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An improved assay procedure for dopamine-B-hydroxylase Holubitsky, Don J. 1981

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AN IMPROVED ASSAY PROCEDURE FOR DOPAMINE-B-HYDROXYLASE by DON J. HOLUBITSKY B. Sc., The Univ e r s i t y of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Anatomy We accept t h i s "thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1980 © Don Holubitsky, 1980 In present ing th is thes is in p a r t i a l fu l f i lment of t h e r e q u i r e m e n t s f o r an advanced degree at the Un ivers i ty of B r i t i s h C o l u m b i a , I a g r e e t h a t the L ibrary sha l l make it f ree ly ava i l ab le for r e f e r e n c e and s t u d y . I fur ther agree that permission for extensive copying o f t h i s t h e s i s for scho la r ly purposes may be granted by the Head o f my D e p a r t m e n t o r by h is representat ives . It is understood that c o p y i n g o r p u b l i c a t i o n o f th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. DON J . HOLUBITSKY Department of ANATOMY The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date December 8, 1980 ABSTRACT Two of the e x i s t i n g assay procedures for dopamine B-hydroxylase were studied with respect to t h e i r s u i t a b i l i t y i n the measurement of low l e v e l s of a c t i v i t y . S p e c i f i c a l l y , the spectrophotometric method and the coupled radioenzymatic methods were assessed r e l a t i v e to t h e i r a b i l i t y to detect DBH a c t i v i t y i n the perfusate of is o l a t e d r a t t a i l a r t e r i e s incubated under conditions known to i n h i b i t the sodium pump. The spectrophotometric method , was the i n i t i a l choice because of i t s low cost, greater ease of performance, and the fac t that DBH i s assayed under saturating conditions. Although i t was found that t h i s method suffered from a lack of s e n s i t i v i t y , attempts were made to improve t h i s . The blank value was reduced and s t a b i l i z e d by the introduction of a -4 -4 mixture of 5 X 10 M f u s a r i c acid and 5 X 10 M EDTA, which produced routine blank l e v e l s of &A= 0.005, equal to the lowest reported l i t e r a t u r e values. Also, the e f f e c t of ADP a c t i v a t i o n was investigated f o r i t s s u i t a b i l i t y i n the procedure. Despite a three-fold increase i n measured DBH a c t i v i t y , however, t h i s method was s t i l l not s e n s i t i v e enough to detect the enzyme a c t i v i t y i n tiss u e incubates, although these improvements made the method more suitable f o r the assessment of lev e l s of DBH a c t i v i t y in the serum of laboratory animals. • The second choice was the coupled radioenzymatic method of Molinoff, - i i i -because of i t s inherently greater s e n s i t i v i t y . While i n i t i a l experiments proved t h i s procedure e f f e c t i v e i n the detection of the release of DBH from r a t vas deferens under conditions known to stimulate maximum exocytosis i t was f e l t that problems could be encountered i n the measurement of DBH a c t i v i t i e s obtained under less than optimal conditiona. Therfore, the procedure was investigated with respect to maximizing the absolute s e n s i t i v i t y . Preliminary studies on the f e a s i b i l i t y of extended incubation i n the f i r s t step, led to a r a t i o n a l e i n which the two enzymic reactions were i s o l a t e d and studied separately. In the second step, i t was found that tyramine as expected, i n h i b i t e d the PNMT rea c t i o n , although with sigmoidal k i n e t i c s . Paradoxically, non-dialyzable and heat stable impurities that i n h i b i t e d PNMT were found i n commercial preparations of catalase, although the addition of ascorbate eliminated t h i s e f f e c t . Bovine serum albumin was found to s e l e c t i v e l y activate PNMT i n a highly concentration dependent manner, with a f i v e - f o l d maximum a c t i v a t i o n r e s u l t a n t from the i n c l u s i o n of 0.14% BSA i n the sample al i q u o t . Blank mixture and reac t i o n time were investigated, and a doubling of s e n s i t i v i t y was found to r e s u l t from l i m i t i n g r eaction time f o r the second step to 25 minutes. The PNMT was also shown to be unsaturated with respect to SAM, and an increase i n the t o t a l concentration of SAM by the addition of l a b e l l e d and unlabelled SAM to a concentration 40 times normal, was found to make the second reaction l i n e a r with respect to octopamine concentration. F i n a l l y , the [PNMT] could be increased up to 20 times - i v -without a f f e c t i n g l i n e a r i t y , and t h i s produced an increase i n s e n s i t i v i t y i n d i r e c t proportion to the increase i n enzyme concentration. These modifications were s u f f i c i e n t l y e f f e c t i v e so as to allow an increase i n the concentration of tyramine i n the f i r s t r e a c t i o n mixture without too much of a loss of a c t i v i t y . This made the DBH r e a c t i o n l i n e a r with respect to both time and enzyme concentration, which greatly improves accuracy and c o r r e l a t a b i l i t y of r e s u l t s . T r i a l experiments proved that the combination of a l l these modificat-ions into an improved procedure resulted i n an assay with an improvement i n s e n s i t i v i t y of at least two orders of magnitude over the standard method, with v a s t l y improved c h a r a c t e r i s t i c s of time and concentration l i n e a r i t y . The s u i t a b i l i t y of t h i s method for our planned t i s s u e release studies was also confirmed. I t i s hoped that the improvement i n s e n s i t i v i t y and l i n e a r i t y of t h i s modified procedure w i l l allow i t s use i n new experim-ental s i t u a t i o n s . - V -TABLE OF CONTENTS T i t l e Page Introduction 1 Materials 36 Methods A. Spectrophotometric assay 3 7 B. Molinoff assay 40 C. Tissue techniques 45 D. S t a t i s t i c s 48 Results 49 Discussion 101 References - v i -LIST OF FIGURES Figure T i t l e Page 1 Pathway of biosynthesis of NE and Epinephrine. 12 2 Basic reactions employed i n the assay of DBH. 27 3 Absorbance values obtained with standard concentrations 52 of octopamine f o r the spectrophotometric assay. 4 Absorbance values obtained with standard concentrations 54 of p u r i f i e d DBH i n the spectrophotometric assay. 5 The e f f e c t of ADP a c t i v a t i o n on DBH . a c t i v i t y as measured 58 by the spectrophotometric method; 6 T y p i c a l t i t r a t i o n curve f o r DBH a c t i v i t y obtained with 63 various concentrations of CuSO. 4 7 Plot of DBH a c t i v i t i e s obtained f o r a ser i e s of 66 concentrations of p u r i f i e d enzyme i n the standard Molinoff method. 8 The e f f e c t of reaction time i n the second step on blank 69 values i n the Molinoff assay. 9 The e f f e c t of extended incubation time on DBH a c t i v i t y 71 obtained with p u r i f i e d enzyme i n the standard Molinoff assay. 10 A c t i v i t i e s produced by standard octopamine concentrations i n 74 the Molinoff assay. 11 The e f f e c t of the i n c l u s i o n of ascorbate and tyramine on 76 the a c t i v i t y obtained with standard concentrations of octopamine i n the Molinoff assay* 12 The e f f e c t of various concentrations of tyramine on the 77 a c t i v i t y obtained with a standard concentriion of octopamine. 13 The e f f e c t of the i n c l u s i o n of catalase, ascorbate, and 80 pargyline i n MRM-1, on the a c t i v i t i e s produced by standard concentrations of octopamine in the Molinoff assay. - V l l -Figure T i t l e Page 14 The s t a b i l i t y of octopamine i n the Molinoff assay over 82 extended incubation times. 15 L i n e a r i t y of a c t i v i t y obtained with a standard concentration 84 of octopamine with respect to incubation time f o r the second step of the Molinoff assay. 16 E f f e c t of,the i n c l u s i o n of BSA i n the reaction mixture 86 for the second step of the Molinoff assay on the a c t i v i t y produced by a standard concentration of octopamine. 17 The e f f e c t of increased concentration of ^ C-SAM on a c t i v i t y 88 produced by a standard concentration of octopamine. 18 L i n e a r i t y of octopamine standards with d i f f e r e n t t o t a l 89 concentrations of SAM i n the second reaction mixture. 19 The e f f e c t of increased concentration of PNMT on the a c t i v i t y 91 obtained with a standard concentration of octopamine jLn the Molinoff assay. 20 L i n e a r i t y of octopamine standards with increased concentra^. ,92 t i o n of PNMT i n the Molinoff assay. 21 The e f f e c t of extended incubation i n the f i r s t r e action step 94 on DBH a c t i v i t y obtained with the p u r i f i e d enzyme i n the Molinoff assay with modified RM2. 22 L i n e a r i t y of extended incubation i n the f i r s t step of the 96 Molinoff .assay with increased concentration of tyramine. 23 L i n e a r i t y of the modified Molinoff assay with respect to 98 concentration of DBH. 24 Extended incubation of high potassium r a t vas deferens incubate without saturating [SAM], 99 V X 1 Z -LIST OF TABLES TABLE 1 A summary of assay methods employed f o r the measurement of Dopamine B-hydroxylase a c t i v i t i e s . 2 Blank values obtained by various methods of DBH i n h i b i t i o n . 3 The .effect of various blank procedures on blank values obtained with the Molinoff method. - o 14 4 C o r r e l a t i o n between time i n storage at 20 C f o r C-SAM versus blank l e v e l . - ix -LIST OF ABBREVIATIONS ADP Adenosine diphosphate ANS . Autonomic nervous system ATP Adenosine triphosphate BSA Bovine serum albumin COMT Catechol-o-methyltransferase DA Dopamine DBH Dopamine B-hydroxylase DEDTC Diethyldithiocarbamate DTT D i t h i o t h r e i t o l EDTA Ethylenediaminetetraacetic acid p-HB p-Hydroxybenzaldehyde HPLC High performance l i q u i d chromatography MAO Monoamineoxidase MRM-1 Modified reaction mixture- 1 (Molinoff method ) NE Norepinephrine OCT octopamine PNMT Phenylethanolamine-N-methyltransferase PS Parasympathetic nervous system PSS Ph y s i o l o g i c a l s a l t s o l u t i o n RM1 F i r s t r e a c t i o n mixture ( Molinoff method ) RM2 Second reaction mixture ( Molinoff method ) SAM S-adenosylmethionine SNS Sympathetic nervous system Acknowledgments I wish to express my sincere appreciation f o r the patience, cheeful enthusiasm, and excellent supervision given to me by my advisor, Dr. V. Palaty, without whose help t h i s t h e s i s could never have been completed. Many thanks also f o r the f r i e n d l y advice and excellent t e c h n i c a l assistance given me by Miss Maryette Mar. The tolerance and patience of the members of the f a c u l t y and s t a f f of the Dept. of Anatomy were prime f a c t o r s i n the completion of t h i s work, and the general enjoyment of my graduate studies. The assistance of a Canadian Heart Foundation Research Traineeship as w e l l as as previous B.C.Heart Foundation support i s i a l s o g r a t e f u l l y acknowledged. To my f r i e n d s , e s p e c i a l l y Dorothy, Mark, Kevin, A r i s , Linda, Susan, 2 Margaret, V i r g i n i a , Fred , Laurie, Klaus, David, Colleen, and Chris: Thank you. And to my A l l i e , thank you more than I can ever say. Introduction The vertebrate nervous system i s the mediator and e f f e c t o r of a l l actions,both .conscious, and unconscious. However, the apparent dichotomy between voluntary and involuntary c o n t r o l seemed hard to r e c o n c i l e with the established anatomical concept of a single integrated nervous system. This c o n f l i c t was r a t i o n a l i z e d by investigators as early as the ancient Greeks, by the d i v i s i o n of the nervous system into two separate but f u n c t i o n a l l y r e l a t e d networks: the somatic and autonomic systems, a concept that has persisted to the present day. Much of the -.early work on the c l a s s i f i c a t i o n of the nervous system, e s p e c i a l l y i n the anatomical sense, was accomplished p r i o r to the 1800*s. However, the terminology of autonomic and somatic, sympathetic and parasympathetic, as well as an understanding of the f u n c t i o n a l s i g n i f i c a n c e of these d i v i s i o n s was proposed by Langley i n the l a t e nineteenth century ( LANGLEY,.1898-). In h i s conception, s t i l l v a l i d today, the autonomic nervous system was s o l e l y responsible f o r the regulation and i n t e g r a t i o n of v i s c e r a l function and metabolism, as w e l l as f o r those p r i m i t i v e body refle x e s t i e d to emotional responses. In t h i s manner, the autonomic nervous system ( ANS ) controls the smooth muscle surrounding a l l of the extracorporal c a v i t i e s ( gut, blood vessels, and glands ) as well as - 2 -the cardiac muscle of the heart. Control i s therefore exerted on such diverse body functions as r e s p i r a t i o n , c i r c u l a t i o n , d i gestion, temperature regulation, and arousal. This l a s t and most prominent feature was termed the " f i g h t or f l i g h t r eaction " by Cannon ( CANNON, 1929 ), and involves the quickened heart r a t e , shallow r e s p i r a t i o n , d i l a t e d p u p i l s , h a i r f o l l i c l e e rection and sweating c h a r a c t e r i s t i c of intense fear or rage. By way of contrast, the somatic nervous system which controls the s t r i a t e d muscle of the s k e l e t a l structure, i s involved p r i m a r i l y with postural c o n t r o l and i n the expression of voluntary motor commands. This system i s capable of highly s e l e c t i v e and c o n t r o l l e d a c t i v a t i o n , while the autonomic nervous system i s e s s e n t i a l l y an a l l or none phenomenon. It can therefore be appreciated that, while a d e f i c i e n c y or t o t a l i n t e r r u p t i o n of the peripheral somatic outflow can be tolerated without threat to l i f e , such a deprivation of peripheral autonomic function would be l e t h a l . By Langley's c l a s s i f i c a t i o n , the ANS i s divided into two f u n c t i o n a l l y and anatomically d i s t i n c t systems, sympathetic and.parasympathetic. Each v i s c e r a l organ commonly receives innervation from both networks, the two being f u n c t i o n a l l y opposed, a f a c t that allows a f i n e measure of c o n t r o l . The parasympathetic system ( PS ) i s the less prominent of the two anatomically, and i s confined to the v i s c e r a of the body core. Its action i s e s s e n t i a l l y concerned with processes involved i n the conservation - 3 -of energy and the recharging of body resources. As such, therefore, PS stimulation leads to a decrease i n the heart rate, i n the rate of r e s p i r a t i o n and basal metabolism, and to bronchiole c o n s t r i c t i o n and peripheral v a s o d i l a t i o n . The sympathetic nervous system ( SNS ) on the other hand, i s anatomically more d i s t i n c t i v e and present throughout the body, both i n the periphery and.'.body-'.core. By i t s action the SNS promotes the u t i l i z a t i o n of body reserves f o r the production of usable metabolic energy. This involves an increase i n the basal metabolic r a t e , heart rate and breathing, a d i l a t i o n of the bronchioles, and perhaps most important i n our studies, peripheral v a s o c o n s t r i c t i o n . As mentioned before, t h i s i s most e a s i l y v i s u a l i z e d i n the f i g h t or f l i g h t response. These e f f e c t s are mediated by a network anatomically and biochemically d i f f e r e n t from the somatic nervous system. The primary s t r u c t u r a l difference l i e s i n the f a c t that there are always two neurons involved i n the efferent pathway from the s p i n a l cord. This necessitates a synapse i n the periphery, usually i n close association with the target organ. The parasympathetic outflow e x i t s from the brainstem and s a c r a l cord l e v e l s as the c r a n i a l nerves and n e r v i erigentes r e s p e c t i v e l y , while the efferent sympathetic neurons a r i s e from the l a t e r a l horn of the thoracic and upper lumbar cord and run with the emerging s p i n a l nerves f o r a - 4 -short distance. The sympathetic component, cons i s t i n g of l i g h t l y myelinated small diameter neurons, separate and extend to the sympathetic trunk, an interconnected chain of segmental ganglia l y i n g l a t e r a l to the cord throughout most of i t s extent. Within these ganglia, synaptic contact i s established with post-ganglionic neurons ( f i n e diameter unmyelinated f i b r e s ) usually i n a r a t i o of one to t h i r t y , although t h i s number va r i e s between ti s s u e s ( BURNSTOGK and COSTA, 1975 ). This efferent bundle t r a v e l s back to the sp i n a l nerve f o r peripheral d i s t r i b u t i o n , usually i n close a s s o c i a t i o n with major blood v e s s e l s . Because of t h e i r c o l o r i n f r e s h specimens, these pre- and post-ganglionic bundles are termed the white and grey rami r e s p e c t i v e l y . With recognition of such anatomical d i s t i n c t i o n s , the next breakthro-ugh i n ch a r a t e r i z a t i o n occurred at the histochemical l e v e l . Although i t was speculated that the transmitters involved at sympathetic and parasympathetic synapses were d i f f e r e n t , the exact chemical i d e n t i t i e s were not proved u n t i l much l a t e r . Both the pre- and post-ganglionic neurotransmitters of the parasympathetic system were found to be a c e t y l -choline, the transmitter at the s k e l e t a l neuromuscular junction. In the case of sympathetic nerves, the s i t u a t i o n i s much more complicated. Although an observed s i m i l a r i t y between the e f f e c t s of i n j e c t i o n of adrenal extract and sympathetic nerve stimulation ( LEWANDOWSKY, 1898; LANGLEY, 1901 ), led E l l i o t t to propose adrenaline ( now more commonly c a l l e d epinephrine ) as the peripheral neurotransmitter i n the SNS ( ELLIOTT, - 5 -1905 ), "later i nvestigators showed this.not to be the case. Despite the demonstration of the release of a sympathomimetic substance, c a l l e d sympathin ( CANNON and ROSENBLUTH^ 1937 ), with pharmacological and chemical s i m i l a r i t y to epinephrine ( LOEWI, 1921; CANNON and URIDIL, 1921 ), sereral differences were noted between the action of administered epinephrine and the e f f e c t s of SNS stimulation ( BARGER and DALE, 1910 ). In f a c t these e f f e c t s were found to be more c l o s e l y p a r a l l e l e d by the a p p l i c a t i o n of norepinephrine ( NE ), which led several investigators to suggest that NE was the peripheral sympathetic neurotransmitter ( BACQ, 1934; STEHLE and ELLSWORTH, 193 7; GREER et a l . , 1938 ). Confirmation of NE as the SNS transmitter was provided when i t was shown that NE was the predominant catecholamine i n mammalian adrenergic neurons ( VON EULER, 1946 ) and the release of NE was demonstrated from splenic nerve ( PEART, 1949 ). The neurochemistry of the SNS i s therefore as follows. Acetylcholine i s found to be the t r a n s m i t t e r i , a l l sympathetic pre-ganglionic neurons, as w e l l as i n a small proportion of post-ganglionic f i b r e s ( t h e SNS serves some peripheral parasympathetic function due to the peripheral absence of the PS ). Epinephrine i s produced by the chromaffin c e l l s of the adrenal medulla f o r general d i s t r i b u t i o n by the c i r c u l a t o r y system, while extra-adrenal chromaffin c e l l s , that serve as interneurons i n the SNS, employ dopamine or NE as neurotransmitters ( BURNSTOCK and COSTA, 1975 ). F i n a l l y , NE i s involved as the transmitter i n the majority of post-ganglionic • - 6 -neurons of the SNS. Since a l l peripheral adrenergic nerurons employ NE, the function of the sympathetic system i s e s s e n t i a l l y i d e n t i c a l to the operation of the peripheral adrenergic system, a f a c t with great c l i n i c a l and pharmacological s i g n i f i c a n c e . Accordingly, i t i s these adrenergic nerves which p a r t i c u l a r l y i n t e r e s t us from the stand point of t h e i r involvement i n cardiovascular problems such as l a b i l e hypertension, as w e l l as other c l i n i c a l l y s i g n i f i c a n t disorders. The remainder of t h i s work w i l l therefore deal e x c l u s i v e l y with peripheral adrenergic neurons. The development of the formaldehyde fluorescence technique by Falck and H i l l a r p (FALCK et a l . , 1962 ) revolutionized the histochemical l o c a l i z a t i o n of catecholamines i n adrenergic neurons, and allowed a more precise c o r r e l a t i o n between neurotransmitter storage and s u b c e l l u l a r structures. T y p i c a l adrenergic neurons have t h e i r c e l l bodies located i n ganglia i n close proximity to the target organ. A short pre-terminal axon extends to the organ where i t develops into a network of f i n e l y branching nerve f i b r e s which widen at i n t e r v a l s into a series of regular swellings, the terminal v a r i c o s i t i e s ( HILLARP, 1946 ). This network i s the so c a l l e d autonomic ground plexus.. The density of adrenergic innervation, as w e l l as the extent of branching of i n d i v i d u a l axons v a r i e s widely between ti s s u e s ( BURNSTO.CK and COSTA, 1975 ). However, i n general, within these e f f e c t o r t i s s u e s the Schwann.cell envelope i s l o s t leaving the terminal v a r i c o s i t i e s naked, and the nerve f i b r e l i e s i n close association with smooth muscle c e l l s . The s i z e of the motor u n i t and the pattern of - 7 -innervation also v a r i e s between t i s s u e s . In most, the innervation i s incomplete and most smooth muscle c e l l s are activated by i n t r a c e l l u l a r e l e c t r i c a l coupling, while i n c e r t a i n organs such as vas deferens, each smooth muscle c e i l receives d i r e c t innervation ( BURNSTOCK and COSTA, 1975). Fluorescence studies have indicated that while a l l portions of an adrenergic nervefctypically contain catecholamine, the terminals, and i n p a r t i c u l a r the terminal v a r i c o s i t i e s concentrate the majority of the transmitter ( IVERSON, 1967; GEFFEN and LIVETT, 1971 ). Electron microscopy showed that w i t h i n these terminal v a r i c o s i t i e s , NE i s contained i n s i d e small membrane l i m i t e d v e s i c l e s that c h a r a c t e r i s t i c a l l y . have electron-dense cores or c e n t r a l i n c l u s i o n s when observed a f t e r glutaraldehyde or osmium tetroxide f i x a t i o n ( TRANZER et a l . , 1969; GEFFEN and LIVETT, 1971; HOKFELT, 1971; BISBY and FILLENZ, 1971; DE POTTER e f i a l . , 1971; SMITH 1972 ). These v e s i c l e s or storage granules occur i n at least two d i s t i n c t populations d i f f e r i n g i n s i z e , with average diameters of approximately 300-600 and. 800-1200 A* r e s p e c t i v e l y , (and a t h i r d population of agranular small v e s i c l e s has been reported by a number of authors ( HOKFELT, 1969; FILLENZ, 1971; TRANZER, 1973 ). In addition to s i z e , these v e s i c l e populations have been found to e x h i b i t a d i f f e r e n c e i n density, a f a c t which has allowed the i s o l a t i o n , p u r i f i c a t i o n , and c h a r a c t e r i z a t i o n of the small and large v e s i c l e s from a number of sources by the use of density gradient c e n t r i f u g a t i o n ( ROTH et a l . , 1968; HORTNAGL et a l . , 1969; CHUBB et a l . , 1970; DE POTTER et a l . , 1970; BISBY et a l . , 1973; KLEIN and TURESON-KLEIN, 1975; LAGERCRANTZ, 1976; FREID et a l . , 1978 ). - 8 -While the r e l a t i v e populations of these two f r a c t i o n s vary-between both t i s s u e s and species ;( BURSTOGK and COSTA, 1975 ), the small dense cored v e s i c l e s tend to predominate i n adrenergic terminals, while the large f r a c t i o n may comprise only a few percent, as i n the vas deferens ( B I S B Y e t a l . , 1973 ) and heart ( FILLENZ and WEST, 1974 ), or may make up almost one t h i r d of the t o t a l , the.case i n s^Lenic nerve terminals (TRANZER, 1973 ). Although i t i s agreed that these v e s i c l e s store the bulk of the c e l l s NE, controversy s t i l l e x i s t s as to the r e l a t i o n s h i p between these d i f f e r e n t v e s i c l e f r a c t i o n s , and the existence of a number of d i s t i n c t but interchangable pools of NE d i f f e r i n g i n t h e i r l a b i l i t y f o r release ( DAHLSTROM, 1973; BURNSTOCK and COSTA, 1975 ). Evidence seems to indicate the d i v i s i o n of NE stores into p r i m a r i l y a small newly synthesized and e a s i l y releasable f r a c t i o n that comprises one to two percent of the t o t a l ( HAGGENDAL and .LINDQVIST, 1964; KOPIN et a l . , 1968; YEN et a l . , 1973 )',• as w e l l as a much more stable storage pool. Although these appear to be the major transmitter pools, a storage s i r e i n the cytoplasmic matrix possibly corresponding to the tubular endoplasmic reticulum has been reported ( TRANZER, 1972, 1973 ). As w e l l , a small pool of free cytoplasmic NE c e r t a i n l y functions under some conditions of pharmacologically induced release, i . e . reserpine or tyramine treatments, and may be of importance i n normal p h y s i o l o g i c a l release, a f a c t to be dealt with more f u l l y l a t e r . However, the v e s i c u l a r pools of NE are of most importance because of t h e i r established p a r t i c i p a t i o n i n release, and because they sequester and protect accumulated NE from the e f f e c t s of the i n t r a c e l l u l a r degradative enzyme, mitochondrial monoamine oxidase ( MAO ). - 9 -I t i s s t i l l uncertain as to whether the two main pools of NE r e l a t e d i r e c t l y or c o i n c i d e n t a l l y to the two v e s i c l e f r a c t i o n s . However, i t appears l i k e l y that the small dense-cored granules, which store the bulk of the c e l l s NE ( BISBY et a l . , 1973; NELSON and MOLINOFF, 1976 ) correspond, to the more stable storage pool, while the large v e s i c l e f r a c t i o n , which contains only a small proportion of the transmitter pool, stores the r e a d i l y releasable, newly synthesized NE ( DAHLSTROM, 1973; FILLENZ and WEST, 1974 ). Also uncertain are the r e l a t i o n s h i p s between the v e s i c l e f r a c t i o n s themselves, i . e . whether the small v e s i c l e s are derived from the larger ones i n a process of f i s s i o n ( SMITH et a l . , 1970) or whether they are the product of membrane r e t r i e v a l following exocytosis or by some other process (HOLTZMAN et a l . , 1973; HOLTZMAN, 1977; DAHLSTROM, 1973 ). The large v e s i c l e s are known to o r i g i n a t e i n the c e l l body ( DE POTTER and CHUBB, 1977 ), and are trans ported to the terminals by active axoplasmic transport at a rate of several mm / hr ( DAHLSTROM, 1973 ). Indeed, non-terminal axons contain almost e x c l u s i v e l y large v e s i c l e s , quite the opposite case to the terminal v a r i c o s i t i e s ( DE POTTER, 1971 ). These storage granules are transported i n almost mature form, and i n a l l cases, are found to contain small amounts of NE ( GEFFEN and RUSH, 1968; LAGERCRANTZ et a i . , 1974 ), due to the presence of both a f u n c t i o n a l amine pump, as w e l l as the intragranular presence of the f i n a l synthetic enzyme f o r NE, dopamine-B-hydroxylase ( DBH ). However, t h i s c o n t r i b u t i o n of NE by axonal v e s i c l e transport i s i n s i g n i f i c a n t compared to the s i z e of the pool i n nerve terminals, and undoubtably does not make a s i g n i f i c a n t contribution - 10 -to the maintenance of transmitter stores ( GEFFEN and RUSH, 1968 ). This process involves primarily a combination of active reuptake of released NE as w e l l as i n s i t u synthesis, which w i l l be discussed s h o r t l y . In additiorito NE, the catecholamine storage granules are known to contain a number of other intragranular components, although again controversy e x i s t s as to t h e i r d i s t r i b u t i o n between v e s i c l e f r a c t i o n s . Contained within the v e s i c l e s are ATP, Mg+^, high concentrations of ascorbate, DBH, and several ( at least 8 ) of a c l a s s of a c i d i c proteins, the chromogranins, of which chromogranin A appears to be the most consistent and important ( DE"POTTER et a l . , 1970; BANKS and HELLE, 1971 ). These molecules appear to be membrane associated and bound together i n a semi soluble matrix i n the v e s i c l e i n t e r i o r , which may represent the dense core seen with glutaraldehyde-0s04 f i x a t i o n ( TRANZER and THOENEN, 1968 ). Of these intragranular molecules, dopamine-B-hydroxylase i s the most important, both from the standpoint of i t s p a r t i c i p a t i o n i n NE synthesis, as w e l l as i t s usefulness:, i n release studies. However, no c l e a r agreement e x i s t s as to the presence of DBH i n the two populations of storage v e s i c l e s . While some investigators f i n d DBH i n both f r a c t i o n s ( BISBY et a l . , 1973; NELSON and MOLINOFF, 1976 ), most locate the bulk of the DBH a c t i v i t y i n the heavier p a r t i c l e s ( DAHLSTROM, 1973; DE POTTER and CHUBB, 1977 ), a f a c t that would seem to be consistent with the postulate that the large v e s i c l e s contain the l a b i l e , newly synthesized - 11 -NE pool. As with most areas of adrenergic neurotransmission, a c e r t a i n discrepancy a r i s e s between t h i s and the f a c t that the l i g h t e r v e s i c l e s appear to be p r e f e r e n t i a l l y depleted of NE during prolonged nerve stimulation ( BISBY et a l . , 1971 ), and that DBH, which i s only releasable by exocytosis, i s l i b e r a t e d i n the same proportion to NE as i s found i n the soluble f r a c t i o n of v e s i c l e homogenates i n t h i s process ( SMITH, 1971; WEINSHILBOUM et a l . , 1971 ). A much more extensive knowledge of the neurochemistry of adrenergic endings w i l l be necessary i n order to assess the implications of these release studies. The. biochemical pathway of synthesis of catecholamines i s now both well characterized and histochemically l o c a l i z e d . I n i t i a l l y suggested by Blaschko as early as 1939 ( BLASCHKO, 1939'), a sequential conversion of tyrosine to NE and epinephrine was confirmed i n the adrenal medulla ( BLASCHKO, 1957; KIRSHNER, 1957; UDENFRIEND and:.W'lJNGAARTEN, 1956 ) and i n sympathetic nerves ( GOODALL and KIRSHNER, 1958 ). Three enzymes ( see f i g . 1 ) are.involved i n the synthesis of NE ( four f o r epinephrine ), i n i t i a l l y proposed to occur i n a single p a r t i c l e ( UDENFRIEND, 1966 ) , though t h i s i s now known to be f a l s e . With the advent of more precise sub-c e l l u l a r f r a c t i o n a t i o n techniques, each step can now be c y t o l o g i c a l l y l o c a l i z e d ( STJARNE and LISHANKO, 1967 ). The i n i t i a l steps, the conversion of 1-tyrosine to 1-dopa by tyrosine hydroxylase, and the subsequent conversion of 1-dopa to dopamine by 1-dopa decarboxylase were found to occur i n the axoplasm. While c e r t a i n studies have indicated that at le a s t 12 L-Tyrosine L-Dopa Dopamine HO-<?V •CH 2-CH-NH 2 COOH Tyrosine hydroxylase HOy v HO-V \\_CH 2-CH-NH 2 COOH L-Dopa decarboxylase H 0 7 — \ H0-(/ V - C H 2 - C H 2 - N H 2 Dopamine -hydroxylase HO-/ Norepinephrine H 0 - < ^ y - C H ( O H ) - C H 2 - N H 2 Phenylethanolamine-N-methyltransferase Epinephrine HO HO ,Mu_CH(OH)-CH 2-NH-CH3 Figure 1. Pathway of biosynthesis of NE and Epinephrine - 13 -a portion of the tyrosine hydroxylase appears to be transported down the axon at a rate comparable to v e s i c u l a r DBH, which may indicate some degree of membrane association ( COYLE and WOOTEN, 1972 ), these enzymes are c e r t a i n l y extravesicular. The f i n a l conversion of dopamine to NE occurs in s i d e the storage granules, due to the exclusive intragranular l o c a t i o n of DBH, a fa c t which i s of great s i g n i f i c a n c e i n release studies. In those tissues that also release epinephrine, the enzyme phenylethanolamine-N-methyltransferase ( PNMT ), which catalyses the methylation of NE to epinephrine, also has an intragranular l o c a t i o n AXELROD, 1962 ). The regulation of synthesis of NE i s under control at a number of l e v e l s , such that the rate of production i s t i e d to the l e v e l of nervous a c t i v i t y ( BURNSTOCK and COSTA, 1975 ). This c o n t r o l i s exerted p r i m a r i l y through two d i f f e r e n t mechanisms, f a s t and slow i n onset. In v i t r o experiments ( UDENFRIEND, 1966 ) have demonstrated, that the rate of the tyrosine hydroxylase reaction i s f a r slower than the other two: t h i s i s the rate l i m i t i n g step. In the f a s t mechanism of regulation, the rate of synthesis of 1-dopa i s held under co n t r o l by the l e v e l of presynaptic NE ( WEINER, 1970 ). Nerve stimulation, therefore, w i l l tend to reduce t h i s concentration, allowing an accelerated rate of synthesis. In, contrast to t h i s mechanism, the slow process causes an increase i n tyrosine hydroxylase a c t i v i t y that manifests i t s e l f a f t e r several hours, and reaches a peak a f t e r several days ( THOENEN et a l . , 1970; AXELROD et a l . 1970 ). This process occurs at the c e l l body and r e s u l t s from an induction - 14 -of enzyme synthesis stimulated by incresed pre-ganglionic nerve a c t i v i t i e s . This has also been reported i n the adrenal medulla, where a s i m i l a r mechanism apparently a f f e c t s the l e v e l of PNMT ( AXELROD, 1970 ). I t can be appreciated that t h i s slow process of enzyme induction i s of great importance i n the reaction of the SNS to stresses such as p h y s i c a l t r a i n i n g or environment, as w e l l as i n such pathological conditions as hypertension, while the rapid c o n t r o l mechanism allows f i n e tuning of the SNS i n response to transient changes i n the l e v e l of a c t i v i t y . These are not the only mechanisms whereby the l e v e l s of NE i n adrenergic terminals can be regulated.. Tyrosine hydroxylase i s under regulation by a number of compounds, i n p a r t i c u l a r those degradative metabolites produced by the action of MAO on cytoplasmic NE, such as DOPEG^C BURNSOCK and COSTA, 1975 ). In addition, DBH may p a r t i c i p a t e i n the c o n t r o l of NE synthesis i t s e l f , although i n v i t r o experiments, might not suggest t h i s . I t i s well established that i n a l l t i s s u e s so f a r investigated, there e x i s t a number of potent endogenous i n h i b i t o r s of DBH a c t i v i t y , which have been determined to be polypeptides or sulphydryl compounds( DUCH and KIRSHNER,.1971 '). What i s not c l e a r , however, i s whether these compounds are present within the i n t e r n a l m i l l i e u of eon5lst«rrf' the catecholamine storage granules . While some studies are^with such access i n vivo ( MOLINOFF et a l . , 1977 ),no conclusive evidence f o r the p h y s i o l o g i c a l r e g u l a t i o n of NE synthesis by endogenous DBH i n h i b i t o r s has yet emerged. 7 - 15 -The f i n a l and perhaps most s i g n i f i c a n t process of con t r o l over l e v e l s of NE i n adrenergic terminals, involves the fate of NE released by nerve stimulation. Unlike c h o l i n e r g i c nerves, no degradative enzymes ex i s t i n the synaptic c l e f t to process released neurotransmitter. Removal of NE from the synapse i s accomplished by two s p e c i f i c uptake processes, into the cytoplasm of neurons and surrounding extraneuronal elements ( prim a r i l y smooth muscle c e l l s ). Reuptake into nerve terminals, termed uptake^ by Iversen ( IVERSEN, 1967 ) i s the more e f f i c i e n t of the< two processes, with estimates of the amount of transmitter recaptured going from 50 - 90 %"( DE POTTER, 1971; BURNSTOCK and COSTA, 1975 ). Uptake-^ i s an active saturable pump, that shows high a f f i n i t y and stereochemical s p e c i f i c i t y f o r 1-NE, and i s capable of concentrating NE by a f a c t o r of 10,000 f o l d above external concentrations . However, at concentrations above 10 ^ g / ml , such as would occur during a nerve impulse, the rate of removal of NE declines r a p i d l y , suggesting that uptake^ may function p r i m a r i l y between impulses ( BURNSTOCK and COSTA, 1975 ). This amine pump can be s e l e c t i v e l y ; i n h i b i t e d by a number of NE r e l a t e d sympathomimetics, as-well as by cocaine and other drugs ( IVERSEN, 1967 ). Several fates may b e f a l l the NE accumulated into the axoplasm of nerve terminals by uptake^. The most s i g n i f i c a n t of these f o r adrenergic transmission involves the subsequent pumping of NE back in t o the depleted v e s i c l e s , i n p a r t i c u l a r the small v e s i c l e f r a c t i o n ( HAGGENDAL and DAHLSTROM, 1971 ). In t h i s manner, the peripheral transmitter pool - 16 -i s conserved as much as possibl e , thus minimizing the need f o r i n s i t u synthesis. A process i n competition with t h i s v e s i c l e reloading mechanism i s the metabolism of free axoplasmic NE by intraneuronal enzymes. The bulk of the NE subjected to t h i s fate i s deaminated by monoamine oxidase located within the outer membrane of mitochondria. One of the major products of t h i s process i s DOPEG, which functions i n feedback regulation of tyrosine hydroxylase a c t i v i t y , and i s p o t e n t i a l l y very important as an ind i c a t o r of cytoplasmic NE release. Much of the deaminated product d i f f u s e s passively from the neuron and i s subsequently methylated i n the l i v e r and other tissues by catechol-o-methyltransferase ( COMT ) p r i o r to excretion. Adrenergic neurons do contain low l e v e l s of COMT i n the axoplasm, so some of the deaminated metabolites are methylated i n s i t u as well ( BURNSTOCK and COSTA, 1975 ). A s i m i l a r uptake process, termed uptakeg ( IVERSEN, 1967 ), i s present i n the extraneuronal elements ( pri m a r i l y i n smooth muscle c e l l s ) surrounding adrenergic terminals. Uptake^ i s a less saturable, low a f f i n i t y process that i s not s t e r e o c h e m i c a l ^ s p e c i f i c f o r NE. ( DAHLSTROM, 1973; BURNSTOCK and COSTA, 1975 ). Unlike neuronal uptake, a l l NE taken up into smooth muscle i s metabolized, again by MAO and COMT, although i n t h i s case o-methylation i s the primary route. This extraneuronal uptake process can be i n h i b i t e d by a number of drugs including normetanephrine, metanephrine, phenoxybenzamine, and ste r o i d s : i n .general d i f f e r e n t than uptake ' i n h i b i t o r s ( IVERS'EN, 1967 ). - 17 -F i n a l l y , although a majority of the released NE i s recaptured e i t h e r by uptake^ or uptake,, , a s i g n i f i c a n t proportion of the transmitter d i f f u s e s away from the neuromuscular junction and escapes into the c i r c u l a t i o n unmodified ( DAHLSTROM, 1973 ). This free NE also has a r o l e i n regulating the stimulation induced release of the transmitter through feedback :activ a t i o n of presynaptic i n h i b i t o r y ©<- receptors ( STARKE, ,1979). This feedback i n h i b i t i o n can be blocked by agents such as phentolamine and phenoxybenzamine. To r e c a p i t u l a t e , therefore, transmitter turnover i n adrenergic nerve terminals i s a rather involved process. Severa^&ifferent pools of NE mainly e x i s t i n the terminal v a r i c o s i t i e s contained^within two main classes of membrane l i m i t e d storage v e s i c l e s , but also found i n the axoplasm as well' as i n a possible axoplasmic storage s i t e . While these pools d i f f e r i n t h e i r l a b i l i t y , interconversion i s possible and c e r t a i n l y of p h y s i o l o g i c a l s i g n i f i c a n c e . These NE pools are maintained p a r t l y by i n s i t u synthesis from t y r o s i n e , a process which comes under complex regulat i o n . Release of NE i n response to nerve stimulation probably occurs by v e s i c l e fusion with the axoplasmic membrane and subsequent exocytosis, with p r e f e r e n t i a l depletion of the l i g h t v e s i c l e f r a c t i o n . This released NE i s p r i m a r i l y -taken up into both neuronal and extraneuronal elements where much, of i t i s metabolized by deamination and O-methylation, and the remainder i s r e -accumulated i n the storage v e s i c l e s . A c e r t a i n overflow of NE d i f f u s e s away into general c i r c u l a t i o n . - 18 -It i s i n the nature of the release process i t s e l f that much of the controversy e x i s t s concerning adrenergic neurotransmission. The obvious s i m i l a r i t i e s between adrenergic terminals and the somatic neuromuscular junction i n the v e s i c u l a r mode of transmitter storage, a w e l l defined and s p e c i a l i z e d synaptic complex, and a quantal nature of release led most investigators to favor an exocytotic mechanism of release, which i s generally accepted today. However, there iis by no means agreement as to whether i n vivo release occurs under a l l p h y s i o l o g i c a l conditions by t h i s same mechanism. For example, c e r t a i n drug treatments may release NE by v e s i c u l a r displacement through a cytoplasmic route, and the persistence of NE release i n the absence of external calcium induced . under c e r t a i n conditions might seem to indicate e i t h e r a. non-exocytotic component to normal release, or the p a r t i c i p a t i o n of endogenous calcium stores. In any event, a great deal of research has been performed i n the past two decades i n order to elucidate the p h y s i o l o g i c a l mechanism of NE release. On the whole, the concensus of early studies was i n agreement with the concept of an exocytotic release mechanism f o r NE. I n i t i a l electron microscopic evidence ( DE ROBERTIS and VAZ FERREIRA, 1957 ) indicated that i n NE release, the granule membrane fused with the axoplasmic membrane, and secretion of the v e s i c l e contents followed. While t h i s was not e n t i r e l y supported by other evidence, l a t e r work ( PYSH and WYLEY, 1974 ) which showed a stimulation induced decrease i n the absolute number of v e s i c l e s , and an enlargement of the axonal surface membrane area and a l t e r a t i o n of structure i n presynaptic terminals, would seem to support t h i s . However, - 19 -! due to d i f f i c u l t i e s i n technique and i n t e r p r e t a t i o n , electron microsocpy studies alone are not conclusive. Perhaps more enlightening, and c e r t a i n l y more p r o l i f i c are studies characterizing the release of NE from perfused or i s o l a t e d t i s s u e s such as the heart, vas deferens, seminal v e s i c l e , spleen, and adrenal medulla. Numerous studies demonstrated the release of chromoganiri A and DBH concommittant with the release of NE ( SMITH, 1972 ). S i m i l a r l y , s u b c e l l u l a r f r a c t i o n a t i o n studies snowed the exclusive intragranular l o c a t i o n of these large non-diffusable proteins .( STJARNE and LISHANKO, 1968; BANKS and HELLE, 1971 ). Such studies also indicated a r e l a t i v e l y constant proportion between NE and DBH found i n the soluble f r a c t i o n of t i s s u e homogenates ( FILLENZ, 1971 ), and other work demonstrated that DBH was released i n s i m i l a r proportions to NE during adrenergic nerve stimulation ( SMITH et a l . , 1970; WEINSHILBOUM e t . a l . , 1971 ). This seemed to i n d i c a t e that i n t e r n a l v e s i c l e proteins might be s u i t a b l e as markers f o r the exocytotic release of. NE. Consistent with t h i s hypothesis were the reports of NE; and DBH depletion i n nerve terminals under prolonged stimulation ( FILLENZ, 1971; FILLENZ and WEST,. 1974 ) , the loss of granular matrix density a f t e r adrenergic stimulation ( KLEIN and THURESON-KLEIN, 1974 ), and the absence of release induced by stimulation or high external potassium i n the absence of external calcium ( VIVEROS et a l . , 1969; SMITH, 1972 ). F i n a l l y , i n reserpine treated nerve terminals, which d i s p l -aces NE from the storage -granules, stimulation produced a release of DBH - 20 -without accompaying NE release, i n a calcium dependent manner ( THOA et a l . , 1975 ). A l l such evidence seemed to point towards an exocytotic mechanism fo r normal NE release. Later work produced much less c l e a r cut and more co n t r o v e r s i a l r e s u l t s that indicated at least under c e r t a i n conditions, the p a r t i c i p a t i o n of alternate mechanisms of release. For example, early studies by Thoa and Kopin which were taken as unequivocal proof f o r exocytosis ( THOA et a l . , 1975 ), also showed a s i g n i f i c a n t release of NE and DBH i n the absence of external calcium. S i m i l a r l y , tyramine and reserpine have been proved to release NE by a cytoplasmic route, p a r t i c u l a r l y i n the presence of MAO i n h i b i t o r s ( SMITH, 1972 ). Of extreme s i g n i f i c a n c e are the controver-s i a l r e s u l t s obtained i n the release of NE by ouabain and conditions which i n h i b i t the sodium pump e.g. low external sodium. Previous studies have shown t h i s ouabain induced NE release to be f u l l y ( KATSURAGI et a l . , 1978; NAKAZATO et a l . , 1978 ) or p a r t i a l l y dependent ( GARCIA and KIRPEKAR,. 1973) i on the presence of external calcium. Others ( VIZI, 1978 ) have demonstrated a calcium independent release which i s consistent with reports of exocytotic release of NE and DBH from adrenals by sodium deprivation i n the absence of external calcium ( LASTOWECKA and TRIFARO, 1974 ). While s t i l l open to controversy, t h i s has been taken by some people to indicate the p a r t i c i p a t i o n of i n t r a c e l l u l a r calcium stores i n an exocytotic release mechanism under these conditions. Blaustein ( 1979 ) - 21 -speculated that t h i s involves mitochondrial and endoplasmic reticulum stores, but t h i s explanation cannot be e n t i r e l y c o r r e c t , because Thoa found the release induced by the calcium ionophore A 23187 to be strongly dependent on external calcium concentrations ( THOA et a l . , 1974 ) S i m i l a r l y , the release of NE from cytoplasmic pools may be f e a s i b l e by a r e v e r s a l of uptake^ or some other process. While not conclusive, these r e s u l t s do point out the relevance of the study of the mechanism of release of catecholamines induced under such nonstandard conditions as the i n h i b i t i o n of the sodium pump. From such studies might emerge a better understandung of the mechanism of NE release under p h y s i o l o g i c a l l y normal conditions. C l e a r l y , therefore, such mechanistic studies demand the use of an accurate and r e l i a b l e marker of v e s i c l e exocytosis. This i s p a r t i c u l a r l y true i n l i g h t of the d i f f i c u l t i e s encountered i n following non-exocytotic release. While ;.-,.?*'. metabolites such as. DOPEG, produced by the intraneuronal conversion of NE accumulated i n the axoplasm by MAO, can be used as i n d i c a t o r s of the l e v e l s of axoplasmic NE, the methodology of such studies i s by no means w e l l established. It should be obvious that the number of p o t e n t i a l exocytotic markers i s quite l i m i t e d . Of the i n t e r n a l contents of the storage - 22 -v e s i c l e s known to be l i b e r a t e d during t h i s process ( SMITH, 1971 ), ATP can be rejected because of the lack of c e l l u l a r e x c l u s i v i t y , which would lead to obvious problems i n the determination of l e v e l s i n t i s s u e homogenates and incubates. Similarly,- chromogranin A i s a biochemically i n e r t molecule, and therefore presents c e r t a i n problems i n detection. While i t i s possible to follow the protein with immunochemical techniques, and indeed, the release of chromogranin A,along with NE and DBH was demonstrated by a number of authors ( SMITH et a l . , 1970; DE POTTER et a l . , 1976 ),cert a i n problems do e x i s t . The r e l a t i v e proportions of.the chr-omogranins are not ne c e s s a r i l y constant between t i s s u e s , the concentrat-ions are quite low ( BANKS and HELLE, 1971 ), and immunological cross-r e a c t i v i t y has been reported between antisera prepared towards DBH and chromogranin A ( RUSH and GEFFEN, 1980 ). It i s therefore-much more a t t r a c t i v e to r e l y upon the t h r i d major intragranular component, DBH, as the marker of choice i n studies of exocytotic release. The primary reason i s that DBH i s contained e x c l u s i v e l y within the catecholamine storage granules, and consequently exocytosis i s the only possible release-route. In support of t h i s are studies which show that tyramine and reserpine, which release NE through the cytoplasm, w i l l not release DBH, and that DBH alone can be released from catecholamine depleted v e s i c l e s ( reserpine treatment-) under conditions known to promote exocytosis ; ( THOA et a l . , 1975 ). Also, DBH possesses enzymatic activity,.making detection r e l a t i v e l y easy through the use of assay techniques. DBH has shown a long term s t a b i l i t y i n serum and perfusate - 23 -( ESHEL et a l . , 1978 ), and as such i s suitable f o r i n vivo studies on adrenergic a c t i v i t y , which can be of great use i n c l i n i c a l i n v e s t i g a t i o n . F i n a l l y , most ind i c a t i o n s are that the release of DBH i s proportional to that of NE, and therefore by following both NE and DBH release, i t should be possible to assess the proportion of e f f l u x occurring by an exocytotic mechanism. Accordingly, numerous studies haveafocused on the i n v e s t i g a t i o n of the release of DBH from various t i s s u e s , under a v a r i e t y of conditions. Dopamine-B-hydroxylase ( E.C. 1. 14. 17. 1. ), because of i t s unique p o s i t i o n as one of the keys to,the understanding of adrenergic neurotransmission, has become one of the most.intensely studied proteins i n the l a s t decade ( FREIDMAN and KAUFMAN, 1965; WEINSHILBOUM, 1979; RUSH and GEFFEN, 1980 ). A copper containing oxygenase, DBH.catalyzes the hydroxylation of a wide v a r i e t y of phenylethylamine derivatives using ascorbate and 0^ as cofactors, showing very l i t t l e substrate s p e c i f i c i t y . In f a c t , tyramine has been shown to be a better substrate than the natural substrate dopamine. With dopamine the optimum pH fo r conversion i s 6.2, which i s i n accord with the a c i d i c i n t e r n a l m i l l i e u of the storage v e s i c l e s , while the optimum f o r tyramine i s approximately 5.5 . Native DBH i s a tetramer of molecular weight 290,000 daltons, c o n s i s t i n g of four i d e n t i c a l subunits, each with one copper atom. The enzyme i s a glycoprotein with 4-67o carbohydrate by weight, and" differences have been reported between the carbohydrate content of soluble and membrane bound forms ( AUNIS et a l . , 1973, 1977 ), although t h i s data i s not conclusive. While early studies - 24 -indicated a c r o s s r e a c t i v i t y between chromogranin A and DBH, l a t e r work has confirmed these proteins as separate e n t i t i e s ( WINKLER, 1976 ). In addition to endogenous polypeptide and sulphydryl i n h i b i t o r s , DBH i s i n h i b i t e d by a wide range of copper chelators such as EDTA and.diethyl-dithiocarbamate, SH compounds such as DTT and glutathione, and p i c o l i n i c , acid d e rivatives l i k e f u s a r i c acid ( RUSH and GEFFEN, 1980 ). Although extremely u s e f u l , studies employing DBH as a marker f o r exocytosis are not without t h e i r problems. One of these originates from the previously noted d i v e r s i t y of storage v e s i c l e s within peripheral adrenergic terminals. In addition to the main large and small dense cored v e s i c l e s , some authors f i n d a t h i r d population of aganular small v e s i c l e s ( which may or may not be f i x a t i o n a r t i f a c t s ), and as discussed before, controversy exists as to the r e l a t i v e NE and DBH contents rof these populations. This d i v i s i o n of catecholamine stores into at least.two v e s i c l e f r a c t i o n s with d i f f e r i n g DBH contents creates obvious problems, and more accurate knowledge i s most c e r t a i n l y required on the p a r t i c i p a t i o n of these pools i n normal p h y s i o l o g i c a l release, i n order to assess the v a l i d i t y of DBH as a r e l a t i v e marker f o r NE release*' However, i f as most authors assume, ei t h e r i m p l i c i t l y or t a c i t l y , the f a c t that only a small f r a c t i o n of NE stores i s released during nerve stimulation, and depletion occurs p r e f e r e n t i a l l y from a sing l e pool might serve to preserve the p r o p o r t i o n a l i t y of NE and DBH release ( DAHLSTROM, 1973; BURNSTOCK and COSTA, 1975. ). This i s c e r t a i n l y an area f o r future research. - 25 -A second problem with t h i s approach i s the f a c t that not a l l of the DBH present i n the storage granules and possibly of that released along with NE, i s enzymically a c t i v e . I t i s w e l l established i n the l i t e r a t u r e that a large proportion of intragranular DBH e x i s t s i n l a t e n t form, although detergent s o l u b i l i z a t i o n appears to release a l l enzyme i n an active form ( KIRKSEY et a l . , 1978 ). Early workers estimated that 80-90 % of the DBH was.membrane bound and therefore unavailable f o r release, as based on studies i n v o l v i n g hypoosmotic shock of adreno-medullary v e s i c l e s ( BELPAIRE and LADURON, 1968; SMITH and WINKLER, 1972 ). While some authors postulate a possible s t r u c t u r a l difference between the soluble and membrane associated variants of the enzyme ( AUNIS et a l . , 1973, 1977 ), disagreement e x i s t s as to whether i n f a c t t h i s insoluble enzyme i s maibrane bound, membrane associated, or merely bound i n an insoluble matrix i n the v e s i c l e core ( KIRKSEY et a l . , 1978; YEN et a l , 1973 ). In addition, i t has been demonstrated that a large proportion of the i n t r a -granular DBH e x i s t s i n a latent form, presumably because of complexation of the enzyme i n the v e s i c l e matrix ( KIRSEY et a l . , 1977 ). This r a i s e s the p o s s i b i l i t y of a p a r t i a l exocytosis mechanism i n which only a c e r t a i n proportion of soluble DBH i s a v a i l a b l e f o r exocytotic release ( DAHLSTROM, 1973 ), a concept c e r t a i n l y a t t r a c t i v e i n terms of conser-vatio n of the c e l l s biosynthetic machinery. However, t h i s latency of i n t r a v e s i c u l a r DBH could also be explained by the i n t e r n a l presence of endogenous i n h i b i t o r s , a point s t i l l i n contention ( MOLINOFF et a l . , 1977). F i n a l l y , studies combining enzyme assays with immunoassays have indicated - 26 -that a s i g n i f i c a n t proportion of c i r c u l a t i n g DBH i s i n a c t i v e ( WEINSHILBOUMy 1979 ), although i t i s by no means c e r t a i n whether t h i s i s true of DBH immediately released, f o r the h a l f l i f e of serum DBH appears to be at l e a s t two days. Clearly., therefore, i n order to be an absolutely e f f e c t i v e marker, c e r t a i n factors governing the l a b i l i t y and p r o p o r t i o n a l i t y of release of the enzyme must be better characterized. F i n a l l y , while the preceding problems deal with the i n t e r p r e t a t i o n of DBH l e v e l s i n the concept of a marker f o r exocytotic release, a t h i r d more fundamental problem a r i s e s from the d i f f i c u l t y of assaying DBH a c t i v i t y under a l l desired conditions. Several d i f f e r e n t assay methods have been reported i n the l i t e r a t u r e ( T a b l e 1 ), and a l l have been v a r i a t i o n s on the two basic r e a c t i o n schemes shown i n F i g . 2. While early methods suffered from a general lack of s e n s i t i v i t y and d i f f i c u l t y of procedure, numerous improvements i n technique and methodology over the years have greatly aided s e n s i t i v i t y and accuracy, although perhaps not as much as might be d e s i r a b l e . The e a r l i e s t method to reach widespread acceptance was the spectro-photometric assay ( NAGATSU and UDENFRIEND, 1972 ). In t h i s procedure, tyramine i s used as a substrate f o r DBH to produce octopamine i n the hydroxylation r e a c t i o n . This i s then chemically converted to p-hydroxy-benzaldehyde. With a high e x t i n c t i o n c o e f f i c i e n t at 333 nm, the l a t t e r i s e a s i l y detected spectrophotometrically. While easy and inexpensive, t h i s method suf f e r s from a low s e n s i t i v i t y , i n part due to high blank - 27 -DBH ASSAY REACTIONS DBH PNMT / = \ ? H -»• HO-(\ /VCI 14,. HO-(\ /> CH2-CH2-NH2 * H 0 \ ^-CH-CH 2NH 2 H <^ y- H-CHg-N- CH 3 14 C-SAM SAH TYRAMINE OCTOPAMINE SYNEPHRINE HO-^ ^-CH 2-CH 2-NH g TYRAMINE DBH 0 H- I 4C-NH, 2 H*. KMn04 FORMAMIDE H ° " \ }~CH~CH2~m2 OCTOPAMINE o . _ it H0- 1 4C-NH 2 "* H0~C + H - C - N H 2 N.xo 4 ' O ^ p -HYDROXBENZALDEHYDE -*• , H C 0 2 + NH 3 Figure 2. Basic reactions employed i n the assay of DBH. - 28 -values, a f a c t which has not li m i t e d the widespread c l i n i c a l use of t h i s procedure. Later modifications employing ether extr a c t i o n , dual wavelength spectrophotometry, and an improved blank ( KATO et a l . , 1978 ). served to increase the s e n s i t i v i t y of t h i s method. In addition, several v a r i a n t s of t h i s procedure using radioisotope l a b e l l e d tyramine as a substrate have been1 •reported using ether extraction of ^C-p-hydroxy-benzaldehyde ( NAGATSU et a l . , 1973; WISE, 1976 ) or i s o l a t i o n and oxidation of the l a b e l l e d formamide and subsequent counting of li b e r a t e d ^CO^ ( JOH et a l . , 1974 ). However, these procedures are not without t h e i r complications, and may su f f e r from the lack of pur i t y of the l a b e l l e d tyramine substrate, which necessitates an involved p u r i f i c a t i o n step. The second major assay method i s the coupled radioenzymatic procedure ( MOLINOFF et a l . , 1971; GOLDSTEIN and BONNAY, 1971 ). Here, tyramine or phenylethylamine i s used as a substrate, with the product, octopamine or phenylethanolamine r e p e c t i v e l y , subsequently to a l a b e l l e d N-metiyl d e r i v a t i v e i n a second enzymatic step involving phenylethanolamine-N-methyltransferase ( PNMT ) and C or H l a b e l l e d S-adenosylmethionine ( SAM ). Although t h i s procedure requires continual t i t r a t i o n of the +2 sample with Cu to optimize the blocking of endogenous i n h i b i t i o n , because PNMT i s i n h i b i t e d by high concentrations of Cu +^ as w e l l ( AXELROD, 1962 ), a l l of the reagents are now av a i l a b l e as commercial preparations, which s i m p l i f i e s the procedure greatly. While t h i s assay i s more involved and expensive than the above methods, and does not measure DBH a c t i v i t i e s under optimal conditions, i t s inherently greater s e n s i t i v i t y has made i t - 2 9 -widely adopted, p a r t i c u l a r l y i n the detection of DBH l e v e l s i n body f l u i d s and i n ti s s u e release studies. Although several modifications to t h i s procedure have been reported, such as the i n c l u s i o n of EDTA into + 2 the second re a c t i o n mixture to negate the Cu i n h i b i t i o n of PNMT ( MOLINOFF et a l . , 1 9 7 4 ) and the use of adenosyl homocysteinase and adenosine deaminase to reduce S-adenosylhomocysteine i n h i b i t i o n ( KARAHASANOGLU et a l . , 1 9 7 5 ), the improvements have not i n general been s u f f i c i e n t l y e f f e c t i v e to warrant deviating.from the published procedure. Several other procedures e x i s t f o r the assay of DBH, but have not received general acceptance f o r a v a r i e t y of reasons. Of these, the most established i s the radioimmunoassay, which promises extremely good s e n s i t i -v i t y ( RUSH and GEFFEN, 1 9 7 5 ; EBSTEIN et a l . , 1 9 7 3 ) a l b e i t with a much more involved and expensive procedure. Correlations between RIA. and conventional assays are excellent ( RUSH et a l . , 1 9 7 5 ), and most c e r t a i n l y t h i s method i s uniquely suited to the measurement of absolute l e v e l s of c i r c u l a t i n g DBH protein rather than only the detectable enzymic a c t i v i t y , a facet u s e f u l i n c l i n i c a l i n v e s t i g a t i o n . However, as mentioned before, the lack of a r e l i a b l e c o r r e l a t i o n between DBH crossreactive protein and enzymic a c t i v i t y i n a l l circumstances, may l i m i t the a p p l i c a t i o n of the method i n c e r t a i n experimental areas such as i n v i t r o release studies. Other assay methods include a phosphorimetric procedure ( YAMAGUCHI, 1 9 8 0 ), at present severly l i m i t e d by the lack of widespread a v a i l a b i l i t y - 30 -of the required instrumentation, and perhaps most promising, a number of reports of the a p p l i c a t i o n of HPLC to detect octopamine i n standard DBH assays ( FUJITA et a l . , 1980; FLATMARK et a l . , 1980 ). WHile s t i l l not completely worked out or widely accepted, the extreme s e n s i t i v i t y promised by t h i s technique, as w e l l as i t s p o t e n t i a l to simultaneously assess the l e v e l s of NE and i t s metabolites, would d e f i n i t d y warrant further development. It can therefore be appreciated that a number of w e l l established assay procedures e x i s t f o r the measurement of DBH; methods tlhat c e r t a i n l y have found a p p l i c a t i o n i n a d i v e r s i t y of experimental s i t u a t i o n s , and of these, several seemed suited to our research aims. The i n i t i a l aim of my research project was to attempt to characterize the mechanism of release of NE under conditions of i n h i b i t i o n of the sodium pump ( 1 mM ouabain, zero external sodium ) by comparison of the release of DBH and NE under these conditions with normal exocytotic release induced by high external potassium or 10""' M v e r a t r i d i n e . This was to be accomplished by incubating t i s s u e samples i n small volumes ( 5 ml ) of PSS containing the appropriate additions, i n a Dubnoff shaker at 37° under carbogen f o r l i m i t e d time periods. Rat t a i l artery was chosen as the substrate t i s s u e because-of i t s pure adrenergic innervation and excellent c h a r a c t e r i z a t i o n by our laboratory. The second choice f o r t i s s u e was the r a t vas deferens, because of i t s r e l a t i v e ease of i s o l a t i o n and handling, and i t s much denser adrenergic innervation. However, i t can - 31 -be appreciated that i n order to measure, DBH release from these t i s s u e s under such conditions, an assay of maximal s e n s i t i v i t y i s required. Accordingly, of the above methods, two were chosen as most sui t a b l e fo r the aims of t h i s project. The i n i t i a l choice was the dual spectro-photometric assay because of ease of performance, lower cost, and the fa c t that DBH i s assayed under saturated conditions. However, i n i t i a l incubation experiments showed a lack of s e n s i t i v i t y to be a problem, due i n part to high blank values. Work was undertaken to reduce and s t a b i l i z e the blank, and blank values were reduced to the lowest reported l i t e r a t u r e l e v e l s . However, s e n s i v i t y was s t i l l i n s u f f i c i e n t to detect the DBH a c t i v i t y released into high K + or v e r a t r i d i n e incubates. A recent report ( TACHIKAWA, 1979 ) of the a c t i v a t i o n of DBH by ADP led to a t r i a l of the i n c l u s i o n of ADP into the re a c t i o n mixture i n the hopes of improving s e n s i t i v i t y . While a th r e e - f o l d increase i n maximum s e n s i t i v i t y r e s u l t e d from the i n c l u s i o n of 6 mM ADP, t h i s was found to be s t i l l too low to detect DBH i n our a r t e r i a l incubate, although i t did make t h i s assay method more s u i t a b l e f o r the assessment of DBH i n the serum of laboratory animals. The alternate choice of procedure f o r the project was the Molinoff assay, because of i t s inherently higher s e n s i t i v i t y . I n i t i a l studies indicated that t h i s method exhibited the necessary s e n s i t i v i t y to detect the release from i s o l a t e d vas deferens perfused with 150 mM K + . However, the measurement of the release of DBH from r a t t a i l artery or vas deferens - 32 -induced by l e s s than optimal conditions such as the i n h i b i t i o n of the sodium pump, was found to be compromised by high blank values r e l a t i v e to perfusate a c t i v i t y . Accordingly an attempt was made to optimize the procedure with respect to maximizing s e n s i t i v i t y . I n i t i a l study disclosed numerous inconsistencies i n the assay that led to further work. A r a t i o n a l e was adopted of separating the two enzymatic steps, and in v e s t i g a t i n g each separately, as w e l l as the i n t e r a c -tions between them. The PNMT step was found to be unsaturated with respect to c e r t a i n key reagents, and BSA was found to give a highly concentration dependent a c t i v a t i o n of t h i s enzyme, quite i n contrast with i t s assumed e f f e c t of non-specific s t a b i l i z a t i o n . The r e s u l t i n g modifications that emerged from t h i s study r a i s e d the s e n s i t i v i t y of the assay to the point where the concentration of tyramine, which i n h i b i t s the second step, could be increased ^ito a l e v e l where the f i r s t r e a c t i o n step was l i n e a r with respect to time and concentration of DBH. The combination of modifications has produced an assay more s e n s i t i v e by more than an order of magnitude, and yet l i n e a r with respect to enzyme and octopamine concentrations, as wel as incubation time. While preliminary experiments ind i c a t e that the improved assay i s now s u f f i c i e n t l y s e n s i t i v e so as to be able to detect the DBH release from ra t t a i l artery into high potassium media, i t i s hoped that i t w i l l f i n d wider a p p l i c a t i o n . C e r t a i n l y the improved l i n e a r i t y of t h i s method w i l l - 33 -make comparisons between r e s u l t s more relevant and accurate, and the improved s e n s i t i v i t y may extend the usefulness of t h i s procedure into new experimental areas. - 34 -Table 1. A summary of assay methods employed f o r the measurement of Dopamine-B-hydroxylase a c t i v i t i e s . AUTHORS SUBSTRATE RATIONALE TECHNIQUES GOODALL and KIRSHNER (1957) LEVIN et a l . , (1960) VON EULER and FLODING (1955) PISANO et a l . , (1960) Dopamine 1 4C-DA DA TYR NAGATSU and TYR UDENFREIND(1972) KATO et al.,(1974) KATO et al.,(1978) " Separation of DA from NE. Cleavage of 14C'-NE by periodate. ion exchange r e s i n . 14 Trapping of C-formamide. Trihydroxyindole fluorescence of NE. U.V. absorption of p-hydroxybenzaldehyde. UV spectrophoto-metry. Conversion of TYR to Ion exchange sep-OCT; periodate cleavage aration of OCT. to p-hydroxybenaldehyde. UV spectrophoto-metry. " dual-wavelength spectrophotometry. " dual-wavelength spectrophotometry, f u s a r i c acid blank. Cu/NEM mix f o r •. endogenous inh i b -i t o r n e u t r a l i z a t i o n . MOLINOFF et a l . , (1971) GOLDSTEIN and BONNAY (1971) TYR 14 C-Me l a b e l l i n g of OCT by PNMT-. Liquid s c i n t i l l a -t i o n counting. MOLINOFF et a l . , (1974) KARAHASANOGLU et al.,(1975) Inclusion of EDTA i n RM2. Adenosine homocys-teinase and adeno^ she deaminase. - 35 Table 1.(cont'd) AUTHORS SUBSTRATE RATIONALE TECHNIQUES FREIDMAN and KAUFMAN (1965) VIVEROS et a l . , (1968) NAGATSU et a l . , (1972) 14 C - T Y R 14 2- C-TYR 14, J O H et al.,(1974) 1- C-TYR WISE (1976) 14 2- C-TYR Conversion of OCT to C-p-hydroxybenzaldehyde. Liquid s c i n t i l l a t i o n counting. Ether extraction. Ion.exchange. L. S . C . OCT formation: periodate Trapping of cleavage to ™C-formamide; 14C02 a n c * L.S.C. [0] to 1 4 C 0 2 . formation of ^ C -p-hydroxyb e zaIdehyde. ether extraction. L.S.C. p u r i f i c a t i o n of C TYR. RUSH and •:. '* DBH GEFFEN (1972) EBSTEIN et a i . , (1973) RUSH et al.,(1975) DBH R A D I O I M M U N O A S S A Y . 1 ?S 1 J I DBH(human). anti-sheep DBH. iCJI.-DBH(human). anti-human DBH. F U J I T A et a l . , T Y R (1977) OCT formation. HPLC with f l u o r -escence det'n. F L A T M A R K et a l . , T Y R (1980) HPLC with electro-chemical det'n.. YAMAGUCHl et a l . , TYR (1980) separation of OCT + periodate cleavage to p-hydroxybezaldehyde. ion exchange. phosphorimetric det'n. - 36 -Materials Catalase ( two times r e c r y s t a l l i z e d ), vanadium and f a t t y a c i d -free ADP, ouabain, pargyline hydrochloride, tyramine hydrochloride, octopamine, S-adenosylmethionine( SAM ), d i t h i o t h r e i t o l ( DTT ), l y o p h i l i z e d PNMT, DBH inCNH^^SO^ suspension, v e r a t r i d i n e , f u s a r i c a c i d , N-ethylmaleimide, Triton-X-100, sodium periodate, sodium b i s u l f i t e , and sodium fumarate were obtained from Sigma Chemical company; diethyldithiocarbamate ( DEDTC ) from Eastman; sodium "ascorbate ( AnalR ) from BDH; DMSO ( AR ) and EDTA ( AR ) from Malincrodt; catalase ( a n a l y t i c a l grade ) was obtained from both Calbiochem and Boehringer Manheim, who also supplied the a n a l y t i c a l grade bovine serum albumin ( BSA ); Sigma also supplied the TRIS and TRIS.-HC1. Radioactive [^ 4C]-S-Adenosylmethionine ( ^4C-SAM ) with an a c t i v i t y of 10 p.Ci/ 0.5 ml was obtained from New England Nuclear, as was Omnifluor. Unless otherwise stated, s c i n t i l l a t i o n grade:methanol and toluene, and A.C.S. d i e t h y l ether and ethyl acetate, as well as a l l common chemicals were obtained from North American Chemical Supply l i m i t e d . Dowex 50-X4 ( H+), 200-400 mesh was procured from Biorad. Methods A. Spectrophotometric assay The spectrophotometric assay was performed according to the procedure d e t a i l e d i n the improved dual wavelength spectrophotometric method f o r DBH reported by KATO ( KATO et a l . , 1978 ). However, the u n a v a i l a b i l i t y of a dual wavelength machine required that absorbances at both wavelengths be taken manually. Stock solutions of octopamine, 20 mM i n 20 mM HC1; pargyline, 40mM; sodium ascorbate, 0.2M; sodium fumarate, 0.2M; tyramine hydrochloride, 0.4M; and f u s a r i c a c i d , 2 mM, were prepared beforehand and stored i n small aliquots frozen at 20°C, and thawed immediately p r i o r to use. The assay was conducted as follows ( f i n a l concentrations of each reagent are given i n parentheses ). An enzyme aliquot of up to 500 i l l was added to a 15 ml glass centrifuge tube ( smaller aliquots were commonly used, with the f i n a l volume made up to 500 i l l with d i s t i l l e d water ). A blank and i n t e r n a l standard by the addition of 50 u l of 2 mM fu s a r i c acid ( 100 uM ) and 50 p.1 f u s a r i c acid plus 20 u l of octopamine ( 100 p.M ) re s p e c t i v e l y to two centrifuge tubes containing s i m i l a r enzyme solutions. Each of these was then treated i n d e n t i c a l l y . - 38 -The following reagents were then added to the tube; lOOul of 2M sodium acetate b u f f e r , pH 5.0 ( 0.2M ); 150 y.l of 0.2M N-ethylmaleimide ( 30 mM ); 50 u l of 200 uM CuS0 4 ( 10 uM ); 25 u l of aqueous 20 mg/ml catalase ( 25,000 units ); and 25 u l of 40 mM pargyline hydrochloride ( 1 mM ), and the so l u t i o n mixed to i n a c t i v a t e endogenous i n h i b i t o r s and MAO i n the crude enzyme so l u t i o n Then the following solutions were added to complete the re a c t i o n mixture; 50 u l of 0.2 M ascorbate ( 10 mM ); 50 u l of 0.2 M fumarate ( 10 mM );j and 50 , u l of 0.4 M tyramine hydrochloride ( 20 mM ). The r e s u l t i n g s o l u t i o n was vortexed and incubated at 3 7° i n a i r under continuous a g i t a t i o n i n a Dubnoff shaker, f o r 45 minutes. At t h i s point, the reaction was'stopped by the addition of 0.2 ml of cold 3M t r i c h l o r o -a c e t i c acid i n an i c e bath and the mixture immediately centrifuged at 3000 rpm f o r 10 minutes. This prevented a gradual r i s e i n blank values noted when the DBH incubation mixture was allowed to stand at room temperature ( KATO et a l . , 1978 ). The supernatant f l u i d was then immediately transferred to a glass microcolumn ( 0.5 X 10 cm, Biorad econocolumn ) packed with 0.2 ml of Dowex-50W-X4 ion exchange r e s i n ( H +, 200-400 mesh ) and eluted through to load the column. The p e l l e t was washed with 1 ml of water and centrifuged again, and the supernatant again loaded on the column. - 39 -The column was then washed twice with two ml aliquots of water, and the octopamine eluted with 1.0 ml of 3 N NH^OH into a graduated glass centrifuge tube. The eluate was then oxidized by the addition of 10 u l of f r e s h l y prepared 27o NalO^ , and the reac t i o n allowed to proceed f o r several minutes ( not c r i t i c a l ). Excess periodate was then eliminated by the addition of 10 u l of 10% Na^S^O,- ( f r e s h ), and the tube was transferred to an ice bath. The s o l u t i o n was a c i d i f i e d by the addition of 0.5 ml cold 6 N HC1, 5 ml of r e d i s t i l l e d d i e t h y l ether were added, and the mixture extracted by vigorous s t i r r i n g on a vortex mixer f o r 30 seconds. The top 4 ml of the organic phase were transferred to another centrifuge tube and extracted with 1 ml of 3N NH^OH, again by vigorous mixing f o r 30 seconds. Then the ether layer was removed by a s p i r a t i o n , and the remaining aqueous phase was incubated at 3 7°C f o r 30 minutes i n a fume hood to remove r e s i d u a l ether, which was found to i n t e r f e r e with the spectrophotometry. . . . . 2 The ammonium hydroxide phase was then transferred to a 1 » quartz cuvette ( v o l . 0.1 ml ) and the absorbance at 333 and 360 nm ( the peak and background absorbance '.. wavelengths.:; r e s p e c t i v e l y ) measured on a G i l f o r d model 250 UV/Visible spectrophotometer equipped with an automatic cuvette changer and chart recorder, under deuterium oxide i l l u m i n a t i o n . - 40 -The absorbance of p-hydroxybenzaldehyde was then determined by subtraction of the absorbance at 360 nm from the value of the absorbance maximum at 333 nm, and the enzyme a c t i v i t y calculated by refence to a standard pl o t of octopamine concentration versus A A valies. A f t e r use, the columns,were regenerated by washing once with 1 ml 3N NH^OH and 2 ml water. This was followed by 1 ml of 6N HC1 and two 2 ml portions of water. B. Molinoff assay a) standard procedure The standard Molinoff assay was performed b a s i c a l l y as reported i n the o r i g i n a l paper ( MOLINOFF et a l . , 1971 ), and i t s introduction was greatly f a c i l i t a t e d by the use of the standard protocol kindly supplied by the Molinoff lab ( Ralph Aarons, personal communication ). Several stock solutions were prepared beforehand and stored frozen i n small aliquots and thawed immediately p r i o r to use; sodium fumarate, 0.5 M, pH adjusted to 5.5 with Na OH; tyramine HC1, 0.03 M, pH adjusted to 5.5 with NaOH; and Octopamine, 5.0 mM i n 6 mM HC1. I t was determined that octopamine "went o f f " during prolonged frozen storage, so t h i s reagent was frequently prepared, and the number of freeze-thaw cycles minimized. - 41 -Other reagents were preweighed and prepared f r e s h , immediately p r i o r to use, including DTT, 1 mg/ml and 10 mg/ml; pargyline HC1, 2.5 mg/ml; and sodium ascorbate, 0.048 M, pH adjusted to 5.5 . Catalase solutions were prepared one hour before the assay was to be conducted, by the 1:4 d i l u t i o n of commercial aqueous catalase preparations with 5 mM T r i s b u f f e r , pH 7.4 and stored on i c e . The assay was conducted as follows. The f i r s t r e action mixture was prepared and*stored on i c e , by the' combination of the following reajentsC amounts given are per assay tube ); 12.5 u l of 0.048 M ascorbate; 12.5 u l offumarate, 0.5..M; 5 u l of 1 M sodium acetate buffer, pH 5.0; 5 u l of d i s t i l l e d water; 5 u l catalase; 5 u l 2.5 mg/ml pargyline; 5 u l of tyramine , 0.03M . 100 u l aliquots of enzyme sol u t i o n were pipetted into 15 ml glass centrifuge tubes, and 10 u l aliquots of CuSO^ so l u t i o n i n a concentration determined by a t i t r a t i o n f o r maximum DBH a c t i v i t y , were added i f found necessary to counteract the e f f e c t s of endogenous i n h i b i t o r s . For r e s u l t s where extreme accuracy was required, the CuSO^ could be pipetted into the tubes the previous night, and allowed to dry, so as not , to a f f e c t the f i n a l r e a c t i o n volume. A blank and i n t e r n a l standard were prepared by the addition of 10 u l of 1 mg/ml DTT, and 10 u l DTT and lOul of 5.0 mM octopamine, r e s p e c t i v e l y . A l l tubes were then treated i d e n t i c a l l y . The r e a c t i o n was i n i t i a t e d by the addition of 50 u l of the f i r s t r e a c t i o n mixture ( RM1 ) to each enzyme tube, the mixture vortexed, and the reac t i o n allowed to proceed f o r 20 minutes i n a i r at 3 7°C. - 42 -During the course of the first reaction, the second reaction mixture ( RM2 ) was prepared by the combination of the following on ice ( amounts are given per assay tube ); 43.25 ill of 1.0 M Tris buffer, pH 8.6 at 37°C; 1 ill 10 mg/ml DTT; 5 pi of 14C-SAM, 10 '-p. Ci per 2.0 ml; and 0.75 p.1 of PNMT solution, 0.25 units/yl . This RM2 was prepared immediately prior to use, as i t was found to be unstable even when stored at 0°tfor longer than 20 minutes ( DJH, unpublished observations ). In addition, the mixing sequence given above was strictly followed with vortexing between each addition, because PNMT was found to be acid labile, and this procedure 14 minimized the degradative effects of the addition of the C- SAM, which is stored in 1 N H-iS'o. solution. 2 4 At the end of the 20 minutes, the first reaction was stopped by the addition of 50 y.1 aliquots of RM2 to each tube, as the DBH in the first reaction is inactivated by alkaline pH. The second reaction was allowed to proceed at 3 7°C for thirty minutes, at which point i t was terminated by the addition of 0.5 ml of sodium borate buffer, pH 10.0, and the mixture • vortexed vigorously for 30 seconds. A- 5.5 - ml portion /.of ethyl acetate was added, the tube covered with parafilm, and the reaction mixture extracted by vortex mixing for a further 30 seconds. At this point, the tubes were then centrifuged for three minutes at 3000 rpm to complete the phase separation, and 4 ml of the organic phase transferred to a glass scintillation vial with 6 ml of a scintillation fluid consisting of 8g of Omnifluor; 267 ml of methanol; 133 ml of Triton-X-100; per 2 1 of toluene. - 43 -Samples were then counted on a Nuclear-Chicago Mark II l i q u i d s c i n t i l l a t i o n counter, model 6847, f o r 10 minutes per channel on a r a t i o mode. b) extended incubation In order to increase s e n s i t i v i t y , t e s t s of the f e a s i b i l i t y of extended incubation times f o r the f i r s t r e a c t i o n mixture as reported by Cubbeddu ( CUBBEDDU et a l . , 1974 ) were performed. The f i r s t r e a c t i o n was i n i t i a t e d b y the addition of RM1 and incubation allowed to proceed fo r a series of times. Af t e r the desired time had elapsed f o r each group of tubes, the rea c t i o n was stopped by quick freezing i n a dry i c e -methanol bath, and the tubes stored frozen at -20° u n t i l a l l the step one incubations were completed. At t h i s point, f r e s h l y prepared RM2 was added to the p a r t i a l l y thawed tubes, melting completed during votex mixing, and the second re a c t i o n allowed to proceed as with the standard assay. Control experiments showed that t h i s method stopped the DBH react i o n completely, that no d e t e r i o r a t i o n occurred during the freeze-thaw c y c l e , and that the course of the second re a c t i o n was completely unaffected by t h i s procedure. c) modified r e a c t i o n mixture 1 In order to better study the assay, a r a t i o n a l e was adopted -44 -i n which the two enzymic reactions were separated and studied independently. The second ( PNMT ) re a c t i o n was i s o l a t e d by the use of a modified f i r s t r e a c t i o n mixture ( MRM-1 ) consi s t i n g only of 12.5 u l of fumarate, 5 u l of 1M sodium acetate b u f f e r , pH 5.0, and water to make up the r e a c t i o n volume. This was added to 100 u l aliquots of octopamine -5 -U -4 standards i n the concentrations, 5.X 10 M, 2.5 X 10 M, and 5 X 10 M, which were chosen to span the desired range of DBH a c t i v i t i e s . This mixture was then used as a substrate f o r the second r e a c t i o n , and the normal procedure followed from the addition of RM2. Each of the other components of the f u l l f i r s t r e a c t i o n mixture were added back into t h i s MRM-1 mixture.,- independently and i n combination, i n order to assess t h e i r e f f e c t on the second re a c t i o n step. The e f f e c t of various concentrations of BSA i n the sample aliquot was also investigated. d) modified r e a c t i o n mixture 2 S i m i l a r l y , the concentrations of reagents i n the second re a c t i o n mixturewere v a r i e d , and the e f f e c t s on f i n a l a c t i v i t y of the octopamine ;:. standards determined. In several experiments, unlabelled SAM was added i n order to r a i s e the o v e r a l l concentration of SAM i n the reac t i o n mixture, and saturate the PNMT with respect to t h i s reagent. - 45 -e) synephrine s t a b i l i t y The s t a b i l i t y of synephrine ( the f i n a l product of the assay ) i n the f i n a l r e a c t i o n mixture was determined by fluorescence spectroscopy. Solutions of 1 iiM synephrine i n 1M T r i s b u f f e r , pH 8.6 were incubated at 37°C f o r t h i r t y minutes, and the fluorescence determined before and 2 a f t e r incubation on a Turner model 430 spectrofluorometer i n quartz 1 cm c e l l s , using the experimentally determined e x c i t a t i o n and > emission wavelengths of 273.2 and 320 nm respectively.' Non-specific scattering was checked at 450 nm. C. Tissue techniques a) i s o l a t i o n Rat t a i l a r t e r i e s were excised from adult male ra t s under sodium pentobarbitone anesthesia ( 50 mg/kg, i n t r a p e r i t o n e a l ). An i n c i s i o n was made on the v e n t r a l surface, from j u s t posterior to the anus to the t i p of the t a i l , through the skin and deep f a s c i a to expose the t a i l artery. The artery was i s o l a t e d by blunt d i s s e c t i o n with a probe t i p and gently removed. Vasadefera were removed from adult male ra t s under sodium pento-barbitone anesthesia. An i n c i s i o n was made i n the perineum through the skin and deep f a s c i a , and the s c r o t a l sacs gently freed from the surrounding - 46 -connective t i s s u e by blunt Dissection. Each scrotum was opened with a pair of f i n e s c i s s o r s , and the epididymis c a r e f u l l y removed. The vas deferens was gently teased f r e e , and cut where i t joined the cauda epididymis as w e l l as at the point where i t entered the inguinal canal. The vasa were then transferred to a p e t r i dish of i c e cold PSS ( see below ), and the outer a d v e n t i t i a , d e f e r e n t i a l blood v e s s e l s , and surrounding connective t i s s u e removed with f i n e forceps. For t i s s u e release studies, the vas* were opened by a l o n g t i t u d i n a l i n c i s i o n to the lumen at t h i s point Adrenals were removed from adult male r a t s under sodium pentobarbitone anesthesia. An i n c i s i o n was made p a r a l l e l to the backbone i n the lumbar? back, approximately 2 cm l a t e r a l to the spine. The kidneys were i s o l a t e d from t h e i r dorsal aspect by blunt d i s s e c t i o n from the surrounding f a t , and the adrenals gently removed from the r o s t r a l pole of the kidney with a p a i r of forceps. b) homogenization Organs, f r e s h l y removed and stored i n i c e cold PSS, were weighed and quickly minced into pieces of approximately 1 mm with a p a i r of f i n e s u r g i c a l s c i s s o r s . The t i s s u e was then transferred to a 10 ml glass centrifuge tube, cooled i n an i c e bath, containing e i t h e r i c e cold EDTA-free Krebs or 50 mM T r i s , pH 7.3 ( with or without 0.27o Triton-X-100 ) i n a volume of 1:25 . Homogenization was then c a r r i e d out with - 47 -a Poytron homogenizer with a PCU-2 c o n t r o l u n i t i n an ice-methanol cooling bath, at set t i n g 8 f o r three periods of 10 seconds each, separated by one minute cooling periods. The Polytron was employed because i t was previously shown to be most e f f e c t i v e i n the homogenization of smooth muscle t i s s u e i n t h i s lab ( AZAD, 1979 ). To provide maximal extraction of soluble DBH a c t i v i t y , t h i s crude homogenate was then transferred to a t i g h t f i t t i n g t e f l o n homogenizer and given three passes utider ice-methanol cooling. T r i t o n extracts were also s t i r r e d inthe cold f o r one hour to y i e l d maximum s o l u b i l i z a t i o n of DBH. The crude homogenate was then transferred to a glass centrifuge tube and centrifuged i n a Sorval RC2-B at 4 ° i n an SS-34 rotor at 10,000 rpm f o r 10 minutes. The supernatant was then used as the crude enzyme preparation. c) incubation experiments Organs, f r e s h l y removed, were transferred to t e s t tubes containing 10 ml of PSS and allowed to recover for 90 minutes at 3 7° with bubbled 957o a i r / 57. CO^ aeration, with several solution changes. Recovered organs were then transferred to i n d i v i d u a l glass s c i n t i l l a t i o n v i a l s containing 5 ml of EDTA-free PSS and incubated f o r f i x e d periods of time at 3 7° i n a Dubnoff shaker under continuous aeration by carbogen. In general, a 45 minute co n t r o l incubation i n normal Krebs was followed by a 45 minute incubation i n Krebs containing the desired additions ( 150 mM hypertonic KC1, 10"^ M v e r a t r i d i n e , 1 mMouaain ). BSA was usually present throughout, i n a concentration of ei t h e r 0.25 °L or 0.147° ( l a t e r determined to be optimal ). - 48 -Incubates were immediately frozen and stored at -20 u n t i l the assay f o r DBH a c t i v i t y could be performed. The p h y s i o l o g i c a l s a l t s o l u t i o n used was a Krebs bicarbonate buffer with the following i o n i c composition i n mEq/litre; Na*, 141.2; K +, 5.0; C a + 2 , 3 .4; Mg + 2, 2.4; C l " , 124.2; HC0 3", 25; H2PC>4~, 1.2; pyruvate or d-glucose, 11.0 ( t o t a l 314.2 ); pH was 7.3 at 37° under aeration. D. S t a t i s t i c s Error bars on the graphs represent the standard error of the points as calculated by the formula; r-_ x - x . ) 2 S = / _ x l J n( n-1 ) Points obtained with the spectrophotometric assay are the r e s u l t of duplicate experiments ( means are presented ) except f o r Figure 3, i n which points are the r e s u l t of f i v e experiments. Results obtained with the Molinoff assay are means of experiments performed i n quadruplicate. Means and standard errors are presented f o r a l l points except f o r those where the calculated error was less than the symbol s i z e . Figures 8 and 17 present the r e s u l t s of duplicate experiments. - 49 -RESULTS A. Spectrophotometric method The spectiophotometric method ( NAGATSU and UDENFRIEND, 1972 ) was the i n i t i a l choice f o r t h i s project f o r several reasons, not the le a s t of which were a lower cost and greater ease of performance than were offered by the competing methods. In addition, recent modifications ( KATO et a l . , 1978 ) raised the s e n s i t i v i t y to the point where i t was thought to be p o t e n t i a l l y applicable to pur ti s s u e release studies. This prompted an i n i t i a l t r i a l of the method. This method involves the conversion of tyramine to octopamine by the target enzyme DBH ( see Figure 1 ), i s o l a t i o n of the product on an ion exchange column, and subsequent periodate cleavage of the octopamine to form p-hydroxybenzaldehyde. which exhibits an intense absorbance at 333 nm. In a biochemical sense, t h i s method i s f a r superior to the others, because i t assays DBH a c t i v i t y under saturated conditions with respect to the substrate, tyramine ( Km=10 mM ), and because i t employs a mixture of N-ethylmaleimide and Cu +^ ( at a concentration which does not i n h i b i t DBH ) to completely counteract the e f f e c t s of endogenous i n h i b i t o r s without the necessity f o r t i t r a t i o n , or any interference with subsequent reactions. Therefore, maximum DBH a c t i v i t i e s are measured by t h i s procedure. - 50 -In preliminary studies, i t was shown that the assay was l i n e a r with respect to octopamine concentrations up to a l e v e l c e r t a i n l y above any a c t i v i t i e s we would be l i k e l y to encounter ( Figure 3 ). In addition, c o n t r o l experiments indicated that the recovery of octopamine standards i n t h i s procedure was ro u t i n e l y better than 957o. However, i t should be noted that inconsistencies i n the absorbance values, and s i g n i f i c a n t deviations from l i n e a r i t y were observed with octopamine concentrations lower than approximately 50 ]iM, quite i n contrast with published reports of l i n e -a r i t y down to 0.5 ]M octopamine with t h i s method ( KATO etj.al. , 1978). Therefore, while detection of a c t i v i t y i n t h i s range i s possible with an extremely low blank value, r e s u l t s obtained might be suspect and of l i t t l e use. The behaviour of p u r i f i e d DBH was also examined i n t h i s procedure ( Figure 4 ), and i t was found that the DBH reaction was l i n e a r up to a concentration of 0.05 units ( OCT/minute ), c e r t a i n l y more than adequate f o r our work. I t was noted, however, that blank values were inconsistent and generally much higher than the AA=0.005 units reported i n the l i t e r a t u r e ( KATO et a l . , 1978 ) f o r the improved method, a f a c t which greatly l i m i t e d maximum s e n s i t i v i t y . Preliminary t i s s u e release studies, which involved monitoring the release of DBH from an is o l a t e d r a t t a i l artery incubated i n 2 ml Krebs f o r 30 minutes, confirmed t h i s as a problem. While DBH a c t i v i t y w.as Ul Figure 3 . Absorbance values obtained with standard concentrations 1 - 1 of octopamine for the spectrophotometric assay - 52 -Figure 4. Absorbance values obtained with standard concentrations of p u r i f i e d DBH ( SIGMA ) i n the spectrophotometric assay - 55 -detectable, with a r e s u l t i n g AA= 0.025 u n i t s , t h i s value was not s i g n i f i c a -n t l y d i f f e r e n t than the blank value produced by f u s a r i c acid addition. Accordingly, an attempt was made to reduce and s t a b i l i z e the blank l e v e l s , i n the hopes of improving s e n s i t i v i t y to the point where absorbance differences i n t h i s range would be s i g n i f i c a n t . As previously mentioned, the blank i n the improved method was produced by the addition of 0.1 mM f u s a r i c acid to the enzyme solu t i o n ( KATO et a l . , 1978 ). Fusaric acid ( 5 - b u t y l p i c o l i n i c acid ) i s known to be an extremely potent and s p e c i f i c i n h i b i t o r of DBH ( NAGATSU et a l . , 1970 ), y i e l d i n g h a l f maximal i n h i b i t i o n of the enzyme at a concentration of 3 X —8 10 M. I n h i b i t i o n occurs p r i m a r i l y as a r e s u l t of copper chela t i o n , as the enzyme bound copper of DBH i s l a b i l e and can be r e v e r s i b l y removed by competitive complexation by a v a r i e t y of chelating agents ( FREIDMAN and KAUFMAN, 1965 ). In addition to f u s a r i c a c i d , a number of other enzyme i n h i b i t o r s have been reported, including the very e f f e c t i v e copper chelator diethyldithiocarbamate ( DEDTC ), which completely i n h i b i t e d DBH at 2 X 10~6 M, disulfuram, CN", CO, EDTA, as w e l l as high excess [ C u + 2 ] ( GREEN, 1964 ). On t h i s b a s i s , therefore, a number of d i f f e r e n t i n h i b i t o r s of DBH were t r i e d at various concentrations, alone and i n combinations, i n the hopes of reducing the blank l e v e l s . As can be seen i n Table 2, f u s a r i c acid alone produced i n e x p l i c a b l y high blanks, even when present i n a concentration ten times above normal. B o i l i n g the enzyme so l u t i o n for f i v e minutes p r i o r to the assay also proved of l i m i t e d effectiveness. - 56 -Table 2 Blank values obtained by various methods of DBH i n h i b i t i o n ( 50 p i aliquots of these concentrated stock solutions were used i n the assayl CONDITIONS M , , , 333 - 360 nm 0.2 units DBH 1.886 Enzyme + re a c t i o n mixture without TYR 0.005 Enzyme +0.1 mM f u s a r i c acid 0.039 Boiled enzyme 0.036 0.071 0.542 0.010 Enzyme + 0.2 mM f u s a r i c acid 0.035 Enzyme + 1 mM f u s a r i c acid 0.014 Enzyme + 10"4M f u s a r i c acid + 5X 10~4M EDTA 0.009 Enzyme + 5X 10 _ 4M f u s a r i c acid + 7oX 10"4M EDTA 0.005 Enzyme + 1 mM EDTA Enzyme + 10 _ 5M DEDTC Enzyme + 10"5M DEDTC + 5 X 10"4M EDTA - 57"-S i m i l a r l y , the copper chelators DEDTC and EDTA did not prove u s e f u l as DBH i n h i b i t o r s , although the combination of the two produced a blank of AA=0.010: low, but s t i l l above the 4A=0.005 l i t e r a t u r e value. F i n a l l y , -4 -4 however, the combination of 5 X 10 M f u s a r i c acid with 5 X 10 M EDTA, was found to produce a r o u t i n e l y reproducible blank of AA=0.005, equal to the best l i t e r a t u r e value. With the improved blank, i t was hoped that the method would then be s e n s i t i v e enough to monitor t i s s u e DBH release. Homogenization experiments were performed i n order to assess the maximum obtainable enzyme a c t i v i t i e s . While crude adrenal homogenate produced a AA of 0.291 , r a t t a i l artery was much less promising, y i e l d i n g a maximum absorbance diffe r e n c e of only 0.029 u n i t s , i n d i c a t i n g a very low density of adrenergic innervation. S i m i l a r l y , the a c t i v i t y i n high K + incubate was barely detectable over the blank l e v e l ( 4A=0.010 ). A recent report i n the l i t e r a t u r e ( TACHIKAWA et a l . , 1979 ) of the a c t i v a t i o n of DBH by ADP and other nucleotides led to a t r i a l of ADP i n the re a c t i o n mixture of the spectrophotometric assay, i n the hopes of again improving s e n s i t i v i t y . Pipeliminary experiments indicated the f e a s i b i l i t y of t h i s , so a study of the e f f e c t s of various concentrations of ADP on DBH a c t i v i t y as measured by t h i s method was performed, the r e s u l t s of which are shown i n Figure 5. As can be seen, the i n c l u s i o n of - 58 -E c o CD CO i CO CO CO 0.4-i 0.2-0 -I r o T [ADP] i 8 m M Figure 5. The e f f e c t of ADP a c t i v a t i o n on DBH a c t i v i t y as measured by the spectrophotometric method. [DBH] = 0.02 u n i t s . - 5 9 -6 mM ADP produced a maximal 2 . 5 f o l d increase i n absorbance l e v e l s . However, while t h i s a c t i v a t i o n was shown to apply with t i s s u e homogenates and incubates, the absorbance differences of 0 . 0 3 5 units and 0 . 0 2 1 units r e s p e c t i v e l y , obtained with r a t t a i l artery homogenate and high K + incubate assayed even with the i n c l u s i o n of 6 mM ADP were s t i l l tooo low f o r our purposes. I t should be noted that t e s t s with r a t serum demonstrated that ADP a c t i v a t i o n was s u f f i c i e n t to r a i s e absorbance differences obtained by the.spectrophotometric assay to AA= 0 . 0 8 4 , c e r t a i n l y above the l e v e l of s i g n i f i c a n c e . Therefore, although t h i s method was found to be of i n s u f f i c i e n t s e n s i t i v i t y f o r our purpose of monitoring DBH release into t i s s u e incubates, even with the improvements d e t a i l e d , these modifications may make t h i s assay more suitable f o r a p p l i c a t i o n to le s s demanding experimental s i t u a t i o n s , such as measurement of DBH l e v e l s i n the serum of laboratory animals. B. Molinoff assay Af t e r the f a i l u r e of the spectrophotometric method, the next choice of procedure was the coupled radioenzymatic assay of Molinoff ( MOLINOFF et a l . , 1 9 7 1 ), whose inherently greater s e n s i t i v i t y had made i t the standard method i n most t i s s u e release studies and other demanding experimental s i t u a t i o n s . Despite the f a c t that i n h i b i t i o n of the PNMT i n the second step by both tyramine and Cu +^ required the use of a concentration of tyramine f a r , l e s s than would be necessary f o r saturation - 60 -of DBH, and required the t i t r a t i o n of each sample with various concentrations of CuSO^ i n order to block endogenous i n h i b i t i o n and maximize a c t i v i t y , the s e n s i t i v i t y of t h i s method i s s t i l l better than the former assay. The radioenzymatic nature of t h i s assay, the e f f i c i e n c y of l i q u i d s c i n t i l l a t i o n counting, as w e l l as the f a c t that r a d i o l a b e l l e d SAM instead of a ra d i o -active DBH substrate i s used i n t h i s procedure, produce a s e n s i t i v i t y of over one order of magnitude improvement over the spectrophotometric method, c e r t a i n l y j u s t i f i c a t i o n f o r the a d d i t i o n a l cost and d i f f i c u l t y of performance. This choice seemed j u s t i f i e d i n view of the success of THOA et al.,(l975 ), i n detecting the release of DBH into bathing media from i s o l a t e d guinea pig vasadefera i n response to high K + using t h i s assay method. v After preliminary investigations i n which the basic performance of the assay was checked with regards to a c t i v i t y of blank and i n t e r n a l standards, by reference to parameters outlined i n the o r i g i n a l paper, i t was decided to attempt d i r e c t a p p l i c a t i o n of the method. An experiment was conducted to monotor the DBH release from 2 r a t t a i l a r t e r i e s into 2 ml of Krebs with 10~4M v e r a t r i d i n e . While DBH a c t i v i t y was measurable, and corresponded to a released a c t i v i t y of 5 nM/g/hr , t h i s was calculated from a counts difference only 20 cpm above the blank value of 104.4 cpm, somewhat less than would be desired f or s i g n i f i c a n c e . In order to determine whether the low a c t i v i t y was a f a u l t of - 61 -procedure, or whether i t was simply a product of low t i s s u e density of adrenergic innervation, a crude homogenate of r a t t a i l a r t e r i e s ( 130 mg/<. ml wet wt. ) was prepared and assayed far DBH a c t i v i t y . After t i t r a t i o n f o r maximum a c t i v i t y with CuSO^ ( see Figure 6 ), i t was found that the measured a c t i v i t y corresponded to only 2.5 pM/ min / mg., or about one - hundredth of the l e v e l reported for' the r a t aorta ( KATO et a l . , 1978 ). It was f e l t , therefore, that the t i s s u e concentration of DBH i n the r a t t a i l artery was simply too low to make i t a vrable substrate f o r t i s s u e release studies, e s p e c i a l l y with less than optimal conditions such as the i n h i b i t i o n of the sodium pump, and a search f o r a suitable t i s s u e was made. The r a t vas deferens seemed the i d e a l choice to replace the t a i l artery as a substrate i n our experiments, because of i t s dense, v i r t u a l l y pure adrenergic innervation ( HOLMAN, 1975 ), the ease of i s o l a t i o n and handling, and the compatibility with other published work. A homogenization performed in'prder to ascertain the t o t a l DBH l e v e l , resulted i n the determination of a s p e c i f i c t i s s u e a c t i v i t y of 13.6 nM/min/g , which was i n good agreement with the value of 25.8 nM/min/g obtained with the spectrophotometric method ( KATO et a l . , 1978). I t was decided, therefore, to undertake another t i s s u e release study i n order to duplicate the r e s u l t s of Thoa i n which the release of DBH into high potassium Krebs was monitored f o r guinea pig vas deferens ( THOA et a l . , 1975 ). Isolated r a t vas defera were incubated i n 150 mM hypertonic K +.Krebs f o r 30 minutes. Measured a c t i v i t i e s f o r duplicate experiments, based on counts differences Figure 6. T y p i c a l t i t r a t i o n curve for DBH a c t i v i t y obtained with ro i various concentrations of CuSO^ ( r a t vas deferens, crude homogenate ). - 63 -- 6 4 -of approximately 4 0 cpm over blank, corresponded to release of 3 5 . 4 and 5 2 . 3 nM/hr/g r e s p e c t i v e l y . This was i n general agreement with the value of 1 5 nM/hr/g reported f o r guinea pig vas deferens incubated i n 75 mM K + ( THOA et a l . , 1 9 7 5 ( ) . Again, however, a problem was envisaged with measurement of the less than optimal DBH a c t i v i t i e s released by i n h i b i t i o n of the sodium pump, due to the low a c t i v i t y r e l a t i v e to the blank value. It was decided, therefore, to examine the assay procedure i t s e l f i n the hopes of improving s e n s i t i v i t y . A study was performed to determine the behaviour of the standard Molinoff method with respect to a series of concentrations of p u r i f i e d DBH. As can be seen i n Figure 7 , the assay was l i n e a r with enzyme concentration only up to 0 . 0 0 2 , u n i t s , and a c t i v i t y t a i l e d o f f r a p i d l y at t h i s point. This i s quite i n contrast with the l i n e a r i t y up to 0 . 5 units exhibited by the spectrophotometric assay. ( Figure 4 , the concentration range covered by Figure 7 i s shown i n the inset ), a f a c t which seemed to indic a t e that the n o n - l i n e a r i t y found i n t h i s procedure could be the r e s u l t of the coupled radioenzymatic method i t s e l f , and c e r t a i n l y j u s t i f i e d further i n v e s t i g a t i o n . An attempt was made at t h i s point to reduce the blank l e v e l . The effectiveness of f u s a r i c acid and boil e d enzyme blanks were compared with the DTT blank used i n the normal procedure. However, as can be seen i n Table 3 , DTT proved to be the most e f f e c t i v e enzyme i n h i b i t o r i n t h i s method. A problem of s t e a d i l y increasing blank values noted over several months, was correlated to the time i n frozen storage f o r the Figure 7. Plot of DBH a c t i v i t i e s obtained for a series of concentrations of p u r i f i e d enzyme ( SIGMA ) i n the standard Molinoff method. - 66 -- 67 -Table 3 The e f f e c t of various blank procedures on blank values obtained 3 with the Molinoff method. ( Subatrate: 2.5 X 10 units DBH ). CONDITIONS , BLANK cpm no RM1 105.3 no DBH 155 Enzyme; + DTT' 146.2 Boiled enzyme 151.4 Enzyme + f u s a r i c acid 178.9 Table 4 - o 14 Correlation between time i n storage at 20 C f o r C-SAM v.s. blank l e v e l . STORAGE TIME ( DAYS ) BLANK cpm  9 113.5 19 111.8 24 119.3 34 233.3 61 359.6 76 395.3 167 448.4 - 68 -C-SAM used i n t h i s melted. Accordingly, hot SAM was subsequently ordered i n the smallest possible quantities ( 10 txCi ), and both the time of storage and the number of freeze-thaw cycles minimized. F i n a l l y , an experiment conducted to determine the r e l a t i o n s h i p between rea c t i o n time i n the second step and the blank value ( Figure 8 ) showed that a sharp break i n a c t i v i t y occurred a f t e r 25 minutes incubation, i n contrast with the o r i g i n a l paper -which reported low blank values u n t i l a f t e r 30 minutes incubation, which was the basis the standard second step re a c t i o n time. Since blank values were found to increase much more r a p i d l y than sample a c t i v i t i e s between 25 and 30 minutes, by reducing, the incubation time i n the PNMT step to 25 minutes, the absolute maximum s e n s i t i v i t y , of the method could be doubled, with routine blank values of approximately 55 cpm. Subsequently a l l assays used a 25 minute second incubation period. The p o s s i b i l i t y of the use of extended incubation times i n the f i r s t step of the Molinoff assay to increase s e n s i t i v i t y , was prompted by l i t e r a t u r e reports of the l i n e a r i t y of the DBH reac t i o n obtained with solutions containing low enzyme a c t i v i t i e s , of up to 2.5 hours incubation, time ( CUBBEDDU et a l . , 1974 ). However, as i n the o r i g i n a l paper by Molinoff et a l . , the rea c t i o n was found to be l i n e a r f o r solutions of higher- a c t i v i t y ( adrenal homogehate ), only up to 20 minutes. As our vas deferens incubation media were c e r t a i n l y of low a c t i v i t y , the f e a s i b i l i t y of t h i s technique was investigated. However, tes t s with - 69 -0 —' T 1 1 1 10 20 30 40 T i m e (min) r o Figure 8. The e f f e c t of reaction time i n the second step on blank values i n the Molinoff assay ( standard conditions ). The e f f e c t of extended step one incubation time on DBH a c t i v i t y _3 obtained with p u r i f i e d enzyme ( [DBH]= 1.25 X 10 units) i n the standard Molinoff assay - 72 -extended incubation of p u r i f i e d enzyme ( Figure 9 ), r a t vas deferens homogenate, and high potassium incubate, a l l demonstrated a marked non-l i n e a r i t y of the DBH reaction with respect to time beyond the i n i t i a l 20 minutes. Reports of the i n s t a b i l i t y of DBH i n the extended incubation of ti s s u e perfusates ( WEINSHILBOUM et a l , , 1971 ) i n the absence of BSA led to a t r i a l of the use of 0.257c. BSA i n the assay mixture , i n the hopes of s t a b i l i z i n g the enzyme. Howvere, contrary to our expectations, while BSA apparently had l i t t l e e f f e c t on the long term s t a b i l i t y of, DBH, i t s i n c l u s i o n appeared to r e s u l t i n an a c t i v a t i o n of measured a c t i v i t i e s , i r r e s p e c t i v e of incubation time. Although i t was thought, that BSA might act v i a some sort of nonspecific protection of octopamine against oxidation, t h i s proved not to be the case. In f a c t , a study of the a c t i v i t i e s produced by a standard series of octopamine concentrations ( chosen to span our desired range of DBH a c t i v i t i e s ), shown i n Figure 10, indicated that the basis f o r t h i s apparent a c t i v a t i o n of measured DBH a c t i v i t y , l a y i n f a c t with an a c t i v a t i o n of the PNMT of the second assay step. At the same time, a marked n o n l i n e a r i t y was observed i n the a c t i v i t i e s of the octopamine standards, which suggested that the problems with non-l i n e a r i t y of the Molinoff assay with respect to time and concentration of enzyme might o r i g i n a t e with the PNMT re a c t i o n . I t was decided that i n order to better study the assay, each of Figure 10. A c t i v i t i e s produced by standard octopamine concentrations i n the Molinoff assay. (a), MRM-1-with 0.25% BSA (b), f u l l RM1 with 0.257o BSA. ( c ) , MRM-1. (d), f u l l RM1. Standard conditions for the second step. - 74 -- 75 -the enzymic reactions should be examined sparately. Accordingly, a r a t i o n a l e was adopted i n which standard concentrations of octopamine were used i n a modified f i r s t r e a c t i o n mixture c o n s i s t i n g only of the acetate bu f f e r and fumarate ( MRM-1 ), chosen to approximate the i o n i c strength of the f u l l RM1, to simulate the products of the f i r s t r e a c t i o n of the assay. This mixture was then used i n the second r e a c t i o n step performed under standard conditions. Each of the reagents i n the f u l l RM1 was then added back, alone and in.combination, i n order to assess the e f f e c t on the second step. ; From Figure 10, i t can be seen that lower a c t i v i t i e s were obtained with the f u l l RM1 as compared with MRM-1, i n d i c a t i n g an i n h i b i t o r or combination of i n h i b i t o r s of PNMT i n the f i r s t r e a c t i o n mixture. In addition, the PNMT a c t i v a t i o n produced by BSA appeared to be quite constant, • ir r e s p e c t i v e of octopamine concentration or composition of the r e a c t i o n mixture. Experiments involving the addition of i n d i v i d u a l reagents back into t h i s modified r e a c t i o n mixture produced the following r e s u l t s . From Figure 11, i t can be seen that ascorbate had no e f f e c t on the PNMT, \ while tyramine produced a marked i n h i b i t i o n . This was expected, as tyramine i n h i b i t i o n of PNMT was responsible f o r the use of concentrations of tyramine f a r below the K of DBH i n the o r i g i n a l method. A study of a c t i v i t y produced by a standard concentration of octopamine i n the - 76 -o -1 • ~l 1 0 . 0 5 0 . 2 5 0 . 5 [OCT] m M Figure 11. The e f f e c t of the i n c l u s i o n of ascorbate and tyramine on the a c t i v i t y obtained with standard concentrations of octopamine i n the Molinoff assay ( standard conditions), (a ) , MRM-1 + ascorbate. (b), MRM-1. ( c ) , MRM-1 + TYR. - 77 -4 0 0 - 1 O 2 0 0 H I—I o o 0 0.5 [ T Y R ] m M Figure 12. The e f f e c t of various conditions of tyramine on the a c t i v i t y obtained with a standard concentration of octopamine ( [0CT]= 5 X 10~5M ) i n the Molinoff assay - 78 -presence of a series of tyramine concentrations ( Figure 12 ) demonstrated the sigmoidal k i n e t i c s of t h i s i n h i b i t i o n . This aspect was of p o t e n t i a l importance because i t implied that the concentration of tyramine might be varied somewhat without greatly a f f e c t i n g s e n s i t i v i t y , and t h i s was l a t e r investigated. S i m i l a r l y , pargyline, alone and i n combination with asxarbate, proved to have no, e f f e c t on the PNMT reaction. Studies with catalase, included i n the assay to break down hydrogen peroxide formed i n the DBH reac t i o n and allow maximum DBH a c t i v i t i e s , produced an unexpected r e s u l t ( Figure 13 ). Commercial preparations of catalase were found to contain a heat stable, non-dialyzable i n h i b i t o r of PNMT a c t i v i t y . Paradoxically, however, ascorbate appeared to protect the enzyme against t h i s e f f e c t , as the combination of catalase and ascorbate i n the f i r s t r e a c t i o n mixture produced no such i n h i b i t i o n . At t h i s point, experiments were performed i n order to eliminate i n s t a b i l i t y of the products of both enzymic re a c t i o n s , octopamine and synephrine, as a cause f o r the observed n o n l i n e a r i t y of the assay. As can be seen i n Figure 14, octopamine was found to be completely stable i n both the modified and f u l l r e a c t i o n mixtures, when incubated for up to two hours at 3 7°C, i n contrast with a reported i n s t a b i l i t y of octopamine during extended incubation at pH 5.0 ( FLATMARK et al.,1978). Figure 13. The e f f e c t of the i n c l u s i o n of catalase, ascorbate and pargyline i n MRM-1, on the a c t i v i t i e s produced by standard concentrations of octopamine i n the Molinoff assay, (a), MRM-1 + catalase, ascorbate, and pargyline. (b), MRM-1. ( c ) , MRM-1, +. dialyzed catalase. (d)-, MRM-1 + catalase. (e)-,MRM-1 + bo i l e d catalase. o - 1 0.05 0.25 0.5 [ O C T ] m M Figure 14. The stability of octopamine ( 5 X over extended incubation times. ( . RM-1. +.0.25 % BSA. (c), MRM-1. 10 M ) i n the Molinoff assay i -i a ), MRM-1 + 0.25 7» BSA. ( b), f u l l ' (d), f u l l RM1 r o T 40 t I 80 m i n u t e s 120 160 - 83 -Also, studies using the fluorescence of synephrine indicated that an incubation at 3 7 ° f o r 30 minutes i n the second r e a c t i o n buffer produced only a 157o drop i n the fluorescence of a 1 pmolar s o l u t i o n , i n s u f f i c i e n t to account f o r any n o n l i n e a r i t y i n the assay. In addition, the l i n e a r i t y of the PNMT re a c t i o n with respect to time was experimentally confirmed ( see Figure 15 ). The nature of the PNMT a c t i v a t i o n exhibited by BSA was examined i n a study of the e f f e c t of a series of concentrations of BSA on the measure a c t i v i t y obtained with an octopamine standard ( Figure 16 ) . Quite s u r p r i s i n g l y , t h i s a c t i v a t i o n was found to be highly s p e c i f i c , and to exhibit a high degree of concentration dependence, quite i n contrast with the supposed nature of i t s action as a nonspecific protein s t a b i l i z e r . Maximum a c t i v a t i o n was found to occur with 0.14 % BSA, and i n c l u s i o n i n the sample aliquot r e s u l t e d i n a three- f o l d increase i n measured a c t i v i t y , c e r t a i n l y of s i g n i f i c a n c e i n the improvement of s e n s i t i v i t y . Despite these r e s u l t s , however, the reasons f o r the observed n o n l i n e a r i t y of the assay with respect to the concentration of enzyme and octopamine, as we l l as time, were not yet c l e a r . Further experiments were therefore conducted i n order to determine them, and a l l e v i a t e t h i s problem i f at a l l possible. - 84 -0 0 1 0 2 0 3 0 4 0 T ime (min) Figure 15. L i n e a r i t y of the a c t i v i t y obtained with a standard concentration of octopamine ( 5 X 10 ) with respect to incubation time for the second step of the Molinoff assay. - 85 -Figure 16. The e f f e c t of the i n c l u s i o n of BSA i n the r e a c t i o n mixture f o r the second step of the Molinoff assay on the a c t i v i t y produced by a standard concentration of octopamine ([OGT]= 5 X-10 M^ ). Standard conditions. - 86 -- 87 -An early study of the properties of PNMT ( AXELROD, 1962 ) indicated that i n order to achieve maximum a c t i v i t i e s of PNMT, high concentrations of the methyl donor, SAM, were required. To determine 14 whether the concentration of C-SAM used in, the Molinoff assay was less than would be required f o r complete saturation, an experiment was performed i n which the a c t i v i t y produced by a standard concentration of octopamine was measured i n the presence of increased concentrations of 14 C-SAM C Figure 17 ). As can be seen, at concentrations normally used i n the Molinoff procedure, the PNMT was f a r from saturation with respect to SAM, and the use of concentrations 20 times higher than normal, yielded an increase i n the measured a c t i v i t y of more than f i v e f o l d . This led to an experiment performed i n order to assess the e f f e c t s of the unsaturation of PNMT with respect to SAM, on the l i n e a r i t y of the rea c t i o n with" respect to the substrate, octopamine. The a c t i v i t y obtained with standard concentrations of octopamine was measured i n the presence of t o t a l concentrations of SAM of 20 and 40 times the normal value of 2.45 X 10 ^ M. In order to reduce the cost of t h i s modification, which otherwise would have made the assay p r o h i b i t i v e l y expensive, the concentrra-14 t i o n of C-SAM was increased by only a fac t o r of f i v e , while the remainder of the t o t a l concentration increase was made with unlabelled. SAM. As can be seen i n Figure 18, even at a t o t a l concentration of SAM of 20 X normal, the assay was s t i l l nonlinear with respect to octopamine. - 88 -8 - i CO O C H I 1 • 1 x1 x10 x20 [ 1 4C-SAM] Figure 17. The e f f e c t of increased concentration of C-SAM on a c t i v i t y produced by a standard concentration of OCT. ( normal [SAM]= 2 . 4 5 X 1 0 _ 5 M ) - 89 -CH 0.0 5 0.2 5 0.5 [ O C T ] mM Figure 18. Linearity of octopamine standards with different total concentrations of SAM in the second reaction mixture. (a), MRM-1; [ 14C-SAM ] X5 ; [ S A M - l t o t a l = x 2 ° . MRM-1 [ 14C-SAM ] X5 ; [SAM] t , = X40. total - 9 0 -However, when the concentration was rai s e d by a f a c t o r of 4 0 , which corresponds to e f f e c t i v e saturation of PNMT as shown by Figure 1 7 , the PNMT reac t i o n became l i n e a r f o r octopamine concentration, although with some s a c r i f i c e of maximum s e n s i t i v i t y . 1 4 The loss of s e n s i t i v i t y due to the d i l u t i o n of the C-SAM a c t i v i t y with unlabelled SAM presented a problem which could p o t e n t i a l l y be overcome i n several ways: by increasing the proportion of "hot" SAM, by increasing the concentration of the transferase i t s e l f , or by extended incubation. Since increasing the concentration of PNMT seemed the most p r a c t i c a l and cheapest of the former two a l t e r n a t i v e s , an experiment was conducted i n which the a c t i v i t y of a standard concentration of octopamine was measured i n the presence of increased PNMT l e v e l s ( Figure 1 9 ) . As i s apparent from the graph, the reac t i o n was l i n e a r with PNMT concentration up to 2 0 times normal, and yielded an a c t i v i t y incease i n d i r e c t proportion to the increase i n concentration. In order to check whether t h i s modification would produce any e f f e c t on the l i n e a r i t y with respect to octopamine obtained with increased [SAM], an experiment was performed i n which the octopamine standards were assayed with normal and increased [PNMT] i n the presence of 3 0 X [SAM] . From Figure 2 0 , i t was found that no such interference with l i n e a r i t y resulted from increased [PNMT], making t h i s a most worthwhile modification to the Molinoff procedure. - 91 -Figure 19. The e f f e c t of increased concentration of PNMT on the a c t i v i t y obtained with a standard concentration of OCT ( 5 X 10"4M ) i n the Molinoff assay. [ 1 4C-SAM ] = X5; [ S A M ] t o t a l " X 3 0 « - 92 -Figure 20. L i n e a r i t y of octopamine standards with increased concentration of PNMT i n the Molinoff assay, ( a ) , MRM-1; [ 1 4C-SAM ] X 5; [ SAM 1 _ .= X 30; [PNMT] X5. t o t a l -(b), MRM-1; [ 1 4C-SAM] X5; [SAM]^^., = X30; normal [PNMT]. - 93 -At t h i s time, an attempt was made to again assess the f e a s i b i l i t y of extended incubation with the now improved method ( Figure 21 ). However, as can be seen, the DBH r e a c t i o n s t i l l t a i l e d o f f d r a s t i c a l l y a f t e r about 30 minutes, implying that n o n l i n e a r i t y with respect to octopamine was not the basis f o r the problem with t h i s technique. The differences i n l i n e a r i t y with respect to enzyme concentration found with the spectrophotometric and Molinoff meltods ( Figures ,3, 7 ), as well as the f a c t that the concentration of tyramine employed i n the MOlinoff method ( 1 mM ) was f a r less than the saturating l e v e l ( 20 mM ) used i n the former method, prompted a t r i a l of increased tyramine concentration. Previous r e s u l t s showed that the k i n e t i c s of i n h i b i t i o n of PNMT were sigmoidal, implying that the concentration of tyramine might be somewhat va r i a b l e without d r a s t i c a l l y a f f e c t i n g DBH a c t i v i t i e s . Preliminary experiments indicated that almost no change i n measured a c t i v i t y r e s u l t e d , from a doubling of the tyramine concentration, while a f i v e - f o l d increase reduced a c t i v i t y by less than a factor of two. Accordingly, an experiment using extended incubation of p u r i f i e d DBH was performed with f i v e times the normal concentration of tyramine. As can be seen from Figure 22, t h i s modification produced a time l i n e a r i t y of the DBH r e a c t i o n , and when incubation time was extended to 2 hours, s i m i l a r a c t i v i t i e s were obtained to those produced i n the 20 minute incubation under normal conditions. Therefore, while not e f f e c t i v e i n further increasing o v e r a l l s e n s i t i v i t y , the use of increased [TYR] i n the f i r s t r e a c t i o n mixture would c e r t a i n l y serve to improve c o r r e l a t i o n between r e s u l t s obtained under d i f f e r e n t conditions and with d i f f e r e n t enzyme a c t i v i t i e s . - 94 -Figure 21. The e f f e c t of extended incubation i n the f i r s t r e a c t i o n step on DBH a c t i v i t y obtained with the p u r i f i e d enzyme i n the Molinoff assay with modified RM2. [DBH]= 2.5 X. 10 un i t s ; [ 1 4C-SAM] X4; [ SAM ] = X40; [PNMT] X5. Figure 22. L i n e a r i t y of extended incubation i n the f i r s t step of the Molinoff assay with increased concentration of tyramineC [DBH] = 2.5 X 10 u n i t s ) , (a), [14C-SAM] X4; [PNMT] X4, f u l l RM1 with normal [TYR]. (b), [ 1 4C-SAM] X4; [PNMT] X4; f u l l RM1 with [TYR] X5. 0 40 80 120 t m i n u t e s - 97 -A t e s t of l i n e a r i t y with respect to enzyme concentration using increased l e v e l s of [SAM]-, [PNMT], and [TYR] i s shown i n Figure 23. As i s apparent, the combination of these modifications makes the Molinoff method l i n e a r f o r enzyme concentration within our desired range of a c t i v i t y . 14 F i n a l l y , since the d i l u t i o n of C-SAM a c t i v i t y by the concentration of cold SAM necessary to achieve PNMT saturation , only served to make the assay l i n e a r to widely d i f f e r e n t concentrations of octopamine, an attempt was made to assess the f e a s i b i l i t y of extended incubation of solutions with low [DBH] such as high potassium incubates, with increased [TYR],and increased but nonsaturating [^C-SAM]. As can be seen i n Figure 24, t h i s produced a l i n e a r response with respect to time over about two hours. Although the use of nonsaturating [SAM] means that such r e s u l t s are not s t r i c t l y c o r r e l a t a b l e with a c t i v i t i e s obtained with higher [DBH], and i n f a c t , p a r a l l e l experiments using the same high K + incubate doped -3 with 2.5 X 10 units of p u r i f i e d DBH exhibited marked time n o n l i n e a r i t y ( not shown ), t h i s technique i s c e r t a i n l y of value i n the assessment of low l e v e l s of DBH a c t i v i t y . - 98 -Figure 23. L i n e a r i t y of the modified Molinoff assay with repect to [DBH];[ 1 4C-SAM] X 4; t S A M ^ t o t a l = x40; [PNMT] x4; [TYR] X2. - 99 -Figure 24. Extended incubation of high potassium r a t vas deferens incubate without saturating [SAM]; [ 1 4C-SAM] X4; [PNMT] X8; [TYR] X5. - 100 -The a p p l i c a t i o n of the modified assay to our desired experimental goals was also examined i n preliminary experiments. In terms of absolute _4 s e n s i t i v i t y , an enzyme concentration of only 2.5 X 10 units produced an a c t i v i t y of over 4000 cpm (0.14% BSA; [PNMT]. X15; [ 1 4C-SAM] X6 ), i n d i c a t i n g an increase i n s e n s i t i v i t y of over two orders of magnitude over the standard procedure. Perhaps more importantly, the DBH release from a sing l e r a t t a i l artery into 5 ml PSS, 150 mM i n K+, over an incubation period of 30 minutes, was assayed and found to produce a measured a c t i v i t y of almost 600 cpm over the blank, even without extended incubation (0.14% BSA; [TYR] X2; [ 1 4C-SAM] X 6; [PNMT] X15 ). - 101 -DISCUSSION The measurement of dopamine B-hydroxylase a c t i v i t y i n incubates of i s o l a t e d r a t t a i l artery or vas deferens under conditions of maximal exocytotic release ( high [ K + ] , v e r a t r i d i n e ), with release induced by less than optimal conditions such as the i n h i b i t i o n of the sodium pump ( 1 mM ouabain , low [Na +] ), requires an assay of maximal s e n s i t i v i t y . Therefore, the ex i s t i n g assay methods f o r DBH were evaluated and two were chosen as most sui t a b l e f o r the aims of t h i s project, the dual spectrophotometric method of Nagatsu ( KATO et a l . , 1978 ) and the coupled radioenzymatic assay of Molinoff ( MOLINOFF et, aL, 1971 ). Both procedures were w e l l developed and widely applied, and each claimed s u f f i c i e n t s e n s i t i v i t y to detect DBH i n t i s s u e perfusates. The i n i t i a l choice was the dual spectrophotometric assay, because of ease of performance, lower cost, and the f a c t that DBH i s assayed under saturated conditions. In addition, a recent paper d e t a i l e d several modifications which raised the s e n s i t i v i t y considerably ( KATO et a l . , 1978) so a t r i a l of the method was c e r t a i n l y warranted. In t h i s method, tyramine i s converted to octopamine by the DBH i n the sample a l i q u o t . This product i s i s o l a t e d by an ion exchange column, s and subsequently converted to p-hydroxybenzaldehyde by periodate oxidation. The high e x t i n c t i o n c o e f f i c i e n t of t h i s compound at 333 nm - 1 0 2 -allows easy spectrophotometric detection. There are several advantages to t h i s procedure which make i t p o t e n t i -a l l y more desirable than the radioenzymatic assay. The primary diffe r e n c e between the two l i e s with the f a c t that chemical rather than ensymatic conversion of octopamine to the f i n a l detectable product i s employed. For t h i s reason, constraints are not placed upon the concentration of reagents i n the f i r s t r e a c t i o n mixture, and concentrations can be optimized with respect to DBH a c t i v i t y . In t h i s method, therefore, the concentration of tyramine used i s 2 0 mM, the value required f o r saturation ( as compared with the 1 mM used i n the Molinoff method ). S i m i l a r l y , a combination of Cu +^ and NEM i s used to in a c t i v a t e endogenous i n h i b i t o r s without a f f e c t i n g DBH a c t i v i t y , a f a r cry from the continual t i t r a t i o n with Cu +^ required with each new sample i n the Molinoff method. In addition, since the conversion of octopamine to p-HB i s p e r f e c t l y l i n e a r , no l i m i t a t i o n i s imposed on re a c t i o n time f o r the f i r s t step. F i n a l l y , a s i m i l a r advantage i n the e f f i c i e n c y of i s o l a t i o n and conversion of the octopamine product, implies an excellent p o t e n t i a l f o r s e n s i t i v i t y : these, processes are over 98 and 95 °L e f f i c i e n t r e s p e c t i v e l y . Therefore, maximum DBH a c t i v i t i e s are measured by t h i s method. The only disadvantage to t h i s procedure i s the spectrophotometric method of detection i t s e l f . While the assay was greatly improved by the use of d i f f e r e n t i a l spectrophotometry, and the adoption of f u s a r i c acid i n the - 103 -blank mixture resulted i n stable blanks with a very low absorbance value, s e n s i t i v i t y was s t i l l somewhat less than could be obtained with ra d i o -metric detection, a f a c t which prompted the numerous radioenzymatic variants of t h i s method ( FREIDMAN and KAUFMAN, 1965; NAGATSU et al.,1972; JOH et a l . , 1974; WISE, 1976 ). In preliminary experiments, the l i n e a r i t y of the assay with respect to octopamine concentration was confirmed up to a l e v e l of 0.2 mM, c e r t a i n l y s u f f i c i e n t f o r our purposes. In addition, i t was found that the DBH reac t i o n was l i n e a r f o r enzyme, concentration up to 0.05 u n i t s , again more than adequate. However, deviations from l i n e a r i t y were observed with otopamine concentrations lower than 50 yM, and blank values were inconsistent and generally-much higher than the AA= 0.005 reported i n the l i t e r a t u r e ( KATO et a l . , 1978 ), which together greatly l i m i t e d maximum s e n s i t i v i t y . Since i n i t i a l studies with t i s s u e perfusates indicated that a c t i v i t i e s w e r e low enough f o r the blank to be a problem, an attempt was made to reduce and s t a b i l i z e blank l e v e l s i n the hopes that s e n s i t i v i t y could be s u f f i c i e n t l y improved to detect these concentrations of DBH within the l e v e l of signifi c a n c e . , A number of d i f f e r e n t compounds were t r i e d i n the blank mixture, with the strategy of enzyme i n h i b i t i o n . b y chelation of the enzyme bound copper necessary f o r a c t i v i t y . Of the various combinations tested, the - 4 - 4 mixture of 5 X 10 M f u s a r i c acid and 5 X 10 EDTA were found to produce a r o u t i n e l y repeatable blank of A= 0.005 u n i t s , equal to the lowest - 104 -l i t e r a t u r e l e v e l . Howver, even with the improved blank, the method was s t i l l not s e n s i t i v e enough to measure DBH release into high potassium incubate. A f i n a l attempt to increase s e n s i t i v i t y was prompted by the l i t e r a t u r e •i report of a c t i v a t i o n of DBH by ADP and other nucleotides ( TAGHIKAWA et a l . , 1979 ). While t h i s approach was successful, with a maximum 2.5 f o l d increase i n a c t i v i t y r e s u l t i n g from the i n c l u s i o n of 6 mM ADP i n the r e a c t i o n mixture, the f i n a l s e n s i t i v i t y waB s t i l l i n s u f f i c i e n t f o r our purposes of measuring DBH i n t i s s u e incubates. Experiments with r a t serum, however , indicated that these modifications might make the procedure more sui t a b l e fo r a p p l i c a t i o n to less demanding s i t u a t i o n s l i k e the assessment of the l e v e l of DBH i n the serum of laboratory animals. With the f a i l u r e of the spectrophotometric method, the alternate choice of procedure was the Molinoff assay, because of i t s inherently greater s e n s i t i v i t y . In t h i s method, the DBH i n the sample aliqu o t i s used to convert the substrate tyramine to octopamine. A second enzymatic step 14 u t i l i z i n g PNMT i s used to l a b e l t h i s product with a C-methyl group to 14 form C-synephrine, which i s detectable by l i q u i d s c i n t i l l a t i o n counting. Despite the f a c t that the c h a r a c t e r i s t i c s of PNMT require the use of a concentration of tyramine f a r below that necessary f o r saturation, and necessitates t i t r a t i o n of each sample with Cu +^ i n order to i n a c t i v a t e endogenous i n h i b i t o r s to obtain maximum a c t i v i t y , t h i s method i s s t i l l an improvement over the l a t t e r . The e f f i c i e n c y of detection made possible - 105 -by the r a d i o a c t i v i t y of the product, as well as the f a c t that the substrate fo r the DBH r e a c t i o n ^ i s unlabelled, as compared with other radiometric assays, produce at l e a s t an order of magnitude improvement i n s e n s i t i v i t y . Although i n i t i a l experiments were promising, as the release of DBH from i s o l a t e d r a t vas defera incubated i n 150 mM K + medium was detectable and produced an estimation, of the release i n good agreement with p a r a l l e l studies reported f o r the guinea pig vas deferns ( THOA et a l . , 1975 ), the low sample a c t i v i t i e s over blank l e v e l s indicated p o t e n t i a l problems with the measurement,of a c t i v i t i e s released by the i n h i b i t i o n of the sodium pump. I t was decided, therefore, to examine the method more c l o s e l y i n the hopes of improving s e n s i t i v i t y . As with the spectrophotometrie method, improvements i n the blank mixture Were attempted. However, the DTT blank employed i n the standard procedure was found to be most e f f e c t i v e . The behaviour of the blank with respect to r e a c t i o n time i n the second step was investigated, and i t was found that due to a sharp increase i n the blank values noted a f t e r the 25 minute mark, r e s t r i c t i o n of the second incubation to 25 minutes reduced the blank to the point where a 2 f o l d gain i n s e n s i t i v i t y could be r e a l i z e d . This i s i n contrast with the reports of stable blanks up to 30 minutes incubation i n the o r i g i n a l paper ( MOLINOFF et a l . , 1971 ). The marked time n o n l i n e a r i t y beyond the i n i t i a l 20 minutes of 1 - 106. -incubation i n the early t e s t s of the f e a s i b i l i t y of extended f i r s t step r e a c t i o n times, as we l l as the n o n l i n e a r i t y with respect to enzyme concen-t r a t i o n found i n studies with p u r i f i e d DBH, indicated that the basis f o r these problems might l i e with the second enzymatic r e a c t i o n . Accordingly, an approach of examining each of the two reactions independently was adopted, and an analysis of the e f f e c t s and interactions of each of the components i n the two reac t i o n mixtures was c a r r i e d out. It was determined that the reason f o r t h i s n o n l i n e a r i t y exhibited by the assay did not l i e with any i n s t a b i l i t y of octopamine or,synephrine over the incubation period, contrary to previous reports ( Flatmark et a l . , 1978 ). Therefore, i t was obvious that the f a u l t must o r i g i n a t e i n the process of enzymatic formation of these two products. In an i n v e s t i g a t i o n of the second step, i t was determined that ascor-bate ( necessary as the oxygen donor to, DBH ), fumarate ( present because d i c a r b o x y l i c acids serve to stimulate DBH a c t i v i t y ), and pargyline ( a MAO i n h i b i t o r ) had no e f f e c t on the a c t i v i t y of PNMT with respect to octopamine standards, e i t h e r alone or i n combination. The substrate tyramine, as expected, produced a marked i n h i b i t i o n of PNMT a c t i v i t y , although the sigmoidal k i n e t i c s had not been previously demonstrated. Quite paradoxically, however, commercial preparations of catalase were shown to contain a heat stable, non-dialyzable i n h i b i t o r of PNMT of an unknown nature. The antioxidant properties of ascorbate seemed to - 107 -protect against t h i s f a c t o r , as no such i n h i b i t i o n was noted when ascorbate was also present i n the re a c t i o n mixture. While BSA was fo\md to increase DBH a c t i v i t i e s measured by the Molinoff assay, studies indicated that the mechanism f o r t h i s e f f e c t was not the nonspecific s t a b i l i z a t i o n of the enzyme reported by a number of authors ( WEINSHILBOUM et a l . , 1971 ). Rather, BSA was found to produce a s p e c i f i c and highly concentration dependent a c t i v a t i o n of the PNMT of the second step. A maximum 3 f o l d increase irimeasured a c t i v i t y was demonstrated with the i n c l u s i o n of 0.14% BSA i n the sample a l i q u o t . However, i t s presence had no e f f e c t on the n o n l i n e a r i t y of the reac t i o n mixture with respect to octopamine. F i n a l l y , experiments conducted to determine the e f f e c t s of increased concentrations of SAM, revealed the basis f o r t h i s n o n l i n e a r i t y . I t was shown that the PNMT was f a r from being saturated with respect to SAM: A concentration of SAM>40 times higher than the standard was required to f u l l y saturate the enzyme. Inclusion of t h i s increased concentration of SAM, composed predominantly of unlabelled SAM to lower the cost of t h i s m odification, produced the desired l i n e a r i t y with respect to d i f f e r e n t . , . 14 concentrations! of octopamine. With the lower l e v e l s of C-SAM used i n the standard method, therefore, the dif f e r e n c e i n re a c t i o n v e l o c i t y produced by the disappearance of t h i s substrate during the course of the r e a c t i o n caused the drop i n measured a c t i v i t i e s found with higher [Oct]. - 108 -The loss of s p e c i f i c i t y produced by the d i l u t i o n of ^ C-SAM a c t i v i t y by the concentration of unlabelled SAM necessary to achieve saturation, could be r e l i e v e d by an increase i n the concentration of PNMT. I t was found that a c t i v i t i e s increased i n d i r e c t proportion to the increase i n [PNMT], without affecting l i n e a r i t y . In t h i s manner, the s e n s i t i v i t y of the assay can be increased according to necessity by adjustment of the 14 proportion of C-SAM and the concentration of PNMT i n the second r e a c t i o n mixture. The f e a s i b i l i t y of extended f i r s t step incubation times was again investigated i n the hopes of increasing absolute s e n s i t i v i t y even fur t h e r . However, as i n the previous t r i a l s , the assay was found to be nonlinear with respect to incubation time past the i n i t i a l 20 minutes, a iproblem found to o r i g i n a t e with the low, unsaturating concentrations of tyramine employed i n the f i r s t r e a c t i o n . While tyramine did i n h i b i t the PNMT of the second step, the sigmoidal nature of t h i s e f f e c t allowed a modest increase i n [TYR] without s i g n i f i c a n t l y a f f e c t i n g measured a c t i v i t y . Therefore, -although a decrease i n s e n s i t i v i t y was produced by t h i s change, the increase i n tyramine concentration to a l e v e l f i v e times higher than standard, produced a l i n e a r i t y of the DBH r e a c t i o n with respect to time over up to two hours. In addition, even though no gain i n s e n s i t i v i t y can r e s u l t from t h i s m odification, the a c t i v i t y of a standard [DBH] incubated f o r two hours i n the presence of 5 X [TYR] was approximately the same as that produced by standard conditions over 20 minutes, with the benefit of much improved c o r r e l a t a b i l i t y between r e s u l t s . - 109 -F i n a l l y , as a technique f o r maximum s e n s i t i v i t y with samples f a l l i n g w ithin a narrow range of a c t i v i t i e s , the p o s s i b i l i t y of the use of increased but nonsaturating [^4C-SAM] i n combination with increased [PNMT] and [TYR] was investigated. While r e s u l t s obtained under these conditions would not be s t r i c t l y c o r r e l a t a b l e , due to the d i f f e r i n g r e a c t i o n v e l o c i t i e s produced with s i g n i f i c a n t l y d i f f e r e n t concentrations of DBH, r e s u l t s obtained f o r samples with s i m i l a r a c t i v i t i e s would c e r t a i n l y be i n reasonable agreement, and c a r e f u l matching of the i n t e r n a l standard would serve to equalize the r e s u l t s . Therefore, f o r applications such as the detection of differences i n the release of DBH occurring under d i f f e r e n t conditions of t i s s u e treatment, t h i s procedure would be i d e a l . A f i n a l check of the s e n s i t i v i t y of the modified method was conduc-ted, and an increase i n s e n s i t i v i t y of at l e a s t two orders of magnitude was shown over the standard method. 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