. H.: The chemical transmitter at synapses in a sympathetic ganglion. J. Physiol. 81:305, 1934. Gyermek, L., Sigg, E. B. and Bindler, B.: Ganglionic stimulant action of muscarine. Am. J. Phys. 204:68, 1963. Innes, J. R. and Nickerson, M.: Antimuscarinic and atropinic drugs. In The Pharmacological Basis of Therapeutics. Ed. by L. S. Goodman and A. Gilman. Macmillan Co., 1970, p. 524. Jenden, D. J., Kamijo, K. and Taylor, D. B.: The action of decamethonium (C10) on the isolated rabbit lumbrical muscle. J. Pharm. Exp. Ther. 103:348, 1951. Koelle, G. B.: Neuromuscular blocking agents. In The Pharmacological Basis of Therapeutics. Ed. by L. S. Goodman and A. Gilman. Macmillan Co., New York, 1970, p. 601. Koketsu, K.: Action of tetraethylammonium chloride on neuromuscular transmission in frogs. Am. J. Phys. 193:213, 1958. Korolkovas, A.: Cholinergic receptors. In Molecular Pharmacology. John Wiley & Sons Inc., New York, 1970. Lewis, J. J. and Muir, T. C.: Drug acting at nerve skeletal muscle junction. In Drugs affecting the peripheral nervous system. Ed. by A. Burger. Marcel Dekker, New York, 1967, p. 237. Long, J. P. and Chiou, C. Y.: Pharmacological testing methods for drugs acting on the peripheral nervous system. J. Pharm. Sci. 59:133, 1970. Nickerson, M.: Receptor occupancy and tissue response. Nature 178: 698, 1956. Paton, W. D. M.: The action of morphine and related substances on contraction and on acetylcholine output coaxially stimulated guinea pig ileum. Brit. J. Pharm. 12:119, 1957. Paton, W. D. M.: The principle of neuromuscular block. Anaesthesia 8:151, 1953. Paton, W. D. M. and Rang, H. P.: The uptake of atropine and related drugs by intestinal smooth muscle of the guinea pig in relation to acetylcholine receptors. Proc. Roy. Soc. 163:1, 1965. Schauman, W.: Inhibition by morphine of the release of acetylcholine from intestine of the guinea pig. Brit. J. Pharm. 12:115, 1957. Taylor, D. B.: The mechanism of action of muscle relaxants and their antagonists. Anesthesiology 20:439, 1959. Thesleff, S.: The mode of neuromuscular block caused by acetylcholine, nicotine, decamethonium and succinylcholine. Acta Phys. Scand. 34:218, 1955. 63 Volle, R. L. and Koelle, G. B.: Ganglionic stimulating and blocking agents. In The Pharmacological Basis of Therapeutics. Ed. by L. S. Goodman and A. Gilman. Macmillan Co., New York, 1970, p. 585. Zaimis, E. J.: The action of Decamethonium on normal and denervated mammalian muscle. J. Physiol. 112:176, 1951. 64 FIGURE 5.1. Organ bath preparation for the frog rectus abdominis. FIGURE 5.2. Typical cumulative dose response curve of frog rectus abdominis to acetylcholine. Muscles were mounted in the organ bath as indicated in Figure 5.1 and allowed to equilibrate for 5-10 minutes, at which point the base line was set. Each muscle was then exposed to 1 X 10'5 M ACh, which pro- duced a maximal response. The sensitivity of the polygraph was then adjusted so that this maximal response produced a suitable (40-60 mm) excursion of the pen. The muscle was then washed twice and allowed to return to the base line. Sequential additions of the required con- centrations of ACh were then made and two minutes were allowed for the contractile response to develop after each addition. 65 To Transducer _.___ Polyvinyl Tube Frog Rectus Abd. __ —. 4‘ . Frog mg... 5..---. __ Rubber Plug (.___ > .7, Outlet FIG.5.l. Organ Bath Preparation for Frog Rectus Abdominis. .<————- I x 164% (.... 2 x Io’°~\ -5 x lOfbM uHom aou menu once u weHBOsm .maOHunnmamua oHomaa mHeHaouom asuumu woum uaououmHu o>Hw suHa moaHuuoo mo>nau uncommon meow uaouommHu 0>Hm 30am mHooshm uHHom o:H .meHumumaoua oHomaa mHaHaounm nauouu woum econoMMHv nuHs poaHmuno mo>uao uncommon omov moo aH aOHumHum> .m.m mmson 67 .UZOU IU( 00L| n v n o n p b F to \@l @\® 0 179 “25:383.-.. + to. ...... a O N S 3 ru< M W ..O.\O w low .octotauonahl v .0 00:03... or: 5 tea oco_< .oc__o;o_.:ou< 0. 2:.Eono< 3.03. not”. .0 03090: {6.0: .UZOU IU( 00.. I x. . e 0 ex 0 e\ .\ C .\ to... x e \ %0 00\0\0 \\\\.\ loo Acctotoaoca 0.032 TEE |0tn< 3.03. Got“. 20.035 5:! $050.30 no>5U 3:033. 300 of E c020C0> 6.0.0: SSNOdSHU lNEIDUSd 68 .oaHaonuo z mtoH N H mo uncommon sou aH 50¢ ou uncommon onu soon moHonHu uHHom one use oaHuouaoonnulu z m:0H N H mo mononoua onu aH sum ou omaoamon onu scam noHouHo vommouo one .no< mo maoHumnuamoaou vooooHuaH ecu ou oHooaa mfiafiaoupm msuouu woum m «o uncommon mam 30am ooHonHo mono ona .oaHaouuu no oaHumnsoonaulm mo uncommon mam aH use oaOHm .oeHHoslouoom ou mHomaa eHaHaovnm mauve» menu «a onaoamom .m.m mMDuHm 69 .UZOU IU< 00.. l v m. 0 h o\o $\ \$\ \ \ \e \o Snug mz_ao~:<+\o l9l® I o m WW Nl BSNOdSBU of 5 tea 6:30.; to octocauooahlp .0 00:03.... oco_<..c__2._:8< o. 2:32 2558.2 .28“ not to 28....“ .n.n. o... 70 FIGURE 5.6. Response of guinea pig ileum to acetylcholine, alone and in the presence of atropine 10’s, 10'9 M and d-tubocurarine 10-5 M. In the left hand panel the solid circles show the response of a guinea pig ileum to the indicated concentrations of ACh and the crossed circles show the response in the presence of 1 X 10"9 M atropine. In the right hand panel the solid circles show the response of a guinea pig ileum to the indicated concentrations of ACh. The open circles and the crossed circles show the response in the presence of 1 X 10'5 M d-tubocurarine or atropine, respectively. FIGURE 5.7. Response of guinea pig ileum.to acetylcholine, alone and in the presence of atropine 10‘9, 10"8 and 10"7 M. The solid circles show the response of a guinea pig ileum to the indicated concentrations of ACh. The crossed circles show the response in the presence of the indicated concentrations of atropine. 71 FIG.5.6. Response olGuinea Pig lleum to Acetylcholine, Alone and in the Presence of Atropine 10*5, lo“9M and d-Tubo ~ curarine 10‘5M. +d—TUBOCURARINE 5 iO— MO 50. _ / I . . fl 2 O (9 I / z 9 - I + ATR.IO"9 {9 3" O 5 3 / 0' o _ ACH / 3 a. ’ I‘ ‘l 3 /. if? AC” a C . O 10.. / .. /. 5 {B I0 + ATR. 10‘ / Q —$"®-$— I l . 5 . E -— lOG ACH CONC. FIG.5.7. Response of Guinea Pig lleum to Acetylcholine, Alone and in the Presence of Atropine t0_9, t0"8 and lO—7M . 40‘ I C ACH 2 / i Z O ... _9 + ATR 107M ‘2 +/ATR lo M ' Q 2 20. . $16} + ATR IO‘BM a}, =2 o’ .. / / / G} / 9 Q9 o.— Ut—l l 4 -lOG ACH CONC. CHAPTER VI ADRENERGIC RECEPTORS OBJECTIVES The objective of these experiments was to demonstrate some aspects of the interaction of drugs with adrenergic receptors. INTRODUCTION Norepinephrine is the neurotransmitter at postganglionic adrenergic nerve endings and in some areas of the central nervous system (Innes and Nickerson, 1970). As with acetylcholine, norepinephrine is_con— sidered to produce its effects by interacting with specific receptors located on the postsynaptic membrane. Early work with epinephrine and norepinephrine showed that these agents produce both stimulatory and inhibitory effects depending on the tissue or organ being studied. In 1948 Ahlquist classified adrenergic receptors as either a or B on the A basis of their response to six different sympathomimetic amines. The a receptors are excitatory and react preferentially with norepinephrine. The 8 receptors are inhibitory and react most readily with epinephrine. There are two exceptions to this rule, the inhibitory a receptors of intestinal smooth muscle and the stimulatory B receptors of the heart. Though initially criticized (Lands, 1949, 1952) the discovery of dichloroisoproterenol, a potent B adrenergic inhibitor, supported Ahlquist'e classification (Furchgott, 1959). 72 73 This classification of adrenergic receptors has been further strengthened by the discovery of specific inhibitors for each type of receptor. Nickerson and Goodman (1947) introduced dibenamine and phenoxybenzamine, which produce a prolonged and very specific blockade of 0 receptors. This type of inhibition has been termed competitive nonequilibrium.blockade by Nickerson (1957). Another group of a receptor blockers are the ergot alkaloids, first reported by Dale (1906). Blockade by the ergot alkaloids tends to be more complete and persistent than that produced by other competitive antagonists, but is much less effective than that produced by the haloalkylamines (Nickerson and Hollenberg, 1967). Simple competitive inhibitors of a adrenergic receptors are tolazoline and phentolamine. Specific blockers for the B receptors were only recently dis- covered. In 1958 Powell and Slater reported on the B blocking action of dichloroisoproterenol, an analog of isoproterenol. This discovery was followed in 1962 by the discovery of pronethalol (Black and Stephenson, 1962) and in 1964 of propranolol (Black et aZ., 1964). These agents are all assumed to produce their effects by simple com- petitive inhibition at the level of the 8 adrenergic receptor. Adrenergic receptors appear to be located on the cell membrane. Early evidence for this location were the observations on the changes in the electrical characteristics of the cell on application of epinephrine. These observations are further supported by the intra- membranoue location of adenyl cyclase, which is probably identical with the 8 receptor (Sutherland and Rall, 1960). 74 MATERIALS AND METHODS Guinea pigs were sacrificed by a blow on the head and the chest opened, the heart taken out and placed on,a petri dish containing Tyrode's solution (NaCl 0.82, KCl 0.02%, NaHCO 0.12, MgCl2 0.012, 3 4 0.0052, glucose 0.1%). The ascending aorta was quickly removed NaHPO and a length of the aorta was then cut into a strip as follows. The aorta was threaded on a fine forceps and gently turned inside out. Then a spiral incision was made in the aorta, gradually rotating the aorta as the incision proceeded. Threads were inserted at both ends of the strip and a fine 100p was made at one end. The strip was then placed in an organ bath con- taining Tyrode's solution at 37° C and gassed with 951 oxygen and 51 carbon dioxide. The loop at one end of the strip was inserted at the hook of the bath and the long thread was attached to the Grass force displacement transducer. The contraction of the aortic strip was recorded on a Grass polygraph. This organ bath is depicted in. Figure 6.1. RESULTS AND DISCUSSION When first isolated and mounted these aortic strips are maximally contracted and a period of up to two hours is required to allow the muscle to relax. During this relaxation period the "base line" falls exponentially and eventually levels out, at which point dose response data may be generated. This relaxation procedure was also required between dose response curves to allow recovery of the pharmacological response. This preparation yielded cumulative dose response curves to norepinephrine without any necessity to block norepinephrine metabolism. 75 This is presumably due to a number of factors. It was noted that in the sympathetic nervous system reuptake and not enzymatic destruction; is the main mechanism for terminating the biological action of circulat- ing (Axelrod at al., 1959) or locally released norepinephrine (Rosell et al., 1963). Norepinephrine reuptake and enzymatic destruction are both relatively slow processes and the capacity of the reuptake process is limited by the small amount of adrenergic tissue present. Thus it appears reasonable that the small amount of tissue present (> 0.5 gm) was unable to significantly reduce the level of norepinephrine in the relatively large bath (30 m1) over the time periods of these experiments. Figure 6.2 shows the response of this preparation to norepinephrine after the two hour relaxation period. The response commences at about 10.-6 M norepinephrine, is maximal at about 10.4 M and is a typical sigmoidal log-dose response curve.r we next tested the effects of‘ phentolamine and dibenamine which are respectively competitive equilibrium and competitive non-equilibrium antagonists to the a adrenergic receptor (Ariene, 1971). Phentolamine shifted the dose response curve to the right about one log unit, the ascending portion of the dose response curves were essentially parallel, and a maximal 3 M norepinephrine. This is the contraction was obtained at about 107 theoretically expected behavior for a simple competitive antagonist. In contrast the action 0f dibenamine was to reduce the maximal height of the dose response curve and the amount of this inhibition depended on both the concentration of the dibenamine and the time for 6 M dibenamine which the tissue was exposed to the drug. Exposure to 10- resulted in a small shift to the right in the dose response curve and a decrease in the maximal response, suggesting that dibenamine had acted to irreversibly inhibit a number of the receptors. If the 76 concentration of dibenamine was increased to 10.5 M the response of the tissue to norepinephrine was completely obliterated and was not recovered after washing. This action of dibenamine was not due to any interference with the contractile mechanism because this tissue still responded fully to histamine. Thus dibenamine interacted irreversibly with a tissue component which resulted in the inhibition of the tissue response to norepinephrine but not to histamine, indicating a degree of selectivity in the action of dibenamine° Figures 6.3 and 6.4 demonstrate the presence in these tissue prepa- rations of histamine receptors and their relative selectivity. At concentrations above 10.6 M histamine elicits a typical sigmoidal dose response curve in these preparations. This action of histamine is partially inhibited by phentolamine but is extremely sensitive to pyrilamine, an antihistaminic drug. Though the interaction between pyrilamine and the histaminergic receptor is usually considered to be equilibrium in nature (Ariens et al., 1964) the data in Figure 6.4 show no tendency for histamine to overcome the action of pyrilamine. This effect might be observable at lower concentrations of pyrilamine, but this was not tested. Pyrilamine had no tendency to interfere with the actions of norepinephrine as shown in Figure 6.30 CONCLUSIONS Norepinephrine and histamine elicited contractile responses in guinea pig aortic strips at concentrations of about 10.5 M. These responses were sigmoidally dependent on dose and were selectively blocked by agents considered to act directly at the binding sites of these drugs. There was minimal cross inhibition between the adrenergic and histaminergic blockers, supporting the concept of specific 77 receptors for these drugs and also indicating that these blockers did not affect the contractile mechanism. The patterns of inhibition by the adrenergic blockers are consistent with the patterns predicted by Michaelis-Menten kinetics, i.e., parallel shifts and simple competitive blockade produced by phentolamine and irreversible (competitive non- equilibrium) blockade by dibenamine. REFERENCES Ahlquist, R. P.: A study of the adrenotropic receptors. Am. J. Phys. 153:586, 1948. Ariens, E. J.: Agonists and antagonists. In Drug Design. Ed. by E. J. Ariens. Academic Press, New York, 1971, p. 162. Ariens, E. J., Simonis, A. M. and van Rossum, J. M.: Drug receptor interaction. In Molecular Pharmacology. Ed. by E. J. Ariens. Academic Press, New York, 1964, p. 199. Axelrod, J., Weil-Malherbe, H. and Tomchick, R.: The physiological disposition of 3H epinephrine and its metabolic metanephrine. J. Pharm. Exp. Ther. 127:251, 1959. Black, J. W. and Stephenson, J. S.: Pharmacology of a new adrenergic 8 receptor blocking compound (nethalide). Lancet 2:311, 1962. Black, J. W., Crowther, A. F., Shanks, R. G. and Dornhorst, A. C.: A new adrenergic beta-receptor antagonist. Lancet 2:1080, 1964. Dale, H. H.: On some physiological actions of ergot. J. Physiol. 34: 163, 1906. Furchgott, R. F.: The receptors for epinephrine and norepinephrine (adrenergic receptors). Pharm. Rev. 11:429, 1959. Innes, I. R. and Nickerson, M.: Sympathomimetic drugs. In The Pharmaco- logical Basis of Therapeutics. Ed° by L. S. Goodman and A. Gilman. Macmillan Co., New York, 1970, p. 478. Nickerson, M. and Goodman, L. S.: Pharmacological properties of a new adrenergic blocking agent: N.N. dibenzyl B chloroethyl amine (dibenamine). J. Pharm. Exp. Ther. 89:16 7, 1947. Nickerson, M. and Hollenberg, N. K.: Blockade of a adrenergic receptors. In Physiological Pharmacology. Ed. by W. S. Rost and F. G. Hofman. Academic Press, New York, 1967, p. 243. 78 Powell, C. E. and Slater, I. H.: Blocking of inhibitory adrenergic receptors by a dichloro analog of isoproterenol. J. Pharm. Exp. Ther. 122:480, 1958. Rosell, S., Kopin, I. J. and Axelrod, J.: Fate of 3H noradrenaline in skeletal muscle before and following sympathetic stimulation. Am. J. Phys. 205:317, 1963. Lands, A. M.: The pharmacological activity of epinephrine and related dihydroxy phenylethyl alkyl amines. Pharm. Rev. 1:279, 1949. Lands, A. M.: Sympathetic receptor action. Am. J. Phys. 169:11, 1952. Sutherland, E. W. and Rall, T. W.: The relation of adenosine 3', 5'- phosphate and phosphorylase to the action of catecholamines and other hormones. Pharm. Rev. 12:265, 1960. 79 .mousdfia e>am pom woumofivaa mm mafiamawnfiv z Ioa no oloa ou vomoexo mes mammfiu onu can? vocmmuno uncommon oafiuomuuaoo one Bonn moaoufio vmmmouo one .m«voa wcwnumn one a“ wonsaoaa mos oeaamaoucond z oloa sons uncommon one oumoavaa moaoufio nfiaom one .moauufio ammo one an woumoawcfi ma muouofiaaafia ca ocfiunmoawdouom ou uncommon maauomuuaou may .moueafia m pom oumunfiaflouo cu vmaoaam new mews Hmuaouwuoz ecu so woumuwvcw oawusmoefiamuoo mo maofiumuuoooaou one on vomomxo mos dauum ofiuuom wad meafisw 4 .2 oloa mafiamHOuaond mam z mica .oIOH mawamconwv mo ouaommum one c« was oaoam .ocaundmnfiao ou muuom wed meadow mo mmaommom .N.e MMDUHM .aoummm sump compo one no coaumucomoumou aflumaonom 4 woman» you coaumumeoua gums compo .eauum owuuom mam .H.o mmoon 80 .WZOU .....me2 00.. IV — .Ie\wv nIO— mZ_<<(Zwm_D+ @\ @\ \ a... 3 \ << lop mZ_<<<._O.—ZwId+ \fi/W . e\ m. 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Von Sole wZ_<<<._OpZmId + ‘ m2:2<»m_r Too— .iolO. sewage—2:28 co iolO— 0550.220 *0 06:29.0 of S pro coo—(6580.2... 2 oto< a... 00:30 .0 02590: .v .0 .0: W W NI ESNOdSBU .u20u .3302 000 I v ... L . ..v z i \O \ 0w :8 o W_Z.<<<20h2m1m + \O \ x a. .. e % \ \K \ EwaOZ iono. mZ_<<<:m>n_ + \ '00. $ \ .2 olo. oEEo ole. 0560...: *0 3:330 o... E van oco_< .octcdocfiotoz o. 125.... to z 0104‘ 03 00:50 *0 oncoanoa 6.0.0.”— WW NI SSNOdSBU CHAPTER VII DIURETICS OBJECTIVES The objective of these experiments was to demonstrate the effects of diuretics with different mechanisms of action on urine formation in rabbits. INTRODUCTION The kidney is the primary organ involved in salt and water homeo- stasis and drugs which modify this function and produce a loss of sodium and water are called diuretics (Pitts, 1958). To understand the mechanism by which diuresis is produced requires a knowledge of the physiology and biochemistry of the kidney. Urine formation begins with formation of a protein-free ultra- filtrate of the blood in Bowman's capsule. This filtrate is physio- logically outside the body and the nephron reabsorbs and adds substances to the filtrate. The final product of these reactions is urine, which may be considered the sum of glomerular filtration, tubular reabsorp- tion and tubular secretion (deSteven, 1963). Filtration, which occurs in Bowman's capsule, is the result of hydrostatic and osmotic pressure differences across the glomerular membrane, the flow of water and solutes being passive in nature (Schanker, 1962). This was shown by walker-at a1. (1941), who observed that the concentration of the electrolytes and organic crystalloids in 83 84 the tubular fluids are identical to their concentration in the aqueous phase of plasma. Wesson (1957) observed that there is a regulatory mechanism that maintains glomerulo-tubular balance so that a rise or fall in glomerular filtration rate is associated with a proportional change in tubular sodium reabsorption. Therefore an increase in glomerular filtration rate alone will not produce a diuresis (Goldberg, 1967). The filtrate formed in Bowman's capsule is partially reabsorbed by the nephron. The filterable solutes of the plasma, i.e., electro- lytes, sugar and amino acids, are reabsorbed in different ways. In the case of reabsorption of electrolytes, the most prominent is the reabsorption of sodium ion. Windhager and Giebisch (1961) showed that sodium is actively reabsorbed from the tubular lumen. It is thought that this active transport is established by the sodium pump which moves sodium ions out of the cells into the blood. Thus a potential site of action of diuretics may be at the transport sites for sodium ions. The site of sodium reabsorption was suggested by Gottschalk (1963), who postulated that sodium reabsorption takes place in the proximal tubule. This reabsorption is followed by the passive move- ment of chloride ions in response to the electrical gradient. Gottschalk further noted that the reabsorption of sodium in the ascending limb of Henle's loop, which is impermeable to water, results in hypertonicity of the medullary interstitium. This osmotic gradient results in the extraction of water from the adjacent'descending limb or, in the presence of antidiuretic hormone, from the collecting duct. In the absence of antidiuretic hormone, solute—free water from the ascending limb will move out of the body without reabsorption at the 85 collecting duct, which has become impermeable to water. A drug which inhibits sodium reabsorption in the proximal tubule will increase this solute-free water and a drug which inhibits sodium reabsorption in the ascending limb of Henle's loop will decrease solute—free water (Goldberg et al., 1964). Bicarbonate ions are reabsorbed by a mechanism which involves the secretion of hydrogen ions coupled with sodium reabsorption (Pitts, 1958). This mechanism is dependent upon the enzyme carbonic anhydrase present in the tubular cell and in the brush border of the tubular epithelium (Rector et aZ., 1965). The inhibition of carbonic anhydrase has marked effects on sodium and bicarbonate reabsorption in the proximal tubule (Goldberg, 1971) and results in diuresis. In the distal tubule there is a cation exchange mechanism which involves sodium reabsorption linked to the secretion of potassium or hydrogen ions (Giebisch and Windhager, 1964). This mechanism is facilitated by the hormone aldosterone. Thus aldosterone inhibitors will have an effect on this mechanism. In the experiments presented below, the effects of several diuretics with different modes of action are demonstrated. Further, since these diuretics have different modes of action, it was possible to demonstrate these actions by their sequential administration to a single animal. MATERIALS AND METHODS Male white New Zealand rabbits, 4-5 kg body weight, were used throughout. Anesthesia was produced by the intraperitoneal administra- tion of 0.6 mllkg Dial-urethane one hour before surgery. When surgical anesthesia was attained the animal was placed supine on an operating table and the neck and abdominal areas were clipped. Immediately 86 before surgery 0.5 ml of Dial-urethane was given intraperitoneally and this was repeated as required during surgery. EKG leads were inserted subcutaneously on each limb and connected to a Grass polygraph. The skin and the fascia on the medial aspect of the neck were opened, the trachea was located and a tracheal tube was inserted. The carotid artery was located, separated from adjacent nerves and tissues and then cannulated and connected to a Statham pressure transducer. The tube and the transducer were filled with heparinized saline. Both blood pressure and EKG were recorded on a Grass polygraph. The jugular vein was located and cannulated with a polyvinyl cannula which was connected to an infusion pump. Normal saline (0.9%) was infused through the jugular vein at the rate of 1 m1/ minute, and this infusion was maintained throughout the experiment. Then, a 3 cm incision was made at the mediocaudal aspect of the abdominal wall. The bladder was located and withdrawn from the abdomen. After emptying the bladder, an opening was made at the tip of the bladder. A fine funnel was inserted into the opening and connected to a 45 cm polyvinyl tube. The end of this tube was inserted into a glass cannula which had an opening of about 0.5 cm. This size of opening allowed the formation of large drops of urine. Urine was allowed to drop from this cannula as it was formed onto a Grass force displacement transducer and thus each individual drop was recorded on the Grass polygraph; Urine flow rates were allowed to stabilize at about 2-3 drops per minute prior to the administration of drugs. This flow rate was taken as the base line rate. All drugs except B.A.L. were administered intravenously at 15 minute intervals and the mean number of drops per 87 minute over five minute intervals was calculated from the drop rate record. B.A.L., which was in an oily base, was administered by intra- muscular injection. Unless otherwise stated all experiments were repeated at least four times and data presented are the means of these four estimations. RESULTS AND DISCUSSION The purpose of the constant saline infusion was to maintain body fluid content during diuresis caused by the drugs, though saline infusion itself depresses the fractional reabsorption of sodium in the proximal tubule (Dirks et al., 1965) and causes a mild diuresis. The rate of urine flow caused by this infusion was taken as the base line for all the experiments. Preliminary experiments were performed with glucose, which pro- duces a rapid transient osmotic diuresis, to test the sensitivity of the system. As shown in Figure 7.2, 20 ml of 10% glucose intravenously produced a marked increase in urine flow. Experiments were then commenced with a number of diuretic agents following the sequence suggested by Dr. J. B. Hook (1972). Figure 7.3 is a demonstration of mercurial diuresis and the various factors affecting it. Chloromerodrin 10 mg/kg was administered i.v. Initially this drug produced a drop in the rate of urine flow and a transient drop in blood pressure immediately after its administra- tion (Figure 7.1). Within the next five minutes, however, the rate of urine flow increased to about twice the base line level and remained at this level for fifteen minutes. At the end of this fifteen minute period 10 ml 0.2% HCl was injected i.v. An immediate increase in the rate of urine flow was noted. This increase continued until the mercurial 88 diuresis was inhibited by the administration of 75 mg/kg B.A.L. intra- muscularly. This drug produced essentially immediate reversal of the chloromerodrin-RC1 diuresis and after 15 minutes urine flow had dropped below control levels. As far as the mechanism of action of mercurial diuretics is con- cerned, it has been suggested that organomercurials release mercuric ions at a rate dependent on the pH of the urine and these ions in turn combine with.cysteine (Mudge and Weiner, 1958). Mudge and Weiner found that mercuric cysteine itself is a potent diuretic. It was suggested that the mechanism of action of mercurial diuretics probably involves a firm attachment of mercury to sulfhydryl groups of renal enzymes involved in the generation of energy for sodium transport or to the sodium carrier system itself (Cafruny, 1968). The potentiation of mercury diuresis with HCl was explained by the lability of the carbon mercury bond in acidic media (Ethridge et aZ., 1936; Grossman et aZ., 1951), i.e., the rate of degradation of the organomercurial is increased in acidic urine. B.A.L. or dimercaprol was reported by Peters et a2. (1945) and Waters and Stock (1945) to be an effective antidote in certain types of heavy metal poisoning. It does this by forming a poorly dissociable chelate with many heavy metals. Its ability to inhibit mercurial diuresis was first demonstrated by Earle and Berliner (1947) and later by Farah at al. (1952). B.A.L. presumably acts by chelating mercury ions in the kidney and preventing their interaction with the mercurial diuretic receptors. Figure 7.4 shows a sequential response to three diuretics which have been classified as weak, moderately effective and strong diuretics (Goldberg, 1967). Mannitol is considered a weak diuretic which causes 89 diuresis due to an increase in osmotic pressure in the tubular lumen. In these experiments mannitol produced a ten fold increase in urine flow above base line levels and this effect was obtained in animals previously treated with the mercurials and B.A.L. Hydrochlorothiazide is considered a moderately effective diuretic. It is a typical member of the thiazide group which induces natriuresis and chloruresis by a mechanism analogous to that of chloromerodrin (Pitts et al., 1958). It also induces kaliuresis and produces an alkaline urine with increased bicarbonate levels and was thought to have the properties of a carbonic anhydrase inhibitor (Beyer et aZ., 1957; Pitts et al., 1958). Its sites of action appear to be in the proximal tubule (Beyer at al., 1957; Heinemann et al., 1959) and in the distal tubule (Heinemann et al., 1959; Cafruny and Ross, 1962). The actions of chlorothiazide and the mercurial diuretics are additive and it was considered that both may interfere with different enzymatic reactions supplying energy to a single reabsorptive mechanism (Pitts et aZ., 1958). Ethacrynic acid, which is considered a potent diuretic,also produces natriuresis and chloruresis (Pitts et al., 1958). It also increases excretion of potassium and abolishes renal concentrating ability (Earley and Friedler, 1964). Earley and Friedler (1964) also noted that the natriuretic effect of ethacrynic acid alone is 3-4 times as great as that observed with chlorothiazide. Ethacrynic acid may have multiple sites of action. These sites of action were thought to be in the ascending limb of Henle's loop (Goldberg at al., 1964; Earley and Friedler, 1964; Beyer at aZ., 1965) and possibly in both the proximal (Beer et al., 1964; Komorn and Cafruny, 1964; Beyer et al., 1965; Gussin and Cafruny, 1966) and 90 distal tubule (Cannon et al., 1963; Baer et al., 1964; Beyer et al., 1965). Komorn and Cafruny (1964) suggested that ethacrynic acid may react with sulfhydryl groups of renal cells of proximal tubules, distal convoluted tubules and the medullary collecting ducts. More recently Gussin and Cafruny (1966) have suggested that there may be receptors in the proximal tubule, ascending limb of Henle's loop and distal tubules which react with ethacrynic acid. SUMMARY The diuretic effects of chloromerodrin, mannitol, hydrochloro- thiazide and ethacrynic acid were demonstrated in a single animal by the sequential injection of these drugs. Hydrochloric acid poten- tiated the action of chloromerodrin, whereas B.A.L. inhibited its diuretic action. The sequential administration of mannitol, hydro- chlorothiazide and ethacrynic acid produced progressive increases in the rate of urine flow. REFERENCES Baer, J. E., Jones, C. B., Michaelson, J. K., Russo, H. F. and Beyer, K. H.: Effect of ethacrynic acid on urine concentration. Fed. Proc. 23:438, 1964. Beyer, K. H., Baer, J. B., Michaelson, J. K. and Russo, H. F.:' Reno- tropic characteristics of ethacrynic acid: a phenoxyacetic saluretic agent. J. Pharm. Exp.T her. 147:1, 1965. Beyer, K. H., Baer, J. H., Russo, H. F. and Haimbach, A.: Chloro- thiazide (6 chloro-7 sulfamiyl- 1,2,4,- Benzothiadiazine 1,1- Dioxide): the enhancement of sodium chloride excretion. Fed. Proc. 16:282, 1957. Cafruny, E. J.: The site and mechanism of action of mercurial diuresis. Pharm. Rev. 20:89, 1968. Cafruny, E. J. and Ross, C.: Involvement of the distal tubule in diuresis produced by benzothiadiazine. J. Pharm. Exp. Ther. 137:324, 1962. 91 Cannon, P. J., Heinemann, H. 0., Stason, W. B. and Laragh, J. H.: Ethacrynic acid, effectiveness and mode of diuretic action in man. Circulation 31:5, 1965. Dirks, J. H., Cirksena, W. J. and Berliner, R. W.: The effect of saline infusion on sodium reabsorption by the proximal tubule of the dog. J. Clin. Invest. 44:1160, 1965. Earley,D. P. and Berliner, R. W.: Effect of 2.3 dimercaptopropanol on diuresis. Am. J. Phys. 151:215, 1947. Earley, L. E. and Friedler, R. M.: Renal tubular effects of ethacrynic acid. J. Clin. Invest. 43:1498, 1964. Ethridge, C. B., Myers, D. W. and Fulton, M. N.: Modifying effect of various inorganic salts on the diuretic action of salyrgan. Arch. Int. Med. 57:714, 1936. Farah, A., Cobbey, T. S. Jr. and Mook, W.: Renal action of mercurial diuretics as affected by sodium load. J. Pharm. Exp. Ther. 104:31, 1952. Giebisch, E. E. and Windhager, G.: Renal tubular transfer of sodium, chloride and potassium. Am. J. Med. 36:643, 1964. Goldberg, M.: The physiology and pathOphysiology of diuretic agents. In Mbdern Trend in Pharmacology and Therapeutics. Ed. by E. Fulton. Butterworths, London, 1967, p. 41. Goldberg, M.: Renal tubular sites of action of diuretics. In Renal Pharmacology. Ed. by J. W. Fisher and E. J. Cafruny. Appleton Century Crofts, New York, 1971, p. 99. Goldberg, M., McCurdy, D. K., Foltz, E. L. and Bluemle, L. W. Jr.: Effect of ethacrynic acid (a new saluretic agent) on renal diluting and concentrating mechanism: evidence for site of action in the loop of Henle. J. Clin. Invest. 43:201, 1964. Gottschalk, C. W.: Renal tubular function: lesson from micropuncture. Harvey lecture Series 58:99, 1963. Grossman, J., Weston, R. E., Lehman, R. A., Halperin, J. P., Ullmann, T. B., and Leiter, L.: Urinary and fecal excretion of mercury in man following administration of mercurial diuretics. J. Clin. Invest. 30:1208, 1951. Gussin, R. Z. and Cafruny, E. J.: Renal sites of action of ethacrynic acid. J. Pharm. Exp. Ther. 153:148, 1966. Heinemann, H. 0., Demartini, F. E. and Laragh, J. H.: The effect of chlorothiazide on renal excretion of electrolytes and free water. Am. J. Med. 26:853, 1959. Hook, J. B.: Personal communication. 92 Komorn, R. M. and Cafruny, E. J.: Ethacrynic acid: diuretic property coupled to reaction with sulfhydryl groups of renal cells. Science 143:133, 1964. Mudge, G. H. and Weiner, I. M.: The mechanism of action of mercurial and xanthine diuretics. Ann. N.Y. Acad. Sci. 71:344, 1958. Peters, R. A., Stockon, L. A. and Thompson, R. A. 8.: British anti lewisite. Nature 156:616, 1945. Pitts, R. F.: Some reflections on mechanism of action of diuretics. Am. J. Med. 24:745. 1958. Pitts, R. F., Kruck, F., Lozano, R., Taylor, D. W., Heidenreich, O. P. A. and Kessler, R. H.: Studies on the mechanism of diuretic action of chlorothiazide. J. Pharm. Exp. Ther. 123: 89, 1958. Rector, F. C., Carter, N. W. and Seldin, D. W.: The mechanism of bicarbonate reabsorption in the proximal tubules of the kidney. J. Clin. Invest. 44:278, 1965. Schanker, L. S.: Passage of drugs across body membrane. Pharm. Rev. 14:501, 1962. deSteven, G.: Diuretics. Chemistry and Pharmacology. Academic Press, New York, 1963, p. 2. Waters, L. L. and Stock, C.: British anti lewisite. Science 102:601, 1945. Walker, A. M., Bott, P. A., Oliver, J. and McDowell, M. C.: The collec— tion and analysis of fluid from single nephron of the mammalian kidney. Am. J. Phys. 134:580, 1941. Wesson, L. G. Jr.: Glomerular and tubular factors in the renal excre- tion of sodium chloride. Medicine, Baltimore, 36:281, 1957. Windhager, G. and Giebisch, E. B.: Micropuncture study of renal tubular transfer of sodium chloride in the rat. Am. J. Phys. 200:581, 1961. 93 FIGURE 7.1. Initial responses of blood pressure (B.P.) and urine flow (U.P.) to chloromerodrin. The upper tracing records urine flow in drops per minute and the lower tracing records blood pressure. At the point indicated by the broken arrow chloromerodrin 10 mg/kg was injected intravenously. FIGURE 7.2. Response of urine flow to intravenous injection of 20 m1 of 10% glucose. Urine flow in drops per minute is plotted on the vertical axis against time in minutes on the horizontal axis. At 15 minutes 20 m1 of 10% glucose was injected intravenously and urine flow recorded as before. 94 FIG. 7.1. Initial Responses of Blood Pressure (8.P. ) and Urine Flow (U.P.) to Chloromerodrin. -+——im$——+. ————— 41m22:_+ ——————— a ——————— v . u! . i__ . u m - ...r.. -« m-n. _l U.F. T I CHLOROMERODRIN FIG. 7.2. Response of Urine Flow to Intravenous Injection of 20 ml of 10% Glucose 10- w ... 3 Z i or w CL (I) 5—1 CL 0 L... a: C) l l l<__10 °/. GLUCOSE (20ml) U I O 15 30 MINUTES 95 .mousefia huufinu as been oflaxuumnuo mo wx\wa CA was mouseaa coouMfim um mamsoao>mnuaw onenmfinuouoanuouvmn wx\ma OH .oaau ouou voumoavcfi um .HOufiocma NOH mo as 0H «0 coaumuumecfiavm msooo>muucfi one means oo>uomno moumu 30am one“: oau msonm mead ofiaom may .A.<.mv meow oeamuumnuo was A.N.e.mv meanmaauouoaeooueae .A.zzuomno one moumofivaa mafia veaom may .maumanomsamuuaw wououweaeeoo ..A.¢.m mo mxxwe mm mo mouoefia on no one H02 Nm.o mo H8 OH an mousofia cm on nosoaaow .mamooco>ouuofi wouoohefi mos w2\we OH A.2.uv oeuvouoa Iouoazo nuances ma u< .AN.N ouswamv %Hm=OH>oum we mean HmueoNfiuos onu so mafiu umcwowo mews Hmoauuo> one no wouuoaa ma ounces use meouv aw scam ocean .omeoamou mean no .A.<.m one How mo muuommo one one afluoouoe iouoano Ou uncommon caucusev one .m.n mmoon 96 mm»32_<< nV Om n— P — a .ZZ<2| .NHI .<.m.|v ‘ mwh32_<< Oo nv lrll . .r .Eu< oExtoofw tco orig—20.5235»: ..ozccoi 0. 33003: 3.0.5.0 0...... 6.50: '9 .0333. 25 co ..(m oco _UI *0 233m of was £50.56 lotoIU o. uncommom 3.955 of 5.5.0: 3 1n NIW 83d SdOHO CHAPTER VIII CENTRALLY ACTING AGENTS: THE NARCOTIC ANALGESICS OBJECTIVES The objectives of these experiments were to demonstrate the hot plate method of testing analgesic drugs and to use it to compare a number of analgesic agents. INTRODUCTION Analgesia may be defined as the state of being relatively insensible to pain. The analgesic drugs presently used in medicine are divided into two major groups, the narcotic and the non-narcotic analgesics. The narcotic analgesics are the most potent and effective and it is with this group that these experiments are primarily concerned. The narcotic analgesics include a large number of drugs which are structurally related to morphine and share its analgesic and narcotic properties and its potential to produce dependence (Bands at aZ., 1965). They have been designated "morphine surrogates" (Way and Adler, 1960). Morphine is used as a prototype for all narcotic analgesics and it is considered the standard against which other analgesic drugs are compared (Martin, 1965). It is believed that the analgesic action of the narcotic analgesics is comprised of three factors, i.e., elevation of the "pain threshold", reduction of the "reaction to pain" and the promotion of somnolence. The "reactive" component is considered to be the most important factor, the subject still perceiving the pain but no longer being affected by it (WOlff at aZ., 1940). 97 98 Though the narcotic analgesics are effective and potent drugs in the reduction of pain, their ability to produce dependence is a severe liability. This problem has resulted in a constant search for new analgesic drugs with no tendency to produce dependence. This search is carried out using animal screening tests; the mouse hot plate method, introduced by Woolfe and MacDonald (1944), is one of these. This method is only effective for the narcotic analgesics and is not effective for the non-narcotic analgesics. In the experiments reported here the relative potency of several commonly used narcotic analgesic drugs was investigated. One non- narcotic analgesic was tested and some aspects of the antagonism and potentiation of analgesia were also examined. MATERIALS AND METHODS The hot plate consists of a thick capper plate held at 55° C by a vapor from a boiling acetone bath. Any excess vapor is trapped in a condenser side arm and returned to the boiling vessel. The experi- ments were carried out by dividing the mice (Swiss Webster mice) into control and test groups, five to ten mice in each group. Mice from each group were injected subcutaneously with 0.2 m1 of normal saline (control) or 0.2 m1 of normal saline containing the test drug. Fifteen minutes were allowed for the absorption and distribution of the drug. In the testing procedure each mouse was placed on the hot plate and a stop watch started. The animal's earliest reaction was to sit on its hind legs and lick or blow on its forepaws. A few seconds later, the animal kicked with its hind paws or attempted to jump out of the cylinder. At this point the stop watch was stopped and the animal removed from the apparatus. Only hind paw reactions were used for 99 evaluation since normal mice often groom their forepaws. Normal mice usually reacted in five to ten seconds. Any changes in the time to reaction produced by drugs were calculated as follows: mean time, drug_treated - mean time, control mean time, control X 100 The increase in reaction times of morphine with the doses of 5 mg/kg, 10 mg/kg and 20 mg/kg were examined so that the dose response curve of morphine was obtained. Similar experiments were carried out with meperidine (15 mg/kg, 30 mg/kg, 160 mg/kg), codeine (40 mg/kg, 80 mg/kg, 160 mg/kg) and salicylic acid (50 mg/kg, 100 mg/kg, 200 mg/kg). Antagonism of the action of morphine by naloxone was tested by calculat— ing the increased reaction time after morphine (20 mg/kg), naloxone (20 mg/kg) and similar doses of naloxone plus morphine. Similar tests were made for meperidine (60 mg/kg) and naloxone. The interaction between chlorpromazine (5 mg/kg) and subanalgesic dose of morphine (5 mg/kg) was tested similarly. RESULTS AND DISCUSSION Figure 8.1 shows the percent increase in the time to reaction on the plate obtained with increasing doses of morphine, meperidine, codeine and salicylic acid. Morphine, which produced analgesia at considerably lower doses than the other drugs, is considered the most potent. Meperidine, which produced an approximately equivalent effect at three times the dose, may be considered about one third as potent as morphine. Similarly, codeine is about one tenth as potent as morphine, but, at least on the mouse hot plate test, produced approxi- mately the same maximal effect as morphine. Wallenstein et al. (1961) have suggested that in man codeine is approximately one fifteenth to 100 one tenth as potent an analgesic as morphine. In this experiment, however, there was a tendency for the higher doses of codeine to pro- duce convulsions. Similar results have been observed in patients receiving very large doses of meperidine who also tend to show con- vulsive phenomena (Isbell and White, 1953). Salicylate was ineffective as an analgesic in this test system and did not produce any significant increase in the reaction time on the hot plate. The dose response curves for morphine and its derivatives are approximately parallel and reach the same maximum. This,as well as the structural relationship between these drugs, suggests that they act on the same type of receptor. If these drugs all act on the receptors, a drug which antagonizes the action of one should antagonize the action of all. Figure 8.2 shows the action of naloxone. Morphine 20 mg/kg was administered to mice and the percent increase in reaction time tested. Naloxone (20 mg/kg) was then administered and the percent increase in reaction time retested. The same procedure was followed with meperidine (60 mg/kg) and the effect of naloxone alone was also tested. The results show that naloxone has little analgesic action of its own but is able to completely antagonize the action of morphine and meperidine. Similarly, it has been shown in humans that naloxone, even at low doses, abolishes the euphoric and analgesic effects of heroin within a half to two minutes after intravenous injection (Fink et al., 1968). An antagonism between morphine and narcotic antagonists by competi- tion for the same receptor sites has been suggested by investigators such as Seevers and Woods (1953) and Seevers (1954). They postulated a competitive displacement of morphine by nalorphine on the surface of the axons or cell bodies of the internuncial neurons. Nalorphine is a 101 narcotic antagonist like naloxone and it should be noted here that naloxone is five to eight times as effective as nalorphine in antagoniz- ing narcotic analgesics in animals (Sadove et aZ., 1963; Jasinki et al., 1967). Other evidence which suggests that nalorphine may antagonize the effect of opiates by competing at common receptor sites was reported by Cox and weinstock (1964) and Grumbach and Chernov (1965). Subanalgesic doses of morphine are reportedly potentiated by ataractic drugs and Figure 8.3 shows such an experiment. Morphine or chlorpromazine alone produced minimal changes in the reaction time of the mice. However, in combination they produced a marked increase. This supra-additive effect of two drugs administered together is called potentiation. CONCLUSIONS The experiments show that morphine, meperidine and codeine pro- duced approximately equivalent increases in time to reaction on the hot plate. However, three times as much meperidine and ten times as much codeine as morphine was required to produce this effect. These ratios indicate the relative analgesic potencies of these drugs. The analgesic actions of morphine and meperidine were obliterated by naloxone, suggesting that these drugs act at the same receptor site. Chlorpromazine increased the percent increase in reaction time of a subanalgesic dose of morphine, i.e., it potentiated the action of morphine. REFERENCES Cox, B. M. and Weinstock, M.: Quantitative studies of the antagonism by nalorphine of some of the actions of morphine—like analgesic drugs. Brit. J. Pharm. 22:289, 1964. 102 Fink, M., Zaks, A., Sharoff, R., Mora, A., Bruner, A., Levit, S. and Freedman, A. M.: Naloxone in heroin dependence. Clin. Pharm. ‘Ther. 9:568, 1968. Grumbach, L. and Chernov, H. 1.: The analgesic effect of opiate-opiate antagonist combination in rat. J. Pharm. Exp. Ther. 149:385, 1965. Houde, R. W., Wallenstein, S. L. and Beaver, W.T.: Clinical measure- ment of pain. In Analgetics. Ed. by G. deSteven. Academic Press, New York, 1965, p. 76. Isbell, H. and White, W. M.: Clinical characteristics of addiction. A. J. Med. 14:558, 1953. Jasinki, D. R., Martin, W. R. and Haertzen, C. A.: The human pharmacol- ogy and abuse potential of N-Allylnoroxymorphone (Naloxone). J. Pharm. Exp. Ther. 157:420, 1967. Martin, W. R.: Strong analgesics. In Physiological Pharmacology. Ed. by W. S. Root and F. G. Hofmann. Academic Press, New York, 1963, p. 275. Sadove, M. S., Balagot, R. C., Hatano, S. and Jobgen, E. A.: Study of a narcotic antagonist N-Allyl-noroxymorphone. J. Am. Med. Ass. 183:666, 1963. Seevers, M. H.: Adaptation to narcotics. Fed. Proc. 13:672, 1954. Seevers, M. H. and Woods, L. A.: The phenomena of tolerance. Am. J. Med. 14:546, 1953. Wallenstein, S. L., Houde, R. W. and Bellville, J. W.: Relative potency and effectiveness of codeine and morphine. Fed. Proc. 20:311, 1961. 103 .wauo oedeuwaoo mo mooov wououwosw onu poems moaoufio vommouo one one ocwovoo noumm moaoueo noum one .oewofiuoeoa woumo oaeu eowuooou ow ommouoafi onu bone ooaoueu veaom osu .oswnmuoa mo momoo wouooevsa onu noumo oeau sowuooou ea oooonucfi uaoouoe one 3020 ooHouflu nomo one .oexo Houaoufiuon onu so wx\we ea ommmov wean umewowo memo Houauuo> one do nouuoae we oafiu eofiuomou ea oomouuce udoouom one .vaoo oeaeoaaom new oceoooo .osaoauoaoa .ooenauoa sues ouoae uon omooa o co oaHu eoeuooou we oooouoaa udoouom .H.m mmsoem 104 .23.. 0:328 9:. 36300 6533022 60.50qu E? Boa so: 382 m :o oEE. :ozomom E ommouofi ucoouoa ._ .m .05 oo— mmoo 82 00. on o. figll 0.u< 2:635. . lmvl l$lK k 0 0 3W”. NOllDVSU OBSVBUDNI 1N3383¢l wZazwsuZ ~51;in 105 FIGURE 8.2. Interactions among naloxone, morphine and meperidine on the mouse hot plate test. Percent increase in reaction time after: morphine 20 mg/kg (A), naloxone 20 mg/kg plus morphine 20 mg/kg (B), naloxone 20 mg/kg (C), meperidine 60 mg/kg (D), naloxone 20 mg/kg plus meperidine 60 mg/kg (E). FIGURE 8.3. Interaction between morphine and chlorpromazine on the mouse hot plate test. Percent increase in reaction time after: morphine 5 mg/kg (A), chlorpromazine 5 mg/kg plus morphine 5 mg/kg (B), chlorpromazine 5 mg/kg (C), morphine 5 mg/kg plus chlorpromazine 5 mg/kg (D). 106 sood I“ E .- 600... PE RC ENT INCREASED REACTION 200.1 o.I FIG. 8.2. Interaction among FIG. 8.3. Interaction between Naloxone, Morphine and Meperidine Morphine and Chlorpromazine on the Mouse HOt Plate Test. on the Mouse Hot Plate Test. CHAPTER IX CARDIAC DRUGS OBJECTIVES The objectives of these experiments were to demonstrate the come parative actions of epinephrine and isoproterenol on cardiac contractile force and coronary flow and to demonstrate the ability of procaine and quinidine to delay the onset of ouabain induced arrhythmias in guinea pig hearts. INTRODUCTION The mechanical, electrical and circulatory properties of cardiac tissues are among the properties of these tissues which are modified by drugs. In this section the actions of a number of drugs on these properties of cardiac tissues will be examined. Drugs which affect the contractile properties of heart muscle are referred to as having inotropic properties, and many of these drugs act on adrenergic receptors in the heart. As outlined previously (Chapter VI) adrenergic receptors are classified as either a or B receptors (Ahlquist, 1948, 1958), and it has been found that the cardiac adrenergic receptors are principally B receptors (Flemming and Hawkins, 1959; Furchgott, 1959). It was noted that B receptors are generally inhibitory (Ahlquist, 1948) except for those in the heart, which are stimulatory (Furchgott, 1959). Therefore, in the first series of the experiments reported here isoproterenol, which is a pure 107 108 8 agonist (Lands and Brown, 1967) was tested as an inotropic agent in the heart. The effects of epinephrine, which has both a and B stimula- tory properties (Lands and Brown, 1967) was also tested. The rate of flow in the coronary system is also of importance for the maintenance of cardiac function. If coronary flow is inadequate compared to the work load of the heart a relative hypoxia occurs, with damage to the cardiac tissues which is experienced as anginal attack by human patients (Harrison, 1970). Such attack may be precipitated by increased sympathetic nervous system activity and these agents may act either by increasing the work load of the heart or by directly constrict- ing the coronary vessels, which have recently been shown to contain both a and B.adrenergic receptors (Hirche, 1966; Isselhard, 1969). We therefore investigated the actions of epinephrine and isoproterenol on coronary flow rates in isolated guinea pig hearts (data presented in Figure 9.2) during the experiments which were presented in Figure 9.1. Normal cardiac function is also dependent on the correct coordina- tion of contractile events and this coordination is maintained by the conducting tissue of the heart (Hoffman and Cranefield, 1960). If the function of the conducting tissue is interfered with, disturbances of the rhythm of the cardiac cycle, known as arrhythmias, develop. Such arrhythmias may be relatively minor, consisting of a few extraventricular beats, or they may be so severe that the cardiac contraction is completely uncoordinated. A heart contracting in this way is said to be fibrillat- ing, and since a fibrillating ventricle does not pump blood, such events are usually terminal. In digitalis toxicity,ventricular fibrillation is the commonest cause of death (Moe and Farah, 1970). A number of drugs have been developed which act to reduce the tendency of cardiac tissues to develop arrhythmias and in the second series of experiments 109 the ability of quinidine sulfate (Sokolow, 1956) and procaine hydro- chloride (Burnstein, 1946) to delay the onset of arrhythmias was investi- gated. Arrhythmias were induced by the exposure of the perfused hearts to digitalis (Farah and Maresh, 1948; Moe and Mendez, 1951). The production of these arrhythmias may involve the inhibition by these glycosides of the (Na+-K+) ATPase of cardiac tissue (Akera et aZ., 1970). MATERIALS AND METHODS Guinea pigs were sacrificed by a sharp blow on the back of the head, their chests opened and their thoracic viscera exposed. Their hearts and lungs were excised immediately, the aorta being cut about 0.5 cm from the heart. The hearts were then allowed to beat in Krebs- Renaeleit (K.H.) solution (27.2 mM NaHCO 118.0 mM NaCl, 4.8 mM K01, 3’ 1.0 mM KHzPO 1.2 mM MgSO4 7 H O, 2.5 mM CaClz, 11.1 mM glucose) 4’ 2 until they emptied of blood. Then the hearts were attached to the Langendorff heart perfusion apparatus (Beckett, 1970) by bringing the aortas up over the cannula and tying them securely. The lungs were then dissected away. The Langendorff heart perfusion apparatus consisted of a reservoir and a thermostatically controlled heating jacket (35’ C). The system contained K.H. solution which was connected to the heart by a small cannula tied into the aorta above the semilunar valves. The height of the reservoir was adjusted so that it provided 75 mm Hg pressure (Beckett, 1970). The pulmonary artery was cannulated and the effluent allowed to drop on a Grass force displacement transducer (FT03) so that coronary flow was recorded as in the renal experiments (Chapter VII), A silk suture was attached to the apex of the heart and connected to a Grass displacement transducer so that the force of contraction could be recorded. 110 Dose-response curves to isoproterenol and epinephrine were made by injecting appropriate drug concentrations into the perfusion apparatus approximately 5 cm above the heart. The maximal increases in contractile force (in grams) and in coronary flow (drops per minute) were taken as the responses to isoproterenol and epinephrine. Dose response curves of cardiac contractile force and coronary flow to the two drugs were then plotted (Figures 9.1 and 9.2). Ouabain-induced arrhythmias were produced as follows. The reser- voir was filled with K.H. solution containing 1 X 10.6 M ouabain and the hearts perfused and EKG's recorded until an arrhythmia was observed. The criterion of arrhythmia in these experiments was the appearance of extrasystoles in the tracing records. The time after the commencement of ouabain perfusion for this event to occur was designated as the time to arrhythmia.r To study the action of the antiarrhythmic drugs, the reservoir was filled with K.H. solution containing 1 X 10-5 M 5 4 ouabain and l X 10- M quinidine sulfate or 1 X 10- M procaine hydro- chloride, and the times to arrhythmia were recorded as before. RESULTS AND DISCUSSION Figure 9.1 shows the changes in cardiac contractile force observed as the concentrations of epinephrine and isoproterenol in the perfusion media are increased. Isoproterenol is a potent drug, producing marked increases in contractile force at concentrations less than 10.8 and is maximally effective at 2 X 10“8 M. In contrast, 10'.7 M epinephrine was required to produce any‘observable increase in contractile force and concentrations higher than 10".6 M were required for maximal effect. Thus, isoproterenol appears to have a higher affinity for the cardiac adrenergic receptors than epinephrine, but in each case the maximal 111 response attained was approximately the same. Since isoproterenol is a pure 8 agonist these data are in agreement with other observations (Isselhard, 1969). However, more detailed experiments using selective a and 8 blockers such as dichloroisoproterenol, pronethalol and propranolol would be required to substantiate this point (Lands and Brown, 1967). Based on the assumption that the adrenergic receptors in the heart are primarily beta, the data show that epinephrine has beta agonist activity but that high concentrations of epinephrine are required to fully activate the cardiac adrenergic receptors. Thus the data agree with the data of Lands and Howard (1952) which showed that isoproterenol is a more potent inotropic agent than epinephrine. The data of Figure 9.1 may be interpreted in terms of the theories of Ariene et al. (Chapter III). On this basis, epinephrine would have full intrinsic activity at the B adrenergic receptor, since it is capable of producing a maximal response. However, its affinity for the adrenergic receptor is almost 100 fold less than that of isoproterenol, as evidenced by the parallel nature of the dose response curves. Thus, from these data at least, epinephrine appears to lack only affinity for the B adrenergic receptor and to be fully active on the receptor I when it does bind. The mechanism by which 8 receptor stimulation produces the ino- tropic response has been partially elucidated. The B adrenergic receptor system is now believed to be an integral part of the adenyl cyclase system (Sutherland et al., 1962), which is an enzyme system located in the cell membrane (Davoren and Sutherland, 1963). When appropriately stimulated adenyl cyclase catalyzes the hydrolyses of adenosine triphosphate (ATP) to an internal anhydride of adenosine 112 monOphosphate (AMP) with the release of pyrophosphate (Rall and Sutherland, 1958). This form of AMP is called 3'5' cyclic AMP and it is a relatively stable molecule whose breakdown is catalyzed by phospho- diesterase (Butcher and Sutherland, 1962). It now appears that most if not all of the effects of beta adrenergic activation are mediated by the formation of cyclic AMP, which mimics all the actions of beta receptor stimulation. The mechanism by which cyclic AMP produces-the inotropic response is not known. One of the first enzymatic changes to be linked to cyclic AMP was the activation of phosphorylase and it was in the search for the mechanism of phosphorylase activation that cyclic AMP was discovered (Sutherland et aZ., 1962). It appears that many of the actions of cyclic AMP are dependent on its ability to catalyze or stimulate the phosphorylation of cellular proteins. In activating phosphorylase cyclic AMP stimulates the phosphorylation of phosphorylase b kinase to an active form which in turn phosphorylates phosphorylase b to give rise to the active phospho-form of phosphorylase (phosphorylase a) (Krebs at al., 1966). Though the activation of phosphorylase apparently occurs too slowly to be causally with the inotropic response (¢ye, 1965), it appears that the basic mechanism of phosphorylation of tissue components is involved in the tissue response to adenyl cyclase. Currently, interest centers on cyclic AMP stimulated phosphorylation of a hypothetical membrane protein involved in the regulation of Ca++ accumulation by the sarcoplasmic reticulum (Sandow, 1965). Figure 9.2 shows the actions of epinephrine and isoproterenol on. coronary flow. In these experiments only the flow from the pulmonary artery was monitored, that from the posterior vena cava being allowed 113 to flow freely. Since the flow through the coronary system may drain through either the pulmonary artery or the posterior vena cava, only a fraction of the coronary flow was being monitored. Preliminary experiments established that the ratios of these two flows were approximately constant, both before and during inotropy. Thus all flow data presented here are those obtained from the pulmonary artery, which was taken to be a constant fraction of the total coronary flow. Figure 9.2 shows the isoproterenol produced marked increases in coronary flow, whereas epinephrine had little effect. These actions could be direct, due to an action of catecholamines on the adrenergic receptors of coronary blood vessels (Paratt, 1965) or.they might be indirect and secondary to the inotropic actions of the catecholamines. This is possible because products of myocardial metabolic activity and in particular lactic acid, act to maximally vasodilate coronary blood vessels (Lundholm, 1956). In comparing the data of Figure 9.1 and 9.2, the observation that maximal changes in contractile force were observed with little changes in coronary flow suggests that coronary vasodilatation due to these drugs is a direct action of these drugs and not secondary to changes in contractile force. As in the previous experiments isoproterenol was the most potent of the drugs and at the doses tested epinephrine produced no changes in coronary flow. These observations are consistent with the data of other investigators, which have demonstrated the presence of beta receptors in the coronary vascula- ture, which are associated with the vasodilatory response (Gaal et aZ., 1966). Figure 9.3 shows the effect of quinidine and procaine on the time to ouabain induced arrhythmias in isolated guinea pig hearts. Perfusion with ouabain alone produced arrhythmias in an average time of 13 114 minutes, and both procaine and quinidine delayed the onset of arrhythmias, to 21 and 43 minutes, respectively. The mechanism by which ouabain induces arrhythmias in the isolated Langendorff heart preparations probably involves inhibition of (Na+FK+) ATPase (ATP phosphohydrolase E 0 3.6.1.3.) (Skou, 1965). The cardiac glycosides and particularly ouabain are potent and highly specific inhibitors of this enzyme, which may be involved in many of their pharmacological effects (Tobin and Sen, 1970). By inhibiting the (Na+;Kf) ATPase or the sodium pump ouabain gives rise to increased intracellular Na+and decreased intracellular K+, as these ions flow down their concentration gradients (Post at aZ., 1960). These ionic changes result in a decreased membrane potential in the cardiac cells, which shifts the membrane potential closer to the value at which spontaneous depolarization occurs. In this way the cardiac glycosides render the heart more susceptible to ectopic foci, i.e., they increase the excitability of cardiac muscle and thus they increase tendency for arrhythmias to develop. In reversing arrhythmias, quinidine depresses the excitability of cardiac tissue by increasing its threshold to electrical stimuli. It has been shown in intact dog heart that the excitation threshold of atrial and ventricular muscle is increased and intraventricular con- duction is depressed by quinidine (wallace et aZ., 1966). Quinidine increases the refractory period as was shown in isolated rabbit atria by West and Amory (1960). The refractory period of the sine-auricular (S.A.) node is slightly prolonged in the presence of quinidine but the vagal actions of quinidine tend to produce the opposite result. Con- duction velocity also is decreased by quinidine (Vaughan Williams, 1958). The action of procaine amide is rather similar to that of 115 quinidine. It depresses excitability, automaticity and conduction velocity either in the atrial and ventricular tissue or in the S.A. and atrioventricular (A.V.) nodes (Woshe et al., 1953). Another well known action of cardiac glycosides is their positive inotropic effect. This effect is not dependent on catecholamine liberation or potentiation (Fawaz, 1967). The mechanism responsible for the positive inotropic effect of the cardiac glycosides is still the subject of investigation. Several proposals have been made and the most acceptable concerns their effects on the excitation-contraction coupling system. The link between the action potential and the contraction is apparently the release of free calcium ions, which then initiate muscle contraction. Digitalis alters the calcium bind- ing characteristic of cellular and intracellular membranes and improves the release of calcium ions (Lee and Klaus, 1971). Digitalis may alter the calcium binding characteristics by inhibiting (Na+FK+) ATPase which has been proposed as the primary inotropic receptor for digitalis (Akera et al., 1970). CONCLUSIONS Both isoproterenol and epinephrine inoreased cardiac contractile force but isoproterenol was markedly more potent than epinephrine. Since the dose response curves were parallel the apparent affinity of isoproterenol for cardiac adrenergic receptors is higher than that for epinephrine. However, the maximal responses to each drug were similar so both drugs appear to have the same intrinsic activity. Isoproterenol increased coronary flow rates whereas epinephrine produced no changes at the concentrations tested. Since cardiac con- tractile force was already maximal before isoproterenol produced any 116 changes in coronary flow, the changes in coronary flow did not appear to be secondary to the changes in contractile force. Ouabain induced arrhythmias in guinea pig hearts developed after a 13 minute infusion period with 10.6 M ouabain. In the presence of 10.6 M quinidine sulfate and 10”4 M procaine hydrochloride, 21 and 43 minutes respectively were required for these arrhythmias to develop. The data show that quinidine and procaine are able to delay the onset of ouabain induced arrhythmias. REFERENCES Ahlquist, R. P.: A study of the adrenotropic receptors. Am. J. Physiol. 153:586, 1948. Ahlquist, R. P.: Adrenergic drugs. In Pharmacology in Medicine. 2nd ed. Ed. by V. 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D.: Membrane ATPase as a participant in the active transport of sodium and potassium in the human erythrocyte. J. Biol. Chem. 235:1796. 1960. Rall, T. W. and Sutherland. E. W.: Mechanism of glycogenolytic action of epinephrine. Pharmacologist 1:41, 1959. Sandow, A.: Excitation-contraction coupling in skeletal muscle. Pharm. Rev. 17:265. 1965. Skou, J. C.: Enzymatic basis for active transport of Na+ and KI across cell membrane. Phys. Rev. 45:596. 1965. Sokolow, M. and Rall, R. E.: Factors influencing conversion of chronic atrial fibrillation with special reference to serum quinidine concentration. Circulation 14:568. 1956. Sutherland, E. W., Rall. T. W. and Menon, T.: Adenyl cyclase. 1. Distribution, preparation and properties. J. Biol. Chem. 237: 1220, 1962. Tobin, T. and Sen. A. K.: Stability and ligand sensitivity of (3H) ouabain binding to (Na+-K+) ATPase. Biochim. Biophys. Acta 198:120. 1970. Vaughan Williams. E. M.: The mode of action of quinidine on isolated rabbit atria interpreted from intracellular potential records. Brit. J. Pharm. Chem. 13:276. 1958. Wallace, A. G.. Cline, R. E., Scaly, W. C., Young. W. G. Jr. and Troyer, W. G. Jr.: ElectrOphysiologic effects of quinidine studies using chronically implanted electrode in awake dogs with and without cardiac denervation. Circulation Res. 19:960. 1966. West. T. C. and Amory. D. W.: Single fiber recording of the effect of quinidine at atrial pacemaker sites in the isolated right atrium of the rabbit. J. Pharm. Exp. Ther. 130:183, 1960. Wohse, H., Belford, J., Fastier, F. N. and Brooks. C. M.: Effect of procaine amide on excitability. refractoriness and conduction in the mammalian heart. J. Pharm. Exp. Ther. 107:135, 1953. 119 FIGURE 9.1. Comparative effects of epinephrine and isoproterenol on the contractile force of isolated perfused guinea pig hearts. Maximal increases in contractile force in grams were plotted against log concentration of isoproterenol (0-0) and epinephrine (.-.) . Each point represents the mean of three separate experiments 1 the standard errors of the means. 120 FlG.9.l.Comparative Effects of Epinephrine and Isoproterenol on the Contractile Force of Isolated Perfused Guinea Pig Hearts . 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