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An investigation into the mechanism and specificity of the antagonism between barbiturates and adrenergic… Friesen, Abram Jacob David 1960

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( i ) AN INVESTIGATION INTO THE MECHANISM AND SPECIFICITY OP THE ANTAGONISM BETWEEN BARBITURATES AND ADRENERGIC AMINES by ABRAM JACOB DAVID FRIESEN B . S . P . , U n i v e r s i t y o f B r i t i s h Co lumbia , 1958 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n t h e Department o f Pharmacology We accep t t h i s t h e s i s as con fo rm ing t o t h e r e q u i r e d s tanda rd THE UNIVERSITY OF BRITISH COLUMBIA September, 1960 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y of B r i t i s h Columbia, Vancouver $, Canada. ABSTRACT Antagonistic inotropic actions of ..barbiturates..and adrenergiccamines have, been demonstrated i n dog hearts. The present work was undertaken to ascertain whether a similar antagonism occurred i n other tissues, and i f so, how sp e c i f i c and potent i t i s . The tissues selected for studying these phenomena were rabbit kidney cortex s l i c e s , uterine horns of previously estrogenized rabbits and guinea pig terminal ileum. The tone and c o n t r a c t i l i t y of the isolated smooth muscle preparation and the active transport of sodium para-aminohippuric acid (PAH) by the kidney cortex s l i c e s , were the processes monitored. No antagonism of the barbiturate induced depression of the active uptake of PAH by kidney cortex s l i c e s on the part of adrenergic amines was observed. Only a weak antagonism between these two groups of drugs was evident under suitable conditions i n the guinea pig ileum. However, i n contrast to these observations, a potent antagonism similar to that demonstrated i n the dog hearts, was evident i n the rabbit uterine horns. The experimental data indicated that adrenergic amines and barbiturates are antagonistic only i n those tissues where barbiturates depress and adrenergic amines stimulate the energy u t i l i z i n g process studied. The antagonism when evident i s not specific and may be c l a s s i f i e d as independent or physiological. ( i i i ) TABLE OP CONTENTS Page I INTRODUCTION 1 II METHODS A. PAH uptake i n rabbit kidney cortex s l i c e s 6 B. Smooth muscle experiments 7 III RESULTS A. PAH uptake i n rabbit kidney cortex s l i c e s 8 B. Smooth muscle experiments 15 IV DISCUSSION 21 V SUMMARY 25 VI BIBLIOGRAPHY 27 (iv) LIST OF TABLES AND FIGURES TABLES Page 1. The ef f e c t of pentobarbital alone and i n the presence of epinephrine or phenylephrine on PAH uptake i n rabbit kidney cortex s l i c e s . 12 2. The ef f e c t of pentobarbital and thiopental alone and i n the presence of a number of adrenergic amines on PAH uptake i n rabbit kidney cortex s l i c e s . 13 3. A summary of the effects of a number of adrenergic amines on guinea pig ileum and rabbit uterine horns i n the presence and absence of pentobarbital as well as various adrenergic blocking agents. 17 FIGURES 1. A time course of PAH uptake i n rabbit kidney cortex s l i c e s i l l u s t r a t i n g the modifying effect of pentobarbital and thiopental. 14 2. Kymograph tracings of contractile a c t i v i t y of terminal guinea pig ileum i l l u s t r a t i n g the a b i l i t y of some adrenergic amines to antagonize pentobarbital i n the presence and absence of various adrenergic blocking agents. 18 3. Same as f i g . 2. 18(a) (v) L i s t of Tables and Eigure3 (contd.) Figures (contd.) Page 4. Part A. Kymograph tracings of guinea pig ileum contractions showing the a b i l i t y of acetylcholine and histamine to antagonize pentobarbital. Parts B and C. Same as f i g . 2 except that here the tissue i s rabbit uterine horn. 19 5. Same as f i g . 2 except that here the tissue i s rabbit uterine horn. 20 (vi) LIST OP ABBREVIATIONS PAH Sodium para aminohippuric acid Pb Sodium pentobarbital Pt Sodium thiopental EPI L-epinephrine bi t a r t r a t e NE DL-norepinephrine hydrochloride INE DL-isopropyl norepinephrine hydrochloride PE Phenylephrine hydrochloride DHE Dihydroergotaraine Ach Acetylcholine bromide Oxy. Oxytocin Microgram mg. Milligram Gm. Gram kg. Kilogram ml. M i l l i l i t r e 1. L i t r e M. Molar °2 Oxygen co 2 Carbon dioxide f i g . Figure Alpha Beta min. Minutes ( v i i ) L i s t of Abbreviations (contd.) lbs. Pounds 0 C Degrees centigrade # Percentage S/M r a t i o Number of mg. of PAH per Gm. of s l i c e s Number of mg. of PAH per ml. of medium I. INTRODUCTION Adrenergic amines have been demonstrated (l) to antagonize the negative inotropic action of barbiturates on dog hearts. This depressant action of barbiturates on heart contractions rather than vasodilation was the predominant cause of the hypotensive effects of barbiturates i n vivo. Norepinephrine (NE), even when i t s vasoconstricting action was prevented by phenoxybehzamine,^allowed the animal to survive four times the l e t h a l dose of pentobarbital (Pb) i n a r t i f i c i a l l y respired dogs. Barbiturates also depress the a c t i v i t y of brain tissues. A number of theories have been postulated as to the biochemical mechanisms underlying the depressant effects of barbiturates on nervous tissue. One of the f i r s t theories presented was that these drugs produced their effects on c e l l s by i n h i b i t i n g oxygen uptake and thereby int e r f e r i n g with energy production. Bain has supported t h i s e a r l i e r theory by demonstrating that barbiturates can depress the oxygen consumption of isolated brain and l i v e r mitochondria (2). More recently, Brody and Bain presented evidence that barbiturates uncouple oxidative phosphorylation (3). The high doses required to demonstrate these two actions, as compared to the dosage required for anesthesia, suggest that i n the central nervous system these mechanisms cannot explain barbiturate action. However, other tissues, such as heart muscle, are less sensitive to these agents and here there appears to be quite a good correlation between dosages required for myocardial depression and the i n h i b i t i o n of respiration or the uncoupling of -2-oxidative phosphorylation. However, i f barbiturates produced their depressant action on the heart by in t e r f e r i n g with energy production through one or both of the discussed mechanisms, one would expect that the l e v e l of the high energy compounds i n the barbiturate treated hearts would be lower than i n the controls. Workers i n t h i s department have found that when heart and diaphragm tissues are exposed to doses of barbiturates s u f f i c i e n t to impair c o n t r a c t i l i t y , the l e v e l of high energy compounds i s very nearly the same as i n the controls (4). A number of other researchers have made similar observations on heart muscle (5, 6, 7 ) . On the basis of t h i s evidence one cannot say that barbiturates produce th e i r effects on the heart solely by impairing energy production. Possibly an i n h i b i t i o n of both energy production and energy u t i l i s a t i o n i s responsible f o r t h e i r action on the heart. Epinephrine (EPI) and related agents on the other hand, activate a number of processes concerned with the expenditure and production of energy. They increase the conversion of glycogen to glucose and favor anaerobic glycolysis i n muscle with the subsequent formation of l a c t i c acid (8). More recently they have been shown to activate phosphorylase i n the heart muscle (9), as well as i n l i v e r and skeletal muscle tissue (10). In addition to these well documented metabolic actions, there are probably a number of. biochemical effects produced which as yet are not well understood. Adrenergic amines also interact with c e l l constituents, which have been c l a s s i f i e d by Ahlquist as -=c and /3 receptors, thereby triggering a chain of events culminating i n a measurable response ( l l ) . The underlying biochemical changes produced by these drug-receptor interactions have not as yet been elucidated. -3-Despite the elucidation of some of the individual biochemical actions of barbiturates and adrenergic amines, the r e l a t i o n of these to their antagonistic actions i n cardiac contractions i s uncertain. In addition, whether a similar antagonism between barbiturates and adrenergic amines occurs i n other tissues has not been determined. The present investigation was prompted by the following questions: (1) Does th i s antagonism occur i n other than heart tissue? (2) Can compounds other than adrenergic amines antagonize the actions of barbiturates? The tissues selected for t h i s study were guinea pig ileum, rabbit uterine horns and kidney cortex s l i c e s . Processes requiring the ultimate u t i l i z a t i o n of energy, namely smooth muscle tone and c o n t r a c t i l i t y , and the active transport of PAH, were monitored. Other workers from t h i s department are experimenting with different tissues on t h i s same general problem. Cross and Taggart demonstrated that rabbit kidney cortex s l i c e s accumulate PAH i n t r a c e l l u l a r l y and that t h i s process requires the expenditure of energy (12). The c l a s s i f i c a t i o n of PAH secretion by the kidney as an active process has also been v e r i f i e d by a number of workers (13, 14, 15, 16). Barbiturates have been shown to depress many processes requiring energy consumption. I t i s not surprising, then, that these same agents were found to depress the tone and c o n t r a c t i l i t y of smooth muscle (8), and to i n h i b i t the active transport of PAH (13, 14). In most instances sympathomimetic amines themselves produce inhibitory effects on i n t e s t i n a l tone and rhythmic contractions. One would therefore be - 4 -inclined to rule out the p o s s i b i l i t y that an antagonism between these two groups of drugs would exist here. However, i f these inhibitory effects could be blocked with a suitable agent, one might show that there was i n fact an antagonism which would otherwise be masked. Bichloroisopropyl arterenol (DCI) has recently been introduced as an agent which selectively blocks the inhibitory actions produced by adrenergic amines on i n t e s t i n a l smooth muscle (17, 18, 19). This agent appeared to provide a useful t o o l for investigation of the p o s s i b i l i t y that an antagonism between adrenergic amines and barbiturates occurs i n i n t e s t i n a l smooth muscle. The uterine horns of previously estrogenized rabbits were used as an example of smooth muscle i n which the predominant effect of sympathomimetic amines i s to enhance tone. The use of two smooth muscle tissues, which d i f f e r i n t h e i r response to adrenergic amines, could provide useful data i n a study such as t h i s . The known actions of adrenergic amines on secretion by the kidney appear to be secondary to th e i r cardiovascular effects rather than di r e c t effects on active transport. However, i f the antagonism were specific or i f the processes involved i n t h i s antagonism are ubiquitous, one might demonstrate that these agents have antagonistic effects on the PAH transport mechanism. Ideally, a greater number of tissues from a number of different species of animals should be used to make such a study more complete and comprehensive. - 5 -Although the present study deals with only a limited number of tissues, certain trends can s t i l l be observed and conclusions drawn. - 6 -I I . METHODS A. PAH Uptake i n Rabbit Kidney Cortex S l i c e s Male rabbits weighing 3 ^ - 5 lbs.were k i l l e d by a blow at the base of the skul l and the kidneys immediately removed and placed i n c h i l l e d normal saline for subsequent s l i c i n g . The methods used for s l i c i n g the cortex, the media used for incubation and the procedure used for the analysis of PAH are similar to those used by Cross and Taggart (12). Approximately 200 mg. of s l i c e s were placed i n 3 ml. of media i n 20 ml. beakers. The beakers were then incubated i n the Dubnoff metabolic shaker at 25° C using a shaking rate of 100/minute. One hundred per cent gaseous oxygen was forced into the incubating chamber at a continuous rate to ensure adequate oxygenation of the s l i c e s . The wet weights of the s l i c e s were taken after incubation following b r i e f b l o t t i n g on dampened f i l t e r paper. The extent to which PAH had been active l y transported was expressed as the ra t i o between the number of mg. of PAH per gram of s l i c e s over the number of mg. of PAH per ml. of media. Henceforth t h i s r a t i o w i l l be referred to as the S/M r a t i o . Percentage recoveries of PAH, as determined by the analytical procedure, were routinely calculated with each determination as a double check on the accuracy of the analysis. I t was found important to compare S/M rat i o s f o r the controls and those with the various combinations of drugs on the same set of kidneys from the same animal, f o r there was considerable variation i n the S/M rat i o s of PAH i n kidney s l i c e s from different rabbits. When t h i s method of comparison was adhered to, consistent results were obtained when the effects of -7-various dosages of drugs were compared with the untreated controls. The auto-oxidation of EPI i s often a problem especially at higher temperatures and over prolonged periods of incubation. Furchgott has suggested the use of ethylene diamine t e t r a acetate ( l x 10 "*) to chelate trace metallic ions which catalyze t h i s oxidation (22). Deionized d i s t i l l e d water should also be used to make up a l l solutions. When these precautionary measures were taken, a b i o l o g i c a l assay showed that there was l i t t l e or no inactivation of EPI. A number of experiments were also conducted with PE because of i t s greater s t a b i l i t y i n the incubating media. B. Smooth Muscle Experiments A l l animals were k i l l e d by a blow at the base of the s k u l l . The desired piece of smooth muscle was immediately removed, placed i n Krebs-Ringer 1s bicarbonate at 37° C and aerated by bubbling 95% 0^ and 5% CO^ i n a fine stream through the media. Segments of terminal ileum were taken from female guinea pigs weighing l\ -2 lbs. Uterine horns were obtained from previously estrogenized rabbits weighing 2 - 3 lbs. The rabbits were estrogenized by injecting 120/^}. of estrogen subcut-aneously per day f o r six days. The animals were then s a c r i f i c e d on the seventh day for their uterine horns. The pieces of uterine horns were opened along the site of the mesenteric attachment before being t i e d to an isotonic lever by their anti-mesenteric border. Conventional kymograph technique was used to record variations i n tone and amplitude of spontaneous rhythmic contractions. -8-III. RESULTS-A. PAH Uptake by Rabbit Kidney Cortex Slices Numerous experiments i n duplicate were carried out under a variety of incubation periods, drug concentrations and different substrates. The S/M ratios f or the controls incubated for 60 minutes i n the presence of added sodium acetate, glucose and i n the absence of added substrate (see table l ) , were similar to those obtained by Cross and Taggart (12). The values which they obtained for t h e i r controls i n the presence of added sodium acetate, glucose and i n the absence of added substrate were: 11.3 + 1.8, 6.1, and 6.5 + 1.8 respec-t i v e l y , i n a 75 minute incubation period. Both Pt and Pb i n adequate doses consistently inhibited PAH uptake. Storen using rat kidney cortex s l i c e s and incubating for 75 minutes with sodium acetate as substrate, obtained approximately 50$ depression of PAH accumulation i n s l i c e s with 9 x 10 M. Pb (14). This compares quite well with our experimental results i n which the s l i c e s were incubated f o r 60 minutes under similar conditions. We got approximately 60$ depression using the same dose of Pb (see table l ) . The active transport of PAH i n kidney s l i c e s appeared to be more sensitive to the action of Pt than i t was to Pb. Pt i n a concentration of 1 x 10 M. i n the presence of glucose as substrate, depressed the active transport of PAH by approximately 50$ i n 60 min. of incubation (see table 2). On the other hand, _4 Pb i n a concentration of 9 x 10 M. under i d e n t i c a l conditions only depressed PAH uptake by approximately 35$ (see table l ) . White, using guinea pig kidney cortex s l i c e s and incubating for 75 minutes -4 i n the absence of added substrate, showed that 1.25 x 10 M. Pt depressed PAH -9-uptake by approximately 45% (13). Under the same conditions but incubating for 60 minutes, we obtained a depression of approximately 60% using 5 x 10 ^ M. Pt (see table 2). When acetate or glucose was added to the medium as substrate a higher concentration of Pt appeared to be necessary to produce the same degree of depressant action (see table 2). A similar relationship did not appear to be true for Pb (see table l ) . This l a t t e r conclusion, however, i s based on only a few experiments i n which substrate was varied. White and Storen, on the other hand, have observed that when acetate was added as a substrate higher doses of both Pb and Pt are required to depress PAH uptake (13, 14). The onset of maximum barbiturate action i n the kidney cortex s l i c e s appeared to be delayed (see f i g . I). Preliminary experiments i n which kidney cortex s l i c e s were allowed to accumulate PAH for 2 hours before the addition of barbiturate also indicated a gradual onset of their action. This gradual onset of action i n the kidney i s i n direct contrast to the almost immediate onset i n smooth muscle tissue, central nervous system and i n the heart. Other preliminary experiments on isolated rat diaphragm and isolated perfused rat gastrocnemius muscle also indicated a gradual onset of barbiturate action and a very slow recovery of the tissue function on washing. In those tissues where the onset of barbiturate action i s prompt, the recovery to i n i t i a l l evels of performance appear to be rapid on washing out the depressant drug. —9 —4 EPI i n concentrations from 1 x 10 M. to 5 x 10 M. was ineffective as an antagonist against the barbiturate induced depression of PAH uptake. In fact, i n a number of experiments EPI i n the higher dosage range ( l x 10**^  to -10-5 x 10 ) appeared to enhance the i n h i b i t i o n of PAH uptake s l i g h t l y (see table 2). A few experiments were also carried out using various dosages of phenylephrine (PE) and isopropyl norepinephrine (INE) but here again there appeared to be no evidence of any antagonism between these agents and barbiturates (see table 2). Most experiments were carried out with EPI, however, for i t has the same qualitative actions that most sympathomimetic amines exhibit. Incubation times were varied from 60 minutes up to 180 minutes on the premise that an antagonism might appear more pronounced i f one incubated for longer periods than 60 minutes. I t can be seen from f i g . I that those s l i c e s which have been treated with barbiturates reach a maximum level of PAH uptake somewhat e a r l i e r than the corresponding controls. I t was f e l t that i f incubation periods were extended to the stage where the controls had reached the maximum level of PAH accumulation, any antagonism between adrenergic amines and barbiturates would be more readily detected. However, S/M ratios at 60 minutes, 120 minutes or 180 minutes s t i l l did not show any indication that an antagonism of the barbiturate e f f e c t had occurred. The maximum S/M ratios for controls incubated i n the presence of sodium acetate appear to be reached between 120 minutes to 150 minutes (12, 20), while those incubated i n the presence of glucose or i n the absence of added substrate appear to reach a maximum S/M level between 90 minutes and 135 minutes (21). Cross and Taggart were the f i r s t to demonstrate the marked stimulatory effect of acetate and some of i t s precursors on the active uptake of PAH i n rabbit kidney cortex s l i c e s (12). They found, however, that the addition of glucose to the media did not a l t e r the a b i l i t y of kidney s l i c e s to accumulate PAH. The use of -11-added acetate or glucose to the media i n our experiments f a i l e d to disclose any antagonism between barbiturates and catechol amines on PAH uptake (see tables 1 and 2). The doses of catechol amines used i n these i n v i t r o experiments are similar to those necessary to produce their characteristic actions i n the intact animal. In addition, however, more potent as well as weaker concentrations were t r i e d without detectable effects. Table 1 . The Effect of Pentobarbital Alone and i n the Presence of Epinephrine or Phenylephrine on PAH Uptake i n Rabbit Kidney Cortex S l i c e s During 6 0 min. of Incubation 8 2 0 mg.% Sodium Acetate 1 0 0 mg.$ Glucose No Substrate No. of S / M % No. of S/M No. of S / M exper. rat i o Controls exper. r a t i o Controls exper. r a t i o Controls Controls 1 2 8 . 9 7 + . 2 8 1 0 0 1 1 6 . 0 1 + . 2 8 1 0 0 ; 6 6 . 4 1 + . 4 6 1 0 0 Pb 9 x 1 0 ~ * M 2 0 5 . 4 4 + . 1 8 6 0 . 6 6 3 . 9 9 + . 3 9 6 6 . 4 - - -Pb 9 x 1 0 M EPI 1 x 1 0 ? M to 1 4 5 . 7 7 + . 2 5 6 3 . 6 1 5 3 . 9 1 + . 2 5 6 5 . 1 - - -1 x 1 0 " 9 M Pb 9 x 1 0 M + _ 3 3 3 5 . 0 9 + . 1 6 5 6 . 7 _ _ _ PE 1 x 1 0 M to 1 x 1 0 - % Table 1 i l l u s t r a t e s the i n a b i l i t y of various dosages of epinephrine and phenylinephrine to antagonize pentobarbital when acetate and glucose are used as substrates. Table 2. The Ef f e c t of Pentobarbital or Thiopental, Alone and i n the Presence of  Several Adrenergic Amines, on PAH Uptake i n Rabbit Kidney Cortex S l i c e s S/M Ratio Controls % Controls Barbiturate io Controls Barbiturate + adrenergic agent Time of incubation Substrate used 9.2 " -4 3x10 M 36.7 EPI 3xlO"^M 39.0 EPI 3xlO~5M 39.1 EPI 3x10 M 38.7 EPI 3xlO"'M 37.8 EPI 3x10*^1 36.2 EPI n 3x10 7M 33.5 60 min. 820 mg. % Sod. acetate 10.2 7 6 -4 9x10 M 40.9 EPI 5x10 M 19.6 EPI 5xlO"3M 37.5 EPI 5x10 M 35.9 EPI 5x10"'M 40.0 EPI o 5x10 M 41.8 - 120 min. 100 mg. # Glucose 5.6 Pt 1x10 M 50.9 PE 1x10 M 45.6 PE lxlO~ JM 55.2 PE , 1x10 M 58.2 PE lxlO"'M 57.6 - - 60 min. 100 mg. % Glucose 6.56 Pt 1x10 M 45.7 INE 1x10 M 45.8 INE 1x10 M 44.4 INE 1x10 M 48.6 - - - 60 min. 100 mg. % Glucose 8.3 Pt , 1x10 M 58.7 EPI l x l O " \ 53.0 EPI 5x10""'M 63.0 EPI 1x10"'M 57.2 EPI 5x10 M 63.9 EPI 1x10 M 55.4 EPI 5xl0~*M 62.2 180 min. 100 mg. % Glucose 7.4 * -5 5x10 3M 56.5 EPI 5x10*^1 66.0 EPI 5x10"'M 54.9 EPI -5xlO"TM 55.0 EPI „ 5x10  yU 60.5 - - 60 min. No Substrate 12.4 -5 2.5x10 ?M 87.9 EPI 2.5x10 M 76.4 EPI , 2.5x10 M 81.5 EPI 2.5xl0~'M 82.3 EPI -2.5x10 M 87.1 - - 120 min. No Substrate Table 2 i l l u s t r a t e s that varying the dose of adrenergic amines, the time of incubation or the substrates, f a i l e d to reveal that adrenergic amines could antagonize the barbiturate induced depression of PAH uptake i n rabbit kidney cortex s l i c e s . Time i n minutes 3(3 ocT 90 1 120 F i g . I. A time course of PAH uptake i n rabbit kidney cortex s l i c e s i l l u s t r a t i n g the modifying e f f e c t of pentobarbital and pentothal. Each point on the graph represents the average of 4 experiments. -15-B. Smooth Muscle Experiments Muscular tone may be d e f i n e d as a sus t a i n e d p a r t i a l degree o f c o n t r a c t i o n . Smooth muscle t i s s u e e x h i b i t s i n h e r e n t tone which may v a r y somewhat from time t o time. The s t a t u s of tone a t any one time was considered t o be the lowest l e v e l of the recorded rhythmic c o n t r a c t i o n s . The extent t o which a drug may i n h i b i t t h i s tone o f t e n depends on the i n i t i a l l e v e l of the i n h e r e n t spontaneous tone (22). I t was found advantageous t h e r e f o r e t o use an agent which would i n c r e a s e tone t o an int e r m e d i a t e l e v e l b efore t e s t i n g the e f f e c t s of b a r b i t u r a t e s . When t h i s procedure was f o l l o w e d adequate doses of Fb i n the same p r e p a r a t i o n produced the same degree of d e p r e s s i o n o f smooth muscle tone. The agents found s u i t a b l e t o i n c r e a s e tone t o the d e s i r e d l e v e l were a c e t y l c h o l i n e f o r guinea p i g ileum and oxytocin- f o r r a b b i t u t e r i n e horn. I n the case of guinea p i g ileum where there i s v e r y l i t t l e rhythmic c o n t r a c t i l i t y , the st a t u s of the tone i s more e a s i l y estimated than i n the r a b b i t u t e r u s . Fb was used throughout a l l of the smooth muscle experiments because of i t s g r e a t e r s t a b i l i t y a t these h i g h e r temperatures. I n a d d i t i o n Pb c o n s i s t e n t l y depresses c o n t r a c t i l i t y and tone w h i l e the e f f e c t s of P t have been r e p o r t e d t o be somewhat v a r i a b l e a t times ( 8 ) . FE, when i n j e c t e d i n t o the medium alone or a f t e r Fb, always i n c r e a s e d guinea p i g ileum tone. T h i s antagonism of Fb by FE c o u l d be blocked by s u i t a b l e doses o f <=<-receptor b l o c k i n g agents (23), e.g. phenoxybenzamine or dihydroergotamine (DHE). When the s t i m u l a t o r y a c t i o n of FE on the tone of t h i s t i s s u e i s blocked, t h i s drug now r e v e a l s t h a t i t possesses d i p h a s i c a c t i o n s , by now r e l a x i n g tone. T h i s r e l a x a t i o n o f tone i n t u r n can be blocked by DCI (see f i g . 2 A and B ) . -16 -EPI differed from PE i n that i t generally caused a further relaxation of the tone of guinea pig ileum. However, i f this relaxing effect produced by EPI i s blocked with DCI, a large dose of EPI can now be shown to antagonize Pb. DHE or phenoxybenzamine can block this antagonism which has been unmasked by DCI (see f i g . 3 A). INE produced effects similar to EPI on guinea pig ileum in the absence of DCI but in the presence of DCI failed to show any significant enhancement of tone (see f i g . 2 C). As might be expected NE exhibited qualitatively the same actions as EPI (see f i g . 3 B and C). EPI, PE and NE i n low doses a l l stimulated the tone of rabbit uterine horn, when used alone or added after Fb. DHE and phenoxybenzamine again demonstrated their ability to block this antagonistic effect. In contrast, INE, whether added alone or after Pb, exhibited only a slight relaxation of tone (see f i g . 4 ) . -17-Table 3. A Summary of the Effects of a Number of Adrenergic Amines  on Guinea Pig Ileum and Rabbit Uterine Horns i n the Presence and Absence  of Pentobarbital as Well as Various Adrenergic Blocking Agents Drug Alone After Pb After Dib + Pb After DCI + Pb After DCI, Dib + Pb GPI Uterus GPI Uterus GPI Uterus GPI GPI EPI 1 I 1 t J 0 t 0 NE J t I t I 0 t 0 INE r l (slight) i I (slight) 1 1 (slight) 0 0 PE r t t t 0 0 t 0 GPI = Guinea pig ileum I = enhancement of tone Dib = Dibenzyline 4- = relaxation of tone 0 = no change i n tone For other abbreviations see l i s t of abbreviations at the beginning of the thesis. -IS-C' I M C i/OA^ A t OCL F i g . 2 These are kymograph tracings i l l u s t r a t i n g the a b i l i t y of some adrenergic amines to antagonize pentobarb depression of guinea p i g ileum tone i n the presence and absence of various adrenergic blocking agents. -18(a)-I V 1K» * t AO. t tvio * bet |1U> ' A<A-. 3 These are kymograph tracings i l l u s t r a t i n g the a b i l i t y of some adrenergic amines to antagonize pentobarb depression of guinea pig ileum tone i n the presence and absence of various adrenergic blocking agents. -19-e F i g . 4 P a r t A Kymograph t r a c i n g s i l l u s t r a t i n g t h a t b o t h a c e t y l -c h o l i n e and h i s t a m i n e as w e l l as phenylephrine can r e v e r s e the pentobarb induced d e p r e s s i o n on guinea p i g il e u m tone. P a r t B and C These a r e kymograph t r a c i n g s i l l u s t r a t i n g the a b i l i t y o f some a d r e n e r g i c amines to antagonize pentobarb d e p r e s s i o n o f r a b b i t u t e r i n e horn tone i n the presence and absence of v a r i o u s a d r e n e r g i c b l o c k i n g agents. -20-A / W W W J W"1 i60/*U&UuJt> 0*^  one Fig. 5 A / V W \ A A A A t These are kymograph tracings illustrating the ability of som« adrenergic amines to antagonize pentobarb depression of rabbit uterine horn tone in the presence and absence of various adrenergic blocking agents. -21-IV. DISCUSSION The f a c t that no antagonism between barbiturates and adrenergic amines was demonstrated on the active transport of PAH favors the view that t h i s antagonism, as witnessed under appropriate conditions i n the dog heart, guinea pig ileum and i n rabbit uterus, occurs i n only those tissues where barbiturates depress and adrenergic amines stimulate the energy u t i l i z i n g process studied. DHE and phenoxybenzamine can block the antagonism produced by PE, EPI and NE i n the smooth muscle tissues studied. This f a c t indicates that their antagonistic action was due to t h e i r combination or interaction with c*c-receptors. In the heart, however, t h i s antagonism i s probably not due to t h e i r interaction with ^ - r e c e p t o r s because i t cannot be blocked with phenoxybenzamine i n t h i s tissue ( l ) . This view i s supported by the observation that INE can antagonize Pb i n the heart-lung preparation of the dog ( l ) but f a i l e d to exhibit a comparable antagonism i n our smooth muscle experiments. The Pb depression of smooth muscle tone appeared unaltered i n the presence of DCI, DHE or phenoxybenzamine or i n the presence of any combination of these adrenergic blocking agents. From these observations one must conclude that Fb i s not acting on receptors and therefore the antagonism observed i n the heart and i n our smooth muscle experiments must be viewed as independent or physiological (24). Other in d i r e c t evidence suggesting the same view i s the f a c t that certain adrenergic amines can also antagonize quinidine and procaine-amide i n the isolated rabbit heart (25). In addition, i t was observed i n the -22-experiments on guinea pig ileum that acetylcholine or histamine also can reverse Fb depression (see f i g . 4 A). Experiments performed on the kidney s l i c e s and preliminary ones conducted on isolated rat diaphragm and isolated perfused rat gastrocnemius muscle indicated that the onset of maximum barbiturate action was gradual. In contrast to th i s , the effects of barbiturates on smooth muscle tissue, on the central nervous system and on the heart are almost immediately maximal. This difference i n the response of various tissues to barbiturates may be due to the different rates at which these agents diffuse to the site of action. On the other hand, the predominant mechanism responsible for barbiturate action may vary from tissue to tissue. A rapid onset of maximum barbiturate action may occur because energy-utilizing processes have been inhibited. Conversely, i f barbiturates produce their action predominantly by impairing energy production, a more gradual onset of maximal action would be anticipated. Experiments correlating the rates at which barbiturates diffuse into different tissues with the onset of barbiturate action and the level of high energy compounds may be helpful i n accounting for the observed va r i a t i o n i n the onset of maximum barbiturate action. A dose of 60 mg. of Pb or Pt per kg. of body weight i n a r t i f i c i a l l y respired dogs produces approximately a 50$ depression of cardiac function ( l ) . A concentration of 200 mg. of Pb per 1. of media ( i n the presence of added glucose) depresses the PAH uptake by approximately 35$. This concentration of Fb compares with a concentration of 25 mg. of Pt per 1. under similar conditions to i n h i b i t PAH uptake by approximately 50$. One can see from the doses l i s t e d that both Pt and Pb were equally effective i n producing cardiac f a i l u r e but that Pt i s about 10 -23-times more potent i n depressing PAH uptake by kidney cortex s l i c e s . Although both of these agents probably produce their action on the heart by the same mechanism there i s a p o s s i b i l i t y that i n the kidney Pt exerts i t s action on PAH uptake v i a a different route. Additional evidence supporting t h i s view i s the fac t that White demonstrated he could i n h i b i t PAH uptake by 80$ without i n t e r --4 fering with oxygen consumption using a dose of 6.25 x 10 M. Pt i n the absence of added substrate (13). In contrast to t h i s , Storen observed a f a i r l y close relationship between a b i l i t y of Fb to depress PAH uptake and i t s i n h i b i t i o n of oxygen consumption i n kidney s l i c e s (14). Doses of Pb i n the range of 50 to 100 mg. per 1. were required to depress tone by 50$ i n the smooth muscle experiments. This dosage range of Fb agrees quite well with that required to i n h i b i t cardiac function i n the dog to a comparable degree. However, two to three times these doses of Fb were necessary to i n h i b i t PAH uptake to the same extent. The a b i l i t y of some catechol amines to antagonize Fb i n the guinea pig ileum appears to be r e l a t i v e l y weak. Concentrations of these agents as high as -5 -4 5 x 10 M. to 1 x 10 M. were required to demonstrate an antagonism of Fb on t h i s tissue. In the rabbit uterine horn experiments FE, NE and EPI demonstrated a potent antagonism of Pb similar to that observed i n dog hearts. In h i s more recent work, Ahlquist, using DCI, has concluded that i n t e s t i n a l smooth muscle has oc as well as/? receptors (19). His e a r l i e r view was that intestine had only ocreceptors ( l l ) . Our observations support his more recent conclusion but d i f f e r i n that our experiments, using adrenergic blocking agents on guinea pig ileum, suggest that PE, EPI and NE, when interacting with <=< -- 2 4 -receptors, enhance tone and when interacting with ft receptors relax tone. Ahlquist, however, states that adrenergic amines always produce relaxation of in t e s t i n a l tone and i n h i b i t rhythmic contractions whether they interact with ^ or /3 receptors (19). He bases t h i s conclusion on experiments conducted on Fb anesthetized dogs pretreated with morphine and atropine. Obviously a more comprehensive study of i n t e s t i n a l receptors i n various species i s required to c l a r i f y the discrepency i n our independent observations. -25-V. SUMMARY 1. Previous studies have demonstrated that barbiturates depress contractions of cardiac muscle and that adrenergic amines antagonize t h i s depression. The actions and interactions of these agents have been examined i n other systems: accumulation of para-aminohippurate (PAH) by rabbit kidney cortex s l i c e s , contractions of i n t e s t i n a l and uterine smooth muscle. 2. Pentobarbital (Pb) and thiopentone (Pt) i n h i b i t accumulation of PAH by rabbit kidney s l i c e s . The inhibitory action of barbiturates on active accumulation of PAH d i f f e r e d from their depressant effect on cardiac contraction i n the following respects: i t was not antagonized by epinephrine, norepinephrine or a number of other adrenergic amines; the dose of Pb required to i n h i b i t PAH accumulation by 50% was two to three times the dose required to depress cardiac contractions to a similar degree; Pt was ten times more potent than Pb i n i n h i b i t i n g PAH accumulation but only equally effective i n depressing cardiac contractions. 3. Pb inhibited uterine tone and spontaneous contractions i n doses similar to those required to i n h i b i t cardiac contractions and adrenergic amines were potent antagonists of the barbiturate-induced depression. Similar doses of Pb were also effective i n i n h i b i t i n g the increase of tone of guinea pig ileum induced by acetylcholine. Adrenergic amines were ineffective antagonists of t h i s depression except i n the presence of dichloroisopropylnorepinephrine (DCI) which prevented their inhibitory actions. Even i n the presence of DCI, the antagonistic action of these amines was weak. Phenoxybenzamine and dihydroergotamine prevented the antagonistic actions of adrenergic amines to barbiturate depression of both types of smooth muscle. In this respect, the antagonism between barbiturates and adrenergic amines i n smooth muscle differed from that i n cardiac muscle. This difference suggested that the antagonistic action of adrenergic amines depended on the i r a b i l i t y to stimulate muscle contractions rather than on the type of receptor with which they combine. The demonstration of a stimulant action of adrenergic amines on i n t e s t i n a l smooth muscle i n the presence of DCI which i s antagonized by phenoxybenzamine suggests that this tissue possesses ^ a s well as/? receptors. There were differences i n the various systems i n the time required to obtain maximal barbiturate actions and to remove the effects of these drugs. In smooth muscle, as i n cardiac muscle, the onset of the maximum action of barbiturates was immediate and t h e i r effects were rapidly removed by washing. In s l i c e s of rabbit kidney cortex, however, at least an hour's exposure to barbiturates was required to obtain maximum effects. This suggested the p o s s i b i l i t y that the mechanism of action of barbiturates differed i n kidney s l i c e s from that i n cardiac and smooth muscle. •27-VI. BIBLIOGRAPHY 1. Daniel, E.E., Fulton, J.B., Hiddleston, M.W., and Foulks, J.G. Arch. Intern. de Pharmacodyn. et de Ther., 1956, 108, 457. 2. Bain, J.A. Fed. P r o c , 1952, 11, 653. 3. Brody, T.M., and Bain, J.A. J . Phann. and Exper. Ther., 1954, 110, 148. 4. Foulks, J.G., and Perry, Florence A. Unpublished observations. 5. Harvey, S.C. Am. J . Physiol., 1955, 183, 559. 6. Furchgott, R.F., and T a i s i j a , G. J . Pharm. and Exper. Ther., 1958, 124, 203. 7. Fawaz, G., and Hawa, E.S. Proc. of the Soc. for Exper. B i o l , and Med., 1953, 84, 277. 8. Goodman, L.S., and Gilman, A. The Pharmacological Basis of Therapeutics, 2nd edition, 1955, pp. 132-133, 489-490. 9. Hess, Marilyn E., and Haugaard, N. J . Pharm. and Exper. Ther., 1958, 122, 169. 10. Sutherland, E.W. In: Symposium of phosphate and phosphorous metabolism. 1951. 11. Ahlquist, R.P. Am. J . Physiol., 1948, 153, 586. 12. Cross, R.J., and Taggart, J.V. Am. J . Physiol., 1950, 161, 181. 13. White, A.G. Am. J . Physiol., 1957, 191, 50. 14. Storen, E.J. Am. J . Physiol., 1958, 195, 343. 15. Foulkes, E.C., and M i l l e r , B.F. Am. J . Physiol., 1959, 196, 86. 16. Forster, R.P., and Copenhaver, J.H. Am. J . Physiol., 1956, 186, 167. 17. Powell, C.E., and Slater, I.H. J . Pharm. and Exper. Ther., 1958, 122, 480. 18. Moran, N.C., and Perkins, Marjorie E... . J.Pharm. and Exper. Ther., 1958r, 124, 223. -28-19. Ahlquist, R.P., and Levy, B. J . Pharm. and Exper. Ther., 1959, 127, 146. 20. Haung, K.C., King, N.B., and Genazzani, E. Am. J . Physiol., 1958, 192, 373. 21. Parah, A., and Bennick, B. J . Pharm. and Exper. Ther., 1956, 117, 428. 22. Purchgott, R.P. Pharm. Rev., 1955, 7, 183. 23. Actions of Epinephrine and Norepinephrine at the Cellular Level and Subcellular Sites. Pharm. Rev., 1959, 429-565. 24. Drug Antagonism. Pharm. Rev., 1957, 9, 211. 25. Drysdale, A.A. Unpublished observations. 

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