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Cardiovascular response to beta-hydroxy thujaplicin and gamma-thujaplicin. Moldowan, Mervin John 1970

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CARDIOVASCULAR RESPONSE TO BETA-HYDROXY THUJAPLICIN AND GAMMA-THUJAPLICIN by M. J . MOLDOWAN A thesis submitted l n p a r t i a l f u l f i l m e n t of the requirements for the degree of MASTER OF SCIENCE i n the D i v i s i o n of Pharmacology of the Faculty of Pharmaceutical Sciences accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1 9 7 0 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree tha permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of British Columbia Vancouver 8, Canada 1 CARDIOVASCULAR RESPONSE TO BETA-HYDROXY THUJAPLICIN AND GAMMA-THUJAPLICIN by M. J . MOLDOWAN ABSTRACT An Investigation was undertaken to determine the ef-fects of beta-hydroxy t h u j a p l i c i n and gamma-thujaplicin, used as the sodium s a l t s , on blood pressure and heart rate i n the r a t . Attempts were made to discover the s i t e s and modes of action of these two compounds, Gamma-thujaplicin 30mg/kg. produced either a vaso-pressor or a vasodepressor response i n anesthetized rats* The pressor response was usually more pronounced, than the vaso-depressor response. Tachycardia occurred with either blood pressure response. To determine whether the central nervous system was necessary f o r the vasopressor and tachycardiac response, pithed ra t s were used. In these preparations gamma-t h u j a p l i c i n produced only a f a l l i n blood pressure and a de-crease In heart rate. The e f f e c t of adrenergic blocking drugs on the response to gamma-thujaplicin was investigated. In the anesthetized ra t the vasopressor response was reduced s i g n i f i c a n t l y by both phenoxybenzamine and pronethalol; however, the heart rate was unaffected. In the pithed r a t the vasodepressor response pro-duced by gamma-thujaplicin was not affected by pronethalol; however, gamma-thujaplicin produced a s i g n i f i c a n t l y greater decrease i n heart rate a f t e r treatment with pronethalol. To determine whether gamma-thujaplicin had adrenergic alpha-receptor or beta-receptor blocking properties i t s ef-fect on.blood pressure responses to noradrenaline and i s o -proterenol was investigated. Gamma-thu-Japlicin was found to reduce the vasopressor e f f e c t of intravenous noradrenaline 0.-5ugAg» but had no e f f e c t on the vasopressor response pro-duced by isoproterenol G.25ug/kg. The pressor response to intravenous physostigmlne s a l i c y l a t e 40ug/kg. was unaffected by gamma-thujaplicin. The Increase i n heart rate and blood pressure produced by gamma-thujaplicin, with i n j e c t i o n s r e -peated every f i f t e e n minutes, was greatly reduced a f t e r the second dose. I t i s concluded that i n the anesthetized rats the vasopressor and tachycardiac response produced by gamma-thuja-p l i c i n were of central o r i g i n while the vasodepressor e f f e c t was a r e s u l t of d i r e c t action on vascular smooth muscle. The vasopressor response could involve the stimulation, v i a sym-pathetic nerves^ of alpha-adrenergic receptors of vascular smooth muscle. Gamma-thujaplicin can produce alpha-adrenergic receptor, blockade to exogenous noradrenaline, but not to endogenously released noradrenaline since the response to intravenous physostigmlne was not affected by the tropolone. i l l Beta-hydroxy thujaplicin iOmg/kg* caused a vaso-pressor response In a l l anesthetized and pithed rats tested,. The vasopressor response was abolished by phenoxybenzamine in both anesthetized and pithed rats. Beta-hydroxy thuja-p l i c i n did not alter Isoproterenol Induced tachycardia or the vasopressor response produced by physostigmlne salicylate. I t i s concluded that beta-hydroxy thujaplicin caused i t s vasopressor effect in rats by acting directly on the alpha-adrenergic receptors of vascular smooth muscle. Signatures of Examiners Iv TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES v i i LIST OF FIGURES v i i i INTRODUCTION 1 LITERATURE SURVEY 2 Tissue Catecholamines 2 D i s t r i b u t i o n 2 Synthesis 2 Tyrosine Hydroxylase 3 DOPA-Decarboxylase 3 Dopamine-Beta-Hydroxylase 3 I n a c t i v a t i o n 4 Monamine Oxidase k Catechol-O-Methyl Transferase 6 Uptake of Catecholamines by Sympathetic Nerves 1 The Adrenergic Receptor 9 Alpha-Adrenergic Receptor 10 Beta-AdrenergiC Receptor 11 Cardiovascular Pharmacology 11 Isoproterenol and Pronethalol \k Noradrenaline and Phenoxybenzamine 16 Tropolones 18 Chemistry 18 Pharmacology 20 V Page EXPERIMENTAL 23 Method 23 Operative Procedure 23 Drugs Employed 24 Tabulation of Heart Rate and,Blood Pressure Data 25 Evaluation of Alpha-Receptor or Beta-Receptor 26 Blockade Results 26 Gamma-Thujaplicin 26 E f f e c t on Blood Pressure and Heart Rate 26 Intact Rats 26 Repeated Doses 29 Pithed Rats 30 Ef f e c t of Adrenergic Blocking Drugs on the Response to Gamma-Thujaplicin 31 E f f e c t of Gamma-Thujaplicin on the Responses to Noradrenaline and Isoproterenol 34 E f f e c t of Gamma-Thujaplicin on the Vasopressor ... . Response to physostigmlne 37 Beta-Hydroxy- Thuijaplicin 38 E f f e c t on Blood Pressure and Heart Rate 38 Anesthetized Rats 38 P-ithed Rats 38 Repeated Doses 39 E f f e c t of Phenoxybenzamine on the Response to Beta-Hydroxy ThuJapilcin 40 E f f e c t of Beta-Hydroxy Thuj a p l i c i n on Isoproterenol Induced Tachycardia 4l E f f e c t of Beta-Hydroxy Th u j a p l i c i n on the Vasopressor Response to Physostigmlne 42 v i Page DISCUSSION ^3 Gamma-ThuJ aplicin Beta-Hydroxy Thujaplicin ^7 SUMMARY AND CONCLUSIONS 8^ BIBLIOGRAPHY 50 v i i LIST OP TABLES Table Page I E f f e c t on Blood Pressure and Heart rate of Repeated Injections of Gamma-Thujaplicin 30mgAg. 30 II The E f f e c t of Pronethalol lOrngAg* and Phenoxybenzamine 5mg A g . on the Response to Gamma-Thujaplicin 30mgAg• 32 -III E f f e c t of Gamma-Thujapllcin on the Vasopressor 1 Response to Physostigmlne. 37 IV E f f e c t on Blood Pressure of Repeated Injections of Beta-Hydroxy Thuj a p l i c i n 10mgAg» ^0 V E f f e c t of Phenoxybenzamine 5mgAg« on the Vasopressor Response to Beta-Hydroxy Thuj a p l i c i n 10mgAg« *rl VI E f f e c t of Isoproterenol 0.25ugAg» Before and A f t e r Treatment with Beta-Hydroxy Thuj a p l i c i n lOmgAff* 2^ v i i l LIST OP FIGURES Figure Page 1. Pathway for Metabolism of Noradrenaline. 5 2. Mechanical^ Forces which Determine the Minute Output of Ventricles* 12 3. Factors which Determine the Resistance to Blood Flow. 13 4. The Two Combinations of Haloalkylamlnes with the Alpha-Receptor. 18 5 . Structure of Tropolones. 19 6. The Vasopressor and Tachycardlac Response Produced by Gamma-Thujaplicin 30mgAg» in Anesthetized Intact Rats 27 7. The Vasodepressor and Tachycardlac Response Produced by Gamma-Thujaplicin 30mgAg» in Anesthetized Intact Rats. 28 8 . Effect of Gamma-Thujaplicin 7mgAg» on Responses to Noradrenaline. 35 9. Effect of Gamma-Thujaplicin ?mg/kg« on Responses to Isoproterenol. 36 10. The Vasopressor Effect of Beta-Hydroxy Thujaplicin 10mgAg« in Anesthetized Intact Rats. 39 ACKNOWLEDGMENT The author Is indebted to Dr. J.E. Halliday for his h e l p f u l guidance throughout the course of thi s work. A spe c i a l note of thanks i s extended to Dr. L.H. Bock of Rayonler Canada (B.C.) Ltd. for samples of beta-hydroxy t h u j a p l i c i n and gamma-thujaplicin. This work was supported by a grant from Medical Re-search Council and a University of B r i t i s h Columbia Graduate Fellowship. 1 INTRODUCTION The natural occurring tropolones found l n the western red cedar tree are alpha-thujaplicin, beta-hydroxy t h u j a p l i c i n , b e t a - t h u j a p l l c i n and gamma-thujaplicin. Limited studies have been done on these tropolones which could indicate sympathetic nervous a c t i v i t y . I t has been found that b e t a - t h u j a p l l c i n caused a f a l l i n blood pressure i n the guinea pig and. rabbit ( 5 2 ) • Gamma-thujaplicin produced a decrease i n blood pressure and heart rate i n the c a t ( 5 6 ) . In small doses be t a - t h u j a p l l -ci n increased the height of contraction of the a o r t i c s t r i p of the rabbit when stimulated with a d r e n a l i n e ( 6 0 ) . Gamma-t h u j a p l i c l n has a p o s i t i v e inotropic action on Isolated a t r i a while beta-hydroxy-thujaplicin has no e f f e c t on t h i s prepara-t i o n (personal communication. Dr. J.E. H a l l i d a y ) . The trop-olones and catechol rings are biochemically i s o s t e r l c . I t would be i n t e r e s t i n g i f they would display an a f f i n i t y f o r a receptor or receptors of the sympathetic nervous system. This would be the only group of compounds known which do not have an amine and have an e f f e c t on an adrenergic receptor. The e f f e c t could be v i a a s l i g h t l y d i f f e r e n t mechanism from that of the catecholamines and perhaps allow a deeper understanding of the s t r u c t u r a l nature of the adrenergic receptor. This study on the cardiovascular response was under-taken In an attempt to determine whether t h u j a p l i c i n s 'in vivo' a f f e c t the adrenergic mechanisms. 2 LITERATURE SURVEY Tissue Catecholamines Research during the last decade has Increased our know-ledge of the tissue catecholamines, dopamine, noradrenaline and adrenaline, with respect to distribution, function, synthesis, storage, release and inactivation. The brief discussion which follows refers to their role In the peripheral sympathetic nerv-ous system. Distribution The identification of noradrenaline as the transmitter substance in the postganglionic sympathetic nerve fibre Was made by von Euler in 1946(1,2). The development of histochemi-cal and biochemical methods made i t possible to show the intra-neuronal localization of the amine. I t was found that the mono-amines were stored in special structures in the nerve cells, called granules, which are concentrated in the nerve terminals. A large number of enlargements or varicosities of the nerve end-ings,, each containing many granules, are localized close to the effector cells(3,4). Noradrenaline i s distributed between a mobile—pool ln-the? cytoplasm and the more stable reserve form within the granules. Synthesis In 1938 the enzyme dihydroxyphenylalanine(DOPA) de-carboxylase was discovered by Holtz, Heise and Ladthe ( 5 ) . In the same year Blaschko proposed a hypothetical series of reac-tions by which noradrenaline and adrenaline might be formed from tyrosine. The hypothetical chain suggested was as follows; L-TYROSINE > DO PA > DOPAMINE > NORADRENALINE > ADRENALINE I t i s now well established that t h i s proposed biosynthesis schema was correct. Tyrosine Hydroxylase The conversion of L-tyrosine to L-DOPA Is catalyzed by the .enzyme--tyrosine hydroxylase(6). This reaction has been shown In v i t r o to require a tetrahydropterldlne cofactor, F e + + and oxygen. This enzyme does not hydrolyze D-tyroslne, tyramine or tryptophan. There are two classes of compounds which I n h i b i t t h i s enzyme, amino acids and catechols(7)• I t was found that catechol i n h i b i t i o n of the—enzyme i s not competitive with the substrate but with the pterldine cofactor. The c a t a l y s i s of L-tyroslne to L-DOPA Is. the rate l i m i t i n g step i n the biosyn-thesis of noradrenaline. DOPA-decarboxylase The decarboxylation of L-DOPA i s catalyzed by the en-zyme DOPA-decarboxylase(8,9)• This enzyme i s not s p e c i f i c and i s widely d i s t r i b u t e d l n the peripheral tissues and the brain. Dopamlne-beta-hydroxylase Dopamlne-beta-hydroxylase Is the enzyme responsible f o r converting dopamine to noradrenaline(10)• The l o c a l i z a t i o n of this enzyme seems to "be In the storage granules(11) • Several other phenylethylamlne derivatives can also be oxidized by this enzyme, for example, tyramine and alpha-methyl-dopamlne(12) • A metal, ion may be important in the activity of this enzyme.. The. ion-seems—tobe Cu*4* although—there Is reason to suspect —Pe+t or Co + +(13). Inactivation Our knowledge of the metabolic degradation of catechol-amines has—l-argely Increased during the last decade. A schema of the metabolic Inactivation pathway of noradrenaline i s given in figure 1. Monoamine Oxidase In 193? Blaschko, Rickter and Scholssman discovered an amine oxidase with the abi l i t y to Inactivate adrenaline(14). Rickter showed that this enzyme was able to oxidize many other amines(15). The enzyme catalyzing the oxidative deamination of monoamines was later called monoamine oxidase (MAO). In 1957 Blaschko(l6) suggested that there might exist metabolic path-ways of monoamine degradation other than oxidadative deamina-tion. Almost at the same time acid catecholamine metabolites were detected in the urine, namely 3,-4-dihydroxymandellc acid and 3,4-dlhydroxyacetic acid(17). The enzyme MAO i s widely distributed In the tissue and i s thought to be largely localized in the mitochondria(18). The deaminated metabolite formed by this enzyme Is an aldehyde(19). which can either be reduced to an ethanol Or a glycol by the enzyme aldehyde dehydrogenase. 2NH 2 HOI Noradrenaline Monoamine oxidase 3atechol-0-Methyl Transferase §HCHO Aldehyde Dehydrogenase 3,4-dihydroxyphenylglycolaldehyde I Aldehyde Reductase CHCH2OH 3,4-dihydroxyphenylglycol CHCH2OH 3-methoxy-4-hydroxyphenylglycol CHCH2NH2 Monoamine oxidase Normetanephrine qH CHCHO Aldehyde Dehydrogenase cliCOOH 3t 4-dihydroxymandellc acid Catechol-O-Methyl Transferase 3-methoxy-4-hydroxy -phenylglycolaldehyde COOH 3-methoxy-4-hydroxy-mandelic acid Figure 1. Pathway for metabolism of noradrenaline (21). — 6 The noradrenaline released from the firmly bound store Is deamlnated by the mitochondrial MAG i n the sympathetic nerves ( 2 0 ) . Thus noradrenaline leaves the nerve as a physio-l o g i c a l l y Inactive deamlnated catechol. Catechol-O-Methyl Transferase The 3 -0-methylating enzyme. catechol-O-methyl transfer-ase (COMT) catalyzes the transfer of methyl groups from S-adenosylmethionlne to the meta-hydroxyl group of catecholamines. The enzyme Is thought to be l o c a l i z e d outside the neuron l n the peripheral t i s s u e ( 2 2 ) . When the p u r i f i e d enzyme COMT was Incubated with adren-a l i n e . 8-adenosylmethlonlne and Mg + +, one mole of metanephrlne was formed f o r each mole of epinephrine metabolized. The en-zyme had an absolute-requtremeht- for Mg + + or other divalent cations such as Mn, Co, Zn, Fe, or-Ni. A l l catechols were O-methylated regardless of the substituent on the aromatic nu-cleus (23) * The enzyme does not show s p e c i f i c i t y toward the d or 1. Isomer. The d i s t r i b u t i o n of COMT Is widespread. I t Is present l n various tissues, glands, blood vessels, sympathetic and parasympathetic nerves and ganglia, and a l l areas of the brain. The main s i t e of O-methylatlon of c i r c u l a t i n g catecholamines i s i n the l l v e r ( 2 ^ ) . The compounds--can-also be O-methylated l o c a l l y In other tissues In v i v o . T h e r e are numerous studies which show that O-methylation i s an Important step In the metab-olism or noradrenaline and adrenaline ( 2 5 t 26, 2 7 ) . The methods 7 used have consisted of i n j e c t i n g a physiological dose of nor-adrenaline or adrenaline and measuring normetanephrine and de-amlnated products formed In the urine or In the whole animal. Almost a l l of the noradrenaline and adrenaline normally formed l n the body Is metabolized and excreted as O-methylated de-amlnated products. The d a i l y excretion of endogenous O-methyl-ated metabolites -range..from 2 to 4 mg. for 3-methoxy-4-hydroxy-mandelic acid (2MA)(28)» 100 to 3 0 0 ug. f o r normetanephrine and 100 to 200_ug. for metanephrlne.. Most of the__VMA presumably arises from the deamlnatiOn of noradrenaline within the_sym-pathetic-nerves followed by 0-methylatlon,-^probably outside the nerves. The VMA l n the urine probably represents the amount of noradrenaline produced and metabolized before I t had a chance to produce a physiological e f f e c t . The normetanephrine larg e l y represents the amount of ph y s i o l o g i c a l l y active noradrenaline that.was-discharged from sympathetic nerves. I n h i b i t i o n of COMT r e s u l t s i n a prolongation of the pressor action of cate-cholamines ( 9 ) • Uptake of Catecholamines by Sympathetic Nerves The p o s s i b i l i t y that exogenous catecholamines might be taken up into storage s i t e s i n the peripheral tissues was sug-gested by Burn(29). I t was not u n t i l r a d l o a c t l v e l y - l a b e l l e d catecholamines of high s p e c i f i c a c t i v i t y became available that experiments could be performed using Injected doses of adren-a l i n e and_noradrenallne small enough to be comparable-to amounts l i k e l y to be encountered under physiological conditions. The 8 f i r s t demonstration of tissue uptake was made by Axelrod, Weil-Malherbe and Tomchick(30)-. Their results showed that after Intravenous administration of -adrenaline a substantlal pro-portion of the Injected dose was inactivated by a rapid trans-fer .from the circulation Into peripheral tissue. In a subse-quent study Whitly, Axelrod and Well-Malherbe(31)» performed similar experiments with H3-noradrenaline. They found that tissue uptake operated to remove^he—lntravenously administered catecholamine from the circulation. The accumulation of nor-adrenaline in the tissue was found to be greater than that of adrenaline. The thought then prevailed that catecholamine up-take might represent an Important mechanism for the physiologi-cal Inactivation of catecholamines. There was evidence that catecholamine uptake occurs in sympathetic nerves ( 3 1 )• Whitly found that after noradrenaline Infusion, uptake of noradrenaline was greatest in the tissue with rich sympathetic innervation such as the heart. In the guinea pig heart and in the isolated perfused cat heart there Is a significant correlation between the amounts of H3-nor-adrenaline taken up by the tissue and the endogenous content of noradrenaline ( 32 ) . In tissues In which a normal sympathetic nerve supply Is lacking, the a b i l i t y to take up exogenous catecholamines Is severely Impaired. After superior cervical gangllonectomy the uptake of catecholamines in tissues innervated by this ganglion i s severely reduoed (33). The uptake of ^-noradrenaline i s also markedly reduced In various tissues of lmmunosympathectomized r a t s and mice, i n which the development of the sympathetic nervous system was suppressed by administration of nerve growth factor antiserum to newborn animals(3*t>). These findings sug-gest that the uptake of catecholamines occurs mainly i n sympa-the t i c nerve terminals, but some caution should be used l n making t h i s Interpretation. In a l l denervation experiments a small.uptake of"noradrenaline has been found i n the denervated ti s s u e s . There are radioautographic and fluorescent histochemi-c a l techniques to show that noradrenaline uptake Is l o c a l i z e d i n postganglionic sympathetic nerve terminals and also In post-ganglionic sympathetic nerve c e l l bodies i n the c e r v i c a l sym-pathetic ganglion. The Adrenergic Receptor Ahlquist ( 3 5 ) i n 19^8 f i r s t combined r e s u l t s of two pro-cedures to c l a s s i f y operationally d i f f e r e n t types of adrenergic receptors. The procedures used f o r d i f f e r e n t i a t i n g types of adrenergic receptors are of two kinds. In the f i r s t procedure, dose-response curves of a given effector system were obtained fo r adrenaline, noradrenaline and c l o s e l y r e l a t e d amines; and the r e l a t i v e potencies of these agonists were compared. In the second procedure, the a b i l i t y of various drugs to antagonize or block the response of the ef f e c t o r system to one or more of the agonists was determined. Ahlquist concluded that most adrenergic receptors were either one of two types which he termed alpha or beta. 10 Alpha-Adrenergic Receptor The alpha-adrenergic receptor Is most responsive to adrenaline and- least responsive to Isoproterenol. I t Is blocked by the classic adrenergic blocking agents such as phenoxybenz-amine and phentolamine. The common effector responses associ-ated with this receptor are:(36) 1. Vascular smooth muscle contraction (vasoconstriction). This response can be obtained In a l l vascular beds but Is most prominent In the skin and kidney. This response has been applied with the greatest success to demonstrate Alpha Adrenergic Activity(37). 2. I r i s radial muscle contraction (mydriasis). Recent evi-dence indicates that only alpha-receptors are involved with this structure. 3« Nictitating membrane smooth muscle contraction. 4. Orbital smooth muscle contraction. This apparent exo-phthalmos Is best seen in the cat. 5 . Splenic smooth muscle contraction. A decrease in spleen size occurs in a l l species but only In some species does an increase in hematocrit occur. 6. Myometrial contraction. This occurs in a l l species but i s a prominent response in female humans, rabbits, dogs and pregnant cats. 7. Retractor penis contraction. 8. Seminal vesicle contraction. 9« Pilomotor muscle contraction. 10. Intestinal smooth muscle relaxation. 11 Beta-Adrenergic Receptor This receptorIs most responsive to adrenaline i f only the naturally occurring catecholamines are considered, In gen-eral noradrenaline i s much less potent. Isoproterenol i s more potent than adrenaline on the beta-receptor. This receptor Is blocked by such agents-as dlst^lor^-soproterenol-and-rpronethaloli, buta=4« unaffected by the alpha-adrenergic- blocking agents• Some of the effector responses assbciated-wi-th beta-adrenergic re-ceptors^ aret (37-)—: 1. Vascular smooth-muscle relaxation (vasodilation). This response occurs in a l l vascular beds but Is most promin-ent, in skeletal muscle. 2. Myocardial positive Inotropic response. 3* Bronchial smooth muscle relaxation. 4. Myometrial relaxation. This occurs in a l l species but is a prominent response in female Tats—and non-pregnant cats. 5.Intestlnal_smooth muscle relaxation. Cardiovascular Pharmacology . . Many factors influence the cardiac output and vascular resistance which In turn Influence blood pressure. A few fac-tors w i l l be de8crlbed(38). Figure 2 shows that the minute output of the ventricle 18 immediately determined by the stroke volume and the fre-quency of contraction of the ventricle. The stroke volume is determined by art e r i a l pressure, strength of the ventricle muscle and diastolic ventricular volume. The diastolic volume 12 Is In _turn determined by venous f i l l i n g pressure, diastolic f illlng._tlme, compliance of the ventricular wall during dia-stole and resistance to blood flow through the atrlo-ventricular valve. I t can be stated that cardiac output i s influenced by several-immediate and remote mechanical factors. Some factors can be influenced by a chemical such as noradrenaline, or by the sympathetic nervous system, postganglionic fibre, the mediator of which i s noradrenaline. Mechanical forces .-involved 1 in f i l l i n g ARTERIAL PRESSURE PILLING PRESSURE STROKE VOLUME VENTRICULAR COMPLIANCE PILLING TIME DIASTOLIC VENTRICULAR VOLUME STRENGTH FREQUENCY .CARDIAC b U T P U T VALVULAR RESISTANCE Mechanical forces involved in emptying •Figure 2. Mechanical forces which determine the minute output of the ventricles(38). 1 3 The resistance to blood flow through the entire systemic vascular bed Influences a r t e r i a l pressure which i n turn a f f e c t s cardiac output (figure 3)'»"' The resistance i s immediately de-termined by the geometric and viscous component of resistance. The vessel radius Is the most Important variable In the geomet-r i c component of resistance. Radius i s influenced by the con-t r a c t i l e state of vascular smooth muscle. A chemical such as noradrenaline or Isoproterenol may a f f e c t resistance by a l t e r -ing the c o n t r a c t i l e state of vascular smooth muscle. CHEMICAL NERVOUS PHYSICAL TRANSMURAL PRESSURE ORGANIC ACTIVE PASSIVE CONCENTRATION SIZE AGGREGATION ADIUS LENGTH PLASMA CELLS GEOMETRY / RESISTANCE •VISCOSITY Figure 3 . Factors which determine the resistance to blood f l o w ( 3 8 ) Noradrenaline l n the Intact animal has l i t t l e regular e f f e c t on the. cardiac output. A f a l l l n contraction frequency Is often balanced by a r i s e i n stroke volume. The f a l l l n f r e -quency In the Intact animal r e s u l t s r e f l e x l y v i a the baroreceptors 14 mechanism and possibly v i a some e f f e c t on the c e n t r a l nervous system.- Administration of noradrenaline l n t r a v e n t r i c u l a r l y into c e r e b r a l l a t e r i a l v e n t r i c l e s produced bradycardia and hypoten-s i o n 38 ) . The Increase i n stroke volume may be a t t r i b u t e d to an increase In c o n t r a c t i l e force. The Increase l n force may r e s u l t from an Increase i n strength of the muscle. Noradrenaline i n the Intact animal a f f e c t s the p e r i -pheral c i r c u l a t i o n by r a i s i n g the t o t a l peripheral resistance. This r i s e In peripheral resistance r e s u l t s predominantly from reduction l n net blood v e s s e l radius due to a c t i v a t i o n on vas-cular smooth muscle. The venous smooth muscle i s also activated which i s Indicated by a decrease i n venous compliance. There i s an elevation_of r i g h t a t r i a l pressure which probably r e s u l t s from generalized decrease In venous radius, compliance or both. The net r e s u l t i s an increase l n blood pressure. Isoproterenol and Pronethalol Isoproterenol i s a drug with primarily beta-mimetic ac-t i o n . The isopropyl group attached to the N-atom seemed to suggest that t h i s catecholamine would not undergo deaminatlon and .would be metabolized exclusively by catechol-O-methyl trans-ferase. Sjoerdsma(39) used the D Isomer of isoproterenol to measure GOMT a c t i v i t y of hypertensive patients by giving Iso-proterenol and I s o l a t i n g the 3-methoxy-isoproterenol. Later Hertting(40) showed that H^-isoproterenol Injected i n the r a t , 65% of the administered a c t i v i t y was excreted i n the urine and 35 to b5% of the administered a c t i v i t y was excreted l n the b i l e . 1 5 The urine contained ^-isoproterenol,aH^-lsoproterenol glucur-onlde, free methoxy-H^-lsoproterenol and 3-raethoxy-H3-lsoproter-enol glucuronide. In the bile only 3-methoxy-H3-isoproterenol glucuronide was found. Tissue studies revealed that most of the activity present In the tissue ten minutes after the Injec-tion of H3-lsoproterenol was already In the 0-methylated form. Tissue uptake of Isoproterenol was confirmed to be of Insig-nificant amount(4l). After Intravenous Injection of Isoproter-enol, small amounts were found In the heart after ten minutes, but most of this disappeared In two hours. Isoproterenol Is poorly bound by Intracellular particles and readily removed by post, perfusion. The beta-adrenergic blocking agents are structurally similar to the beta-adrenergic agonist isoproterenol. The side chain of pronethalol Is similar to that of Isoproterenol. One of the most important features of the beta-adren-ergic blocking drugs i s their-relatlvely high-degree of speci-ficity.-. For example although: they block the posi-tive lhotropic and chronotropic effects of adrenergic stimuli, they do not block the cardiac stimulating effects of calcium, methyl xanthine-or d i g i t a l i s glycosides. Similarly whereas vasodila-tation i n response to injected isoproterenol i s blocked, vaso-dilatation In response to histamine, nitroglycerine or acetyl-chO'llna_is unaffected(42). The vasodllatatlon-and cardlac acceleratlon effects of isoproterenol were effectively-blocked by pronethalol, but there was no Inhibition of the vasocon-strictor response to noradrenaline (43), These findings were 1 6 consistent with the hypothesis that pronethalol i n h i b i t e d only beta-adrenergic receptors. The h a l f l i f e f o r the block of isoproterenol tachycar-d i a produced by pronethalol l n rabbits was 36 to 45 minutes, whereas the metabolic h a l f l i f e was 60 to 70 minutes(44). In addition to pronethalol several other beta adrenergic receptor blocking agents have been developed(44)• A l l these compounds have been found to exert a competitive type of block-ade;, that Is, the dose-response curve of the agonist Is s h i f t e d progressively to the r i g h t with increasing concentration of the agonist. The beta-adrenergic blocking agents dlchloro-1sopro-terenol and pronethalol prevent_the uptake of noradrenaline In-fused tnto organs(45). Noradrenaline and Phenoxybenzamine Noradrenaline causes bradycardia of r e f l e x o r i g i n . There Is a r i s e i n both s y s t o l i c and d i a s t o l i c blood pressure during i n j e c t i o n of noradrenaline, a r e f l e c t i o n of generalized vasoconstriction. Cardiac output remains unchanged or f a l l s ( 2 2 ) . Various drugs have been shown to abolish the pressor action of noradrenaline i n anaesthetized animals by antagoniz-ing i t s vasoconstrictor action. This has been demonstrated f o r ergot a l k a l o i d s , yohimbine, to l a z o l i n e , phentolamine, phenoxy-benzamine and dibenamine. I t has also been demonstrated that phenoxybenzamine i n doses which abolish the pressor action of noradrenaline i n normal anaesthetized cats and rat s f a l l to ~ abolish the pressor action of noradrenaline i n preparations that have been pithed* This i s due to the Increase i n cardiac output (46)• Another action of alpha-adrenergic blocking agents such as phenoxybenzamine has been shown to prevent the uptake of Infused n o r a d r e n a l i n e ( 4 7 ) • The term I r r e v e r s i b l e competitive antagonism or non- . equilibrium antagonism have both been used to designate the type of antagonism exerted by phenoxybenzamine against drugs acting on a number of d i f f e r e n t types of receptors. In t h i s type..of-antagonlsm the f i n a l complex formed between receptor and antagonist does not rever s l b l y dissociate ( 4 8 ), and therefore mass action equilibrium, such as i s assumed to occur In c l a s s i -c a l competitive antagonism i s not possible. However, two r e -ports noted that dlchlorisoproterenol could antagonize the blocking action of some alpha-adrenergic blocking agents on the pressor e f f e c t of adrenaline and noradrenaline. In 1 9 6 5 another report appeared"which stated that pronethalol reversed the Inhibitory e f f e c t produced by phenoxybenzamine on the,pres-sor response to adrenaline-and n o r a d r e n a l i n e ( 4 9 ) . This e f f e c t i s p a r t i c u l a r l y Intriguing i n the case of phenoxybenzamine, " i ' • -since blockadeJby. this^drug-is of the-non-equilibrium type, with-very-prolonged duration of action. ^The pharmacological active form of the molecule i s the ethylene lmmonlum Ion which; f I T S t combines with the anionic s i t e of the alpha receptor by an i o n i c bond, competing with noradrenaline and then rearranges to alkylate the receptor, thereby producing an i r r e v e r s i b l e prolonged antagonism. These mechanisms, which are shown diagramatically i n figure 4 , were f i r s t postulated by B e l l e a u ( 5 0 ) , 18 R > CH R » ^ p R R» NCHgCHgCl CH 2 I Receptor ethylene immonlum ion; i o n i c bond to anionic s i t e of alpha-receptor covalent bond to receptor Figure 4. The two combinations of haloalkylamines with the alpha-receptor. The f i r s t combination i s rev e r s i b l e and the antagonism of noradrenaline i s competitive; the second combination i s i r r e v e r s i b l e . In phenoxybenzamine. Tropolones Sanders(51) wrote an excellent review of the t h u j a p l i -cins which are the natural occurring isopropyl derivatives of tropolone (I) to the year 1961. Except f o r some information pertinent to t h i s thesis the l i t e r a t u r e survey Included i n -formation from 1961 to the present time. Chemistry The structures of various tropolones are shown l n figure 5. The natural occurring tropolones which are referred to as th u j a p l i c i n s are found i n the western red cedar tree; alpha-t h u j a p l l c i n ( I I ) , beta-hydroxy t h u j a p l i c i n (V), b e t a - t h u j a p l i c i n ( I I I ) , and gamma-thujaplicin (IV). The inv e s t i g a t i o n of betar hydroxy t h u j a p l i c i n (7-hydroxy-4-isopropyltropolone) (V) and gamma-thujaplicin (5-1sopropyltropolone) (IV) Is described l n t h i s t h e s i s . 19 R^ R 2 H3 H«j I, H H H H H II, CH(CH3)2 H H H H III. H CH(CH3)2 H H H IV, H H CH(CH3)2 H H V, H CH(CH 3) 2 H H OH VI, H CH3 H H H Figure 5. Structure of tropolones(52). A chemical property which the tropolone and catechol ring.have in common is the a b i l i t y to form stable chelates with divalent ions(53)• It has also been shown that the two rings are isosterlc(52). The thujaplicins are weak acids with tit r a t i o n curves typical of monobasic acids. The acidity increases from alpha to beta-to gamma-thujaplicin,-with f K a values of 7»8, 7*3 and 7«1 respectively. In solution, beta-thujaplicin chelation w i l l be accompanied by a drop in pH. The stability of chelation of beta-thujapllcin decreases in the order..copper, iron (III, nickel, zinc and cobalt (II). Magnesium and zinc do not form detachable chelates with beta-thujaplicin 10"2* molar (54). 20 Beta-thujaplicin Is moderately stable at 10~3 molar solution i n water at room temperature(54). After two days about \$% decomposed and at fourteen days about 40# of the b e t a - t h u j a p l i c i n decomposed. Pharmacology The pharmacology of b e t a - t h u j a p l i c i n was investigated f i r s t by Lee i n 1 9 5 K 5 D . In h i s f i r s t paper, he reported on the t o x i c i t y and l o c a l action of b e t a - t h u j a p l l c i n and i t s s a l t . In h i s second paper Lee found that b e t a - t h u j a p l l c i n caused a f a l l i n blood pressure In guinea pigs and i n r a b b i t s . He also noticed a decrease i n pulse rate and respiratory depression. The pharmacological properties of gamma-thujaplicin were f i r s t Investigated by Halliday ( 5 5 ). These studies consis-ted of t o x i c i t y determinations i n mice. In addition, the ef-f e c t of gamma-thujaplicin was studied on the central and p e r i -pheral nervous system, blood pressure and heart rate of the cat and dog. He found that gamma-thujaplicin 25 to 50mgAg« pro-duced a temporary f a l l In blood pressure and a decrease In heart rate; while 50mg/kg» produced complete paralysis of r e s -p i r a t i o n . The hypotensive e f f e c t remained unchanged a f t e r a t r o p l n l z a t i o n or b i l a t e r a l ; section of the vagus. Responses to c a r o t i d occlusion and to injected adrenaline were not a l -tered by an- Immediately previous i n j e c t i o n of gamma-thujaplicin. Belleau(52) was the f i r s t to show i n v i t r o that trop-olones were i n h i b i t o r s of COMT. He had also shown that the r e -action was of a competitive nature, l a t e r confirmed by 21 D ,Jario(56). The i n h i b i t o r y mechanism of COMT i s thought to Involve the enzyme bound magnesium and the tropolone. Belleau thought^that a 1:1:1 complex i s formed between COMT, magnesium and I n h i b i t o r . 4-methy1,1 tropolone was found to be the most ac-ti v e COMT i n h i b i t o r whereas beta - t h u j a p l i c i n was of moderate a c t i v i t y . I t was l a t e r found that beta-thujaplicin does not form chelates with magnesium(55)• Gamma-thujaplicin and beta-hydroxy t h u j a p l i c i n showed moderate COMT i n h i b i t i o n . Ross(57) found that 4-methyltropolone (VI) lOmg/kg. and beta-thujapllcin 20mg/kg.~,could cause COMT i n h i b i t i o n In mice. Doses- as high as lOOmg/kg. produced no adrenergic blockade i n these animals. Studies i n mice indicated that 4-methyltropolone i n small doses Increased the percent mortality of mice Injected with adrenaline? t h i s may be due to In h i b i t i o n of COMT. Large doses exert a protective e f f e c t which might be due to a t r a n s i -tory blockade of the alpha-receptors. In the same study-it was also found that methyltropolone reduced the vasodepressor ef-f e c t of isoproterenol(58). Murnaghan i n 1964(59) showed that 4-methyltropolone and beta-thujaplicin i n small doses increased the height of contraction of the a o r t i c s t r i p of the rabbit when stimulated with adrenaline and potentiated the toxic ef-f e c t of adrenaline i n mice; i n large doses they antagonize the adrenaline Induced death i n mice. Methyltropolone also poten-t i a t e s and antagonizes respectively potassium and barium i n -duced contractions on the ao r t i c s t r i p . They therefore con-cluded that the mechanism of the adrenaline potentiation cannot be due to I n h i b i t i o n of COMT. They f e l t that antagonism and 22 potentiation of adrenaline were due to non s p e c i f i c e f f e c t s of 4-methyltropolone and bet a - t h u j a p l l c i n . Gamma-thujaplicin as mentioned previously i s a chelat-ing agent which complexes with various divalent ions, but i s most stable with copper. The tropolones have been shown to be potent i n h i b i t o r s of dopamine hydroxylase l n v l t r o ( 1 3 ) » Since t h i s enzyme Is located i n the storage s i t e of noradrenaline, i t would seem that these tropolones could enter t h i s part of the neurone. Wyse and H a l l l d a y ( 6 0 ) reported that gamma-thuja-p l i c i n l n vivo lowered the copper content of r a t heart tissue. They had shown that the responses of the r a t a t r i a to tyramine had been reduced when treated with gamma-thujaplicin. They then concluded that t h e i r data was consistent with the hypothe-s i s that chelating agents lower tissue catecholamines due to chelation of heavy metals necessary for the a c t i v i t y of enzymes Involved i n catecholamine synthesis. 23 EXPERIMENTAL Method Male Wistar rats weighing 300 to 500 grams were used for these experiments. The operative technique used was simi-l a r to that o r i g i n a l l y described by Landgrebe(61) and modified by Dekanskl(62). I n i t i a l l y pentobarltone sodium 50mg/kg. was employed as an anesthetic, but anesthesia l e v e l s varied greatly from animal to animal and sometimes a supplemental dose was r e -quired. Urethane 1.3 to 1.5gm/kg. was found to be a more sat-i s f a c t o r y anesthetic and was used f o r the majority of experi-ments. The r a t was chosen for these experiments because the limited- quantities of gamma-thujaplicin and beta-hydroxy t h u j a p l i c i n available d i d not permit the use of a larger experi-mental animal. Operative Procedure The fore and hind legs were secured to a warmed table and r e c t a l temperature was maintained from 33 to 35 degrees centigrade. The l e f t c a r o t i d artery and the trachea were d i s -sected, ready f o r cannulation. The trachea was cannulated with a length of polyethylene tubing of size PE 240. A femoral vein was. cannulated with polyethylene tubing of size PE 60, which was connected by-a hypodermic needle to a three way stopcock. One of the remaining arms of the stopcock was connected to a 25 ml. burette, the other to a syringe used for the Injection 24 of chemicals. Heparin 1.5mg/100gm. of body weight was injected intravenously via the femoral vein cannula. The l e f t carotid artery was then cannulated with a polyethylene tube of size PE 90, which was connected through a s a l i n e - f i l l e d tube to a Condon mercury manometer, or to a Statham model P 23 series pressure transducer. A model 5 Grass Polygraph, sensitivity 10, or a Kymograph was used for recording blood pressure. After the operative procedure, one hour was allowed for the blood pressure and heart rate to stabilize. Pentolinium tartrate, a ganglionic blocking drug, was administered in.the dose of 0.125mg/rat to prevent--fluctuation of blood pressure(63). Pentolinium tartrate was not administered to rats -ln experi-ments conducted to-find•the•effect of gamma-thujaplicin or beta-hydroxy thujaplicin on the vasopressor response to physo-stlgmlne. In pithed animals, pithing was done under ether accord-ing to the method of Shipley and Tilden(64), prior to exposure and cannulation of blood vessels. Pithed animals were main-tained on a Harvard Series 680 Rodent Respiratory pump. Res-piration was maintained at 60 cycles per minute, the volume of air per stroke being one ml. per 100 grams of body weight. Heart rate was monitored by a Sanborn Viso Cardlette, model 51 with readings taken every minute. Later a model 5 Grass Polygraph was used with a model 5P6 EKG preamplifier. Drugs Employed The following drugs were employed; noradrenaline b i -tartrate, isoproterenol hydrochloride, gamma-thujaplicin sodium, 2 5 beta-hydroxy t h u j a p l i c i n sodium, phenoxybenzamine hydrochlor-ide, pronethalol hydrochloride, pentollnium ta r t r a t e , heparin sodium and physostigmlne s a l i c y l a t e . The doses used are ex-pressed In terms of s a l t s employed. A l l solutions were made with normal saline p r i o r to each experiment. The sodium s a l t of beta-hydroxy t h u j a p l i c i n was pre-pared according to the method of H a l l l d a y ( 5 5 ) • Tabulation of Heart Rate and Blood Pressure Data Measurements tabulated are the maximum changes, either Increases or decreases of blood pressure (mm.Hg.) or heart rate (beats per minute), from the baseline. The baseline and base-rate respectively Is taken as the blood pressure or heart rate Immediately before an i n j e c t i o n of a drug. The duration of response indicates the time In minutes from the i n i t i a l r i s e u n t i l the blood pressued had returned to within 5 mm.Hg. of the baseline and the heart rate to within 2 0 beats per minute of the baserate. In a l l s t a t i s t i c a l data the " t " t e s t was used with a l e v e l of si g n i f i c a n c e of 0 . 1 0 and the standard error of the mean tabulated. The + or - refer s to an increase or a decrease of blood pressure or heart rate from the baseline. Control i n j e c -tions of normal saline 0 . 5 ml, were made In each animal. Values for the ef f e c t s of these control Injections are given only f o r animals i n which a response occurred. 26 Evaluation of Alpha-Receptor or Beta-Receptor Blockade In experiments where pronethalol lOmg/kg. was used. Isoproterenol was used In the following manner to test the ef-fectiveness of beta-receptor blockade. A test response to 0.25ugAg« of Isoproterenol was obtained before administration of pronethalol. Following pronethalol, the absence of a res-ponse, or presence of only a slight response to 0.5ug/kg. of isoproterenol was considered to indicate the presence of an adequate beta-receptor blockade. The same procedure using noradrenaline as the test drug was used for evaluating the effectiveness of alpha-receptor blockade by phenoxybenzamine 5mg/kg. —Results.-Gamma-Thujaplicin Effect on_ Blood-Pressure and Heart Rate A. Anesthetized Rats Gamma-thujaplicin 30mg/kg» produced either a vasodepres-sor or a vasopressor response. Tachycardia occurred in a l l ex-periments following gamma-thujaplicin administration. A vasopressor response was produced In six rats out of twelve animals ( f i g . 6). The average increase In blood pressure was 29.5 mm.Hg. or 55% of control pressure, which occurred one-half—minute after an Injection of gamma-thujaplicin. The aver-age duration of this response was two minutes. The magnitude 27 of the pressor response varied between animals, ranging from 6 to 60.mm.Hg. In conjunction with the pressor response tachy-cardia occurred. The-average increase-In heart rate was $0 beats._.per-..mlnute after an Injection of gamma-thujaplicin. The peak maximum increase in heart rate occurred one-half minute after the peak change In blood-pressure was reached. The dura-tion of the tachycardia was three minutes. 0 blood pressure O heart rate Tstandard error n=6 .400 300 4) 4) •p -P as 0 « c 4-4 as co a> - P W as © fi 200 w 2 '3 Time (minutes) Figure 6. The vasopressor and tachycardlac response induced by gamma-thujaplicin 30mg/kg. in anesthetized intact rats • 28 In the remaining s i x rat s gamma-thujaplicin produced a vasodepressor response ( f i g . 7)• The average decrease i n blood pressure was 12 mm.Hg. or 16% and occurred one-half minute a f t e r an Injection of -gamma-thujaplicin. The duration of th i s res-ponse was d i f f i c u l t to determine because the blood pressure did not return to within 5 mm.Hg. of the i n i t i a l blood pressure which was 6 l . 5 ± 5 mm.Hg. The magnitude of the depressor res-ponse varied from minus 5 to minus 28 mm.Hg. Vasodepression was accompanied by tachycardia. The average increase i n heart rate was 45 beats per minute. This response occurred one to two minutes a f t e r gamma-thujaplicin was injected. This Increase i n heart rate followed the vasodepressor response by one-half minute. The duration of the tachycardia was four minutes. # Blood pressure O heart rate T standard error n =6 T ?3 '4 Time (minutes) Figure 7 . The vasodepressor and tachycardiac response produced by gamma-thujaplicin 30mgAg« i n anesthetized Intact r a t s . Gamma-thujaplicin apparently produced complex and var-i a b l e cardiovascular changes i n these r a t s . Although the i n i -t i a l blood pressures and heart rates were similar* either a decrease or an Increase i n blood pressure occurred. In rats i n which gamma-thujaplicin increased the blood pressure, the aver-age I n i t i a l blood pressure was 6 2 . 6 mm.Hg. and the average i n i t i a l heart rate was 2 9 0 beats per minute. In rats l n which the blood pressure was-decreased, the average i n i t i a l values for blood pressure and heart rate were 6 1 . 5 mm.Hg. and 3 1 6 beats, per minute. Whether there was an increase- or a decrease l n blood pressure* the maximum increase i n heart rate was of the same magnitude. I t would seem that gamma-thujaplicin could influence the heart rate independently of the change i n blood pressure. The tachycardiac response was more prolonged when—vasodepression occurred. I t was also observed that the vasodepressor response was of a greater duration than the vaso-pressor response, although both were s h o r t - l a s t i n g . Since a ganglionic blocking drug was employed i t seemed u n l i k e l y that the responses could be mediated v i a the preganglionic autonomic neurons. Doses of gamma-thujaplicin lower than 3 0 m g A g » d i d not produce a response consistently. B. Repeated Doses To determine whether changes i n blood pressure and heart rate could be rep l i c a t e d i n the same animal, four i n j e c -tions of gamma-thujaplicin 30mg/kg»---were-admlnistered f i f t e e n minutes apart. Table I Ef f e c t on Blood Pressure and Heart Rate of Repeated Injections of Gamma-Thujaplicin 30mg/kg. F i r s t Dose Second Dose Third Dose Fourth Dose Blood Pressure Increase (mm.Hg.) 23+3.2 27.6+6.3 13.3+6.7 -6.6+6.6 Heart Rate Increase (beats per minute) 80+20 -80+30 6.6+6.7 26.6+23.6 n=4 In a l l four animals an Increase In blood pressure and heart rate were observed. The maximum Increase i n blood pres-sure and heart rate were s i m i l a r a f t e r the f i r s t two doses of gamma-thujaplicin (table I) but were greatly reduced a f t e r the t h i r d and fourth dose of gamma-thujaplicin. A degree of tachyphylaxis appeared to have developed following the second dose of gamma-thujaplicin. C. Pithed Rat Four experiments were done i n rats i n which the brain and sp i n a l cord were destroyed by pithing according to the method of Shipley and Tilden(65). The average i n i t i a l blood pressure of the pithed rats was 34.5+1*6 mm.Hg. which was con-siderably lower than that of the anesthetized r a t s . The aver-age I n i t i a l heart rate of the pithed rats was 290+17.3 beats per minute which was sim i l a r to that In the anesthetized r a t s . 31 In a l l four pithed rats gamma-thujaplicin produced a f a l l In "blood pressure and decrease In heart rate. The aver-age decreases In blood pressure and heart rate were respectively minus 16+2 mm.Hg. and minus 46+6 beats per minute. Since a l l central Influences on the cardiovascular system are absent In the pithed r a t , the eff e c t s of gamma-thujaplicin could not have.been of cen t r a l o r i g i n and must be due to action at p e r i -pheral s i t e s . E f f e c t of Adrenergic Blocking Drugs on the Response to Gamma-Thujapllcln Gamma-thujaplicin 3 0 m g A g . was administered to obtain a control response. This was followed by administration of the appropriate blocking agent. The blocking agents used were pronethalol lOmg/kg. and phenoxybenzamine 5mg/kg. The proced-ure described e a r l i e r (see Experimental p.26) was employed to establ i s h the effectiveness of receptor blockade and gamma-t h u j a p l i c i n 30mg/kg. was again administered. Both Intact and pithed r a t s were used. Since tachyphylaxis developed a f t e r two doses of gamma-thujaplicin the e f f e c t of only one blocking agent was tested i n a single animal. 32 Table II The E f f e c t of Pronethalol lOmg/kg. and Phenoxybenzamine 5mgAg» on the Response to Gamma-Thujaplicln 30mgAg» Gamma-Thuj a p l i c i n Intact Rats Maximum Change i n B • P • H.R. mm.Hg.+S «E. Pithed Rats Maximum Change i n B. P . H • R • mm.Hg.+S. E. Control Response +15+6.6 +36+21.6 A f t e r +3.3+3.5* +8+13*5 Phenoxybenzamine n=5 n=5 Control Response +10+5.3 +60+5.1 -13.6+3.3 -47+6.6 A f t e r Pronethalol -5+9.5* +15+9.4 -14.5+0.5 -70+10.1* n=4 n«4 n=3 n=3 * " t " test (paired), p=0.10, s i g n i f i c a n t B.P. = blood pressure H.R. « heart rate, beats per minute n = number of animals I t can be seen from table II that the vasopressor ef-fect of gamma-thujaplicin i n anesthetized rats was s i g n i f i c a n t l y reduced by phenoxybenzamine 5mg/kg« The control vasopressor response to gamma-thujaplicin was +15.4 mm.Hg. which was reduced to +3*3 mm.Hg. by phenoxybenzamine. The control Increase i n heart rate produced by gamma-thujaplicin was +36 beats per minute. This response was reduced to +8 beats per minute l n the presence of phenoxybenzamine but, th i s change was not s t a -t i s t i c a l l y s i g n i f i c a n t . The i n i t i a l blood pressure which was 58.5 mm.Hg. before phenoxybenzamine was administered was reduced to 40 mm.Hg, a f t e r phenoxybenzamine 5 m g A g . The I n i -t i a l heart rate was Increased from 3 2 0 beats per.minute to 370 beats per minute by phenoxybenzamine. In anesthetized rats pronethalol lOmg/kg. reversed the vasopressor e f f e c t produced by gamma-thujaplicin 30mgAg. to one of vasodepresslon. The increase i n blood pressure of 10.mm.Hg. produced by gamma-thujaplicin was reduced to a de-crease, of 5 mm.Hg. i n the presence of pronethalol, a change which was s t a t i s t i c a l l y s i g n i f i c a n t . Although pronethalol reduced the Increase i n heart rate produced by gamma-thuja-p l i c i n from 60 beats per minute to 15 beats per minute the reduction was not s t a t i s t i c a l l y s i g n i f i c a n t . Pronethalol I t s e l f caused l i t t l e change i n Vblood pressure. The blood pres-sure before pronethalol was 54 mm.Hg. whereas a f t e r administra-tion of pronethalol the blood pressure was 55 mm.Hg. Prone-t h a l o l d i d decrease the heart rate s l i g h t l y , from 375 to 300 beats per minute. In the pithed r a t pronethalol lOmg/kg. dld.not a l t e r the vasodepressor response produced by gamma-thujaplicin 30mg/kg. The decrease i n blood pressure produced by gamma-t h u j a p l l c l n before pronethalol was 1 3 . 6 mm.Hg., whereas af t e r -pronethalol the decrease was 14.5 mm'.'Hg. I t can then be as-sumed that the decrease i n blood pressure produced by gamma-t h u j a p l i c i n i n the pithed rats was not due to beta-receptor stimulation. In the pithed r a t gamma-thujaplicin produced a s i g n i f i c a n t l y greater decrease i n heart rate a f t e r treatment with, pronethalol. The control decrease In heart rate produced by gamma-thujaplicin was 47 beats per minute whereas the de-crease In heart rate a f t e r treatment with pronethalol was 70 beats per minute. This greater decrease In heart rate pro-duced a f t e r pronethalol administration might be explained as being due to additive e f f e c t s of the two drugs since prone-t h a l o l has been shown to produce bradycardia by d i r e c t myo-c a r d i a l depresslon(65) and beta-adrenergic blockade. Effect, of Gamma-.Thujaplicin on Responses to Noradrenaline and Isoproterenol The following procedure was used on anesthetized r a t s . Two doses of noradrenaline 0.5ug/kg. were given twenty minutes apart .followed by two Injections of Isoproterenol 0.25ug/kg. twenty minutes apart. Gamma-thujaplicin 7mg/kg. was then ad-ministered. Ten and t h i r t y minutes a f t e r the administration of gamma-thujaplicin, noradrenaline 0.5ug/kg. and Isoproter-enol 0.25ug/kg. respectively, were again given. The r e s u l t s obtained are recorded In figure 8 and 9 as maximum changes In blood pressure. Two control i n j e c t i o n s of noradrenaline (fig... 8) and Isoproterenol ( f i g . 9) were given per animal. The average of t h e i r responses was calculated. The control vasopressor response to noradrenaline was 32+6.8 mm.Hg. The vasopressor responses to noradrenaline ten and t h i r t y minutes a f t e r gamma-thujaplicin were 18+5.01 mm.Hg. and 14.2+5.9 mm.Hg. respectively, both of which are s i g n i f i -cantly lower than the control responses. 3 5 • bO W • • < 9 U CM CQ CQ <D CO rH o PM •d •d 1 o o © H c pq H C as •H C 0) <D rH CO •d ai as <D rH U O o a c H ~ Tstandard error 16 Minutes A f t e r GT 3 0 Minutes A f t e r GT Figure 8. Eff e c t of gamma-thujaplicin 7mg/kg. on responses to noradrenaline• * " t " test, n= 5 , p=0.1, s i g n i f i c a n t . GT - gamma-thujaplicin The vasodepressor response to isoproterenol before gamma-thujaplicin was minus 21.5+1*5 mm.Hg. Responses of minus 21.4+3.2 mm.Hg. and minus 24.5+2 mm.Hg. were obtained ten and t h i r t y minutes a f t e r the administration of gamma-t h u j a p l i c i n . faO a . • § to < 60 u V\ (0 CM CO • <D o A* H O -ti c O O h H 0) PQ +3 O C IH *H P. O <D CO CO M CO OJ U O 09 Q -40--30_ -20 -10. T standard error X Before GT 10 Minutes After GT 30 Minutes A f t e r GT Figure 9. E f f e c t of gamma-thujaplicin 7mg/kg. on responses to isoproterenol. n=5 I t would appear from these r e s u l t s that gamma-t h u j a p l l c i n can I n h i b i t the action of noradrenaline on the rat blood pressure but has l i t t l e or no e f f e c t on the action of Isoproterenol. 3 7 E f f e c t of Gamma-Thujaplicin on the Vasopressor Response to Physostigmlne The pressor response to intravenously Injected physo-stigmlne i n the r a t arises from a c t i v a t i o n of the central sympathetic mechanism ( 6 6 , 6 7 , 6 8 ) and i n many ways i s s i m i l a r to the e f f e c t of sympathetic nerve stimulation. The pressor response i s blocked by bretyllum, guanethidine and syrosingo-p i n e ( 6 9 ) . Physostigmlne s a l i c y l a t e 40ug/kg. when injected intravenously produced a pressor e f f e c t which was constant when repeated i n the same r a t but was variable between r a t s . Two control responses to physostigmlne 40ug/kg. were obtained t h i r t y to forty minutes apart. A t h i r d i n j e c t i o n of physostigmlne was given twenty minutes following administration of gamma-thujaplicin. Pentolinlum t a r t r a t e was not administered i n these rat s since blockade of sympathetic ganglia would i n -h i b i t the pressor response to physostigmlne. Table III Ef f e c t of Gamma-Thujaplicin on the Vasopressor Response to Physostigmlne. Maximum Change i n Blood Pressure (mm.Hg.+s.e.) Before Gamma-Thujaplicin A f t e r Gamma-Thujaplicin Physostigmlne +28.5+4.3 + 3 1 + 8 . 6 n=2 The physostigmine response before gamma-thujaplicin was very s i m i l a r to the response obtained a f t e r gamma-thuja-p l i c i n (table I I I ) . Since gamma-thujaplicin did not change the response to physostigmine i t can be assumed that i t does not antagonize the central stimulant e f f e c t of physostigmine or a f f e c t peripheral sympathetic transmission of impulses to the cardiovascular system. Beta-Hydroxy Thu j a p l i c i n E f f e c t on Blood Pressure and Heart Rate A. Anesthetized Rats Beta-hydroxy t h u j a p l i c i n lOmg/kg. caused a vasopressor response l n a l l nine of the anesthetized i n t a c t rats i n which i t was tested. Figure 10 i l l u s t r a t e s t h i s response. The average increase l n blood pressure was 16 mm.Hg. and occurred three minutes a f t e r an Injection of beta-hydroxy t h u j a p l i c i n . The duration of the pressor response was seven minutes. There was no change l n heart rate l n any of the animals. The I n i -t i a l blood pressure was 68+4.1 mm.Hg.j the heart rate was 340+18 beats per minute. B. Pithed Rats Beta-hydroxy t h u j a p l i c i n 10mg/kg. produced a pressor response l n pithed rats also (table IV). There was no change i n heart rate. The I n i t i a l blood pressure was 34+6.2 mm.Hg. which was less than the i n i t i a l blood pressure of the in t a c t r a t s . The i n i t i a l heart rate was 273+10.6 beats per minute 39 which also was less than the i n i t i a l heart rate of the Intact r a t s . Since i n the pithed rat central responses which control the cardiovascular system were abolished, the response to beta-hydroxy t h u j a p l i c i n In these animals must be of peripheral o r i g i n . T - standard error m ' 5 0 I  1 1 1 t 1 r- rt I 11 0 1 2 3 4 5 6 7 8 1 5 Time (minutes) Figure 1 0 . The vasopressor e f f e c t of beta-hydroxy t h u j a p l i c i n lOmg/kg. l n anesthetized Intact rats (mm.Hg.+s.e,) n=9 C. Repeated Doses To determine whether r e p l i c a t i o n of changes inl b l o p d pressure could be obtained l n each animal, Injections of beta-- hydroxy t h u j a p l i c i n lOmgAg. were administered every t h i r t y minutes to three intact-and three pithed r a t s . The res u l t s obtained appear i n table IV, 40 Table IV Eff e c t on Blood Pressure of Repeated Injections of Beta-Hydroxy Thujaplicin lOmg/kg. Maximum Change i n Blood Pressure (mm.Hg.+s.e.) F i r s t Dose Second Dose Third Dose +8+0 +7.3+1-4 +6.6+1.4 Intact Rat n=3 Pithed Rat n=3 +7+0.58 +7+1.7 +5-3+3.2 I t can be seen that values i n Table IV are simi l a r for Intact and pithed rats upon repeated administration of beta-hydroxy t h u j a p l i c i n . Tachyphylaxis did not appear to develop i n either i n t a c t or pithed rats when three successive doses were given. E f f e c t of Phenoxybenzamine on the Response to Beta-Hydroxy Thujaplicin. Beta-hydroxy t h u j a p l i c i n lOmg/kg. was administered to fi v e anesthetized rats, followed by phenoxybenzamine 5mg/kg. After the effectiveness of the alpha-receptor blockade was eatabllshed, i n the manner previously described, beta-hydroxy t h u j a p l i c i n lOmg/kg. was again administered. The r e s u l t s appear In Table V. Administration of phenoxybenzamine caused a marked reduction i n the ef f e c t of beta-hydroxy t h u j a p l i c i n on blood pressure. Since the response to beta-hydroxy t h u j a p l i c i n f o l -lowing the blocking agent was no d i f f e r e n t than that caused by 41 saline, complete I n h i b i t i o n of the ef f e c t of beta-hydroxy t h u j a p l i c i n appears to have occurred. Table V Ef f e c t of Phenoxybenzamine 5mgAg. on the Vasopressor Response to Beta-Hydroxy Thuj a p l i c i n lOmgAg. Increase i n Blood Pressure (mm.Hg.+s.e.) Before Phenoxybenzamine After Phenoxybenzamine Beta-Hydroxy Thu-j a p l i c i n +24+7.7 +6+2. 6* Normal Saline +5.5+1.3 * " t M test (paired), n=5, p=0.05, s i g n i f i c a n t E f f e c t of Beta-Hydroxy Thujaplicin on Isoproterenol Induced Tachycardia The experiment consisted of two inj e c t i o n s of isopro-terenol 0 . 2 5 u g A g . twenty minutes apart. Beta-hydroxy t h u j a p l i * n c l n lOmgAg* was then given followed at ten and t h i r t y minutes by isoproterenol 0.25ugAg« Heart rate was monitored. The average Increase i n heart rate and duration of the tachycar-diac response are shown i n Table VI. Ross(57) demonstrated that COMT i n h i b i t i o n by a drug could be shown by the following technique. Using an ele c t r o -cardiogram to record heart rate of anesthetized mice, Ross found that the tachycardlac e f f e c t of intravenous isoproterenol was prolonged by a previous i n j e c t i o n of 4-methyltropolone. He also found that tissue COMT a c t i v i t y was reduced by treatment with tropolones and concluded that the prolongation of Isopro-terenol action was due to i n h i b i t i o n of thi s enzyme. However, as can be seen from Table VI, beta-hydroxy t h u j a p l i c i n did not appear to influence the tachycardlac response to isoproterenol i n these experiments l n anesthetized r a t s . Table VI Eff e c t of Isoproterenol 0 . 2 5 u g / k g . Before and After Treatment with Beta-Hydroxy Thujaplicin lOmg/kg. Isoproterenol Induced Tachycardia Before Beta-Hydroxy Thujaplicin After Beta-Hydroxy Thujaplicin Heart Rate Duration Heart Rate Duration beats/minute minutes beats/minute minutes +85+5.7 5.7+1.5 + 9 0 + 1 6 . 3 + 6 . 5 + 1 . 8 E f f e c t of Beta-Hydroxy Thujaplicin on the Vasopressor Response to Physostigmine Using the same procedure as described under gamma-th u j a p l i c i n i t was found that administration of physostigmine 40ug/kg. increased the blood pressure by 47+13.7 mm.Hg. before beta-hydroxy t h u j a p l i c i n and 46.5+12 mm.Hg. aft e r administra-t i o n of beta-hydroxy t h u j a p l i c i n . Four animals were used. I t can be concluded that beta-hydroxy t h u j a p l i c i n had no ef f e c t on the central stimulant action of physostigmine. 43 DISCUSSION A survey of the l i t e r a t u r e suggests that various t r o -polones may produce, at d i f f e r e n t dose l e v e l s , a diverse number of effects i n animals. Some of these effects appear to involve adrenergic mechanisms such as COMT i n h i b i t i o n ( 5 7 » 5 2 ) , i n h i -b i t i o n of dopamine h y d r o x y l a s e ( 1 3 ) , transitory blockade of a l p h a - r e c e p t o r s ( 5 8 ) and reduction of the vasodepressor e f f e c t produced by i s o p r o t e r e n o l ( 5 8 ) . In the study which has been described here, i t was found that gamma-thujaplicin and beta-hydroxy t h u j a p l i c i n had certain actions on the cardiovascular system of the r a t . In the case of gamma-thujaplicin the ef-fects were not consistent i n a l l animals. G amm a-Thu j apl 1 c i n In experiments using anesthetized rats gamma-thujaplicin 30mgAg» produced an Increase l n blood pressure l n some animals and a decrease i n others. The type of response did not appear to. depend on the l e v e l of the I n i t i a l blood pressure, since th i s was s i m i l a r i n a l l r a t s . The heart rate was increased to the same degree i n a l l animals regardless of whether the blood pressure was increased or decreased. Gamma-thujaplicin must have some Influence on the heart either through the central nervous system or through peripheral mechanisms which i s inde-pendent of changes i n blood pressure. 44 Halllday(55) reported that gamma-thujaplicin produced central nervous stimulant a c t i v i t y i n the form of convulsions which were abolished when the r a t was decerebrated. This i s evidence that gamma-thujaplicin can pass through the blood brain b a r r i e r and Into the central nervous system. The pithed r a t was used to investigate the possible r o l e of the central nervous system In the actions of gamma-thujaplicin i n i n t a c t animals. In the pithed rats^ gamma-thujaplicin slowed the heart and lowered the blood pressure. In these rats, i n which the brain and spin a l cord are destroyed, I t must be assumed that these e f f e c t s are due to peripheral actions on the heart and blood vessels. The magnitude of the decrease i n blood pressure i n the pithed r a t was of the same order as that i n the anes-thetized i n t a c t r a t . I t would appear that the vasodepresslon i n the i n t a c t r a t could be due to peripheral action on the blood vessels. I t also must be assumed that the Increase i n heart rate and vasopressor actions produced by gamma-thujapli-c i n i n the anesthetized rats were of central o r i g i n since they do not occur af t e r destruction of the brain and spin a l cord. I t would seem then that gamma-thujaplicin can exert both central and peripheral actions which oppose one another on the cardio-vascular system. In the Intact anesthetized rats, where both of these mechanisms would be operational, the ef f e c t s produced must represent the balance of the opposing actions. . This would explain the fact.that i n some anesthetized rats the blood pres-sure was depressed, while In others i t was Increased. I t 45 could be assumed that i n the former the peripheral action on the vessels overcame the central action. The occurrence of central e f f e c t s i n the presence of a ganglionic blocking agent would indicate that ganglionic blockade was not complete. I t i s possible that varying degrees of sympathetic ganglionic blockade may be partly responsible for the d i f f e r e n t blood pressure responses. I n h i b i t i o n of the vasopressor action of gamma-thujapli-c i n by phenoxybenzamine suggests that t h i s action i s mediated through the postganglionic- sympathetic f i b r e to the blood ves-sels since phenoxybenzamine Is an alpha-adrenergic blocking agent. The reduction of the vasopressor response to gamma-t h u j a p l i c i n by pronethalol was unexpected and cannot rea d i l y be explained. Interference with the action of gamma-thujapli-c i n at whatever receptors i t i s acting on ce n t r a l l y may be a p o s s i b i l i t y . The reduction l n blood pressure produced by gamma-th u j a p l i c i n l n pithed rats apparently did not Involve an ac-ti o n on beta-adrenergic receptors since i t was not antagonized by pronethalol. The i n t e n s i f i c a t i o n by pronethalol of the bradycardia produced by gamma-thujaplicin inethese rats Is probably due to additive effects of the two drugs, since prone-t h a l o l i t s e l f , by v i r t u e of beta-adrenergic blocking a c t i v i t y and d i r e c t myocardial depressant effects can cause bradycardia. Gamma-thujaplicin could cause d i r e c t myocardial depression since 30mg/kg. does not af f e c t the vasodllatory response to isoproterenol and thus does not appear to have beta-receptor adrenergic blocking a c t i v i t y . 46 The r e s u l t s of the experiments i n which gamma-thujapli-c i n was tested for adrenergic blocking action suggests that the tropolone derivative can block receptors acted on by c i r c u l a t -ing noradrenaline but not those occupied by noradrenaline re-leased from sympathetic nerves. This Is i l l u s t r a t e d by the reduction, by gamma-thujaplicin, of the vasopressor response to injected noradrenaline ( f i g . 8) and the absence of e f f e c t by gamma-thujaplicin on the response to physostigmlne which produces a vasopressor e f f e c t mediated through sympathetic nerves. The I n a b i l i t y of gamma-thujaplicin to a f f e c t the physostigmlne response i s l i k e l y due to Its i n a b i l i t y at lower doses to gain access to the receptors at the neuro-effector junction of the vascular smooth muscle. Tachyphylaxis, to the vasopressor ef f e c t , resulted from.-repeated administration of gamma-thujaplicin. This action i s produced c e n t r a l l y and appears to be mediated through sym-pathetic nerves. Upon repeated administration of gamma-thuja-p l i c i n the drug could eventually gain access to and block the receptors at the neuro-effector junctions on vascular smooth muscle. This e f f e c t could r e s u l t i n p a r t i a l blockade of the adrenergic alpha-receptor and tachyphylaxis to gamma-thujaplicin would occur. In single doses, gamma-thujaplicin blocked only responses to exogenous noradrenaline; however, the development of tachyphylaxis suggests that endogenous noradrenaline may be blocked by repeated administration. 47 Beta-Hydroxy Thujaplicin Beta-hydroxy t h u j a p l i c i n lOmg/kg. caused a mean i n -crease i n blood pressure of 16 mm.Hg. i n anesthetized i n t a c t r a t s . The duration of the vasopressor response was longer l a s t i n g than the vasopressor response caused by gamma-thujapli-cin.-.Unlike gamma-thujaplicin, beta-hydroxy t h u j a p l i c i n did not cause any change l n heart rate or a decrease Jft iblood pres-sure i n any of the animals used. Beta-hydroxy t h u j a p l i c i n elevated the blood pressure i n both Intact and pithed rats but had no ef f e c t on heart rate l n either preparation. I t i s concluded therefore that beta-hydroxy t h u j a p l i c i n acts d i r e c t l y on the vascular smooth muscle. Since t h i s action was opposed by phenoxybenzamine i t appears to be mediated through the alpha-adrenergic receptors. This i s l n agreement with r e s u l t s obtained i n t h i s laboratory from experiments with beta-hydroxy t h u j a p l i c i n on i s o l a t e d rabbit a o r t i c s t r i p s . The chronotropic e f f e c t of isoproterenol was the same before and a f t e r an i n j e c t i o n of beta-hydroxy t h u j a p l i c i n . I t would appear that beta-hydroxy t h u j a p l i c i n i n the dose used did not exert beta-receptor blockade or COMT In h i b i t i o n . 48 SUMMARY AND CONCLUSIONS 1 . Intravenous administration of gamma-thujaplicin sodium 3 0 m g / k g . to anesthetized rats caused a moderate increase i n blood pressure i n one-half of the animals used and a smaller decrease In blood pressure In the remaining r a t s . 2. The vasopressor action i n anesthetized rats was of central o r i g i n and was mediated v i a sympathetic nerves to the alpha-adrenergic receptors i n vascular smooth muscle. 3 . The vasodepressor action was the r e s u l t of a d i r e c t action of gamma-thujaplicin on vascular smooth muscle not involv-ing beta-adrenergic receptors. 4. In a l l of the anesthetized rats t h i s dose of gamma-thuja-p l i c i n produced an Increase i n heart rate which was be-li e v e d due to central stimulation. 5 . In pithed rats gamma-thujaplicin 3 0 m g / k g . caused a reduc-tion i n heart rate which suggests that the compound has a d i r e c t action of myocardial depression. 6. In pithed rats the only blood pressure response observed was that of vasodepression. ?. Tachyphylaxis to the vasopressor action of gamma-thujapli-c i n developed i n the anesthetized r a t afterttwo doses were administered. 8. 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