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Chapter 27. Regulation of Cell Metabolism: Role of Cyclic AMP
Chapter 27. Regulation of Cell Metabolism: Role of Cyclic Cnarles G. Smith, The Upjohn Company, Kalamazoo, Michigan Cyclic 3',5'-adenylic acid (cyclic AMP), the structure of which is shown in Figure 1, was isolated from animal tissue by Sutherland and Pall''', while investigating the mechanism by which epinephrine exerts its glycogenolytic effect in liver and muscle. These and subsequent investigations in various laboratories suggested a role for cyclic AMP as a modifier or mediator of pho~phorylase~'", UDFG-~-transglucosylase7 phosphofructokinases, lipasel' tryptophan pyrrolase12, steroid~genesisl~-~~ '*a J15b ,ketogenesis'" , amino acid uptake into liver proteins17, acetate incorporation into liver fatty acids and cholesterol16, lactate conversion to glucoseLs, release of amylase'@, water and ion permeability in the toad bladder20'21, sugar transport in thyroid tissue22 and acid secretion in the gastric mucosa23. In addition, this nucleotide has been implicated in the actions of g l ~ c a g o n ~ACTH3, ~, vasopressin=, luteinizing hormones , thyroid stimulating hormone=, serotonina, acetyl cholinem, prostaglandin1'", histamine%, melanocyte-stimulating and i n s ~ l i n X ~ ' ~ ~Several C. recent papers have appeared in which the role of cyclic AMP as a hormone mediator or "second messenger" has been described in some detai131-3s and a suggestion that adenyl cyclase may It is the actually be the adrenergic receptor site has also been made3'". purpose of this communication to center discussion on the possible value of cyclic AMP, and test systems for measuring its effects, to the medicinal chemist. Synthesis and Degradation of Cyclic AMP Adenyl cyclase - The synthesis of cyclic AMP from ATP is catalyzed by the enzyme adenyl cyclase, which itself is responsive to hormone stimulation in various tissues3'. Catecholamines have been shown to stimulate the formation of cyclic AMP in a variety of tissues3' (Table 133), and adenyl cyclase has been shown to be wide-spread in various tissues39. Since procedures for the isolation and assay of this enzyme and measurement of the products of its reaction are available3' direct effects of analogues of ATP and/or cyclic AMP (or hormones) on the enzyme can be determined in an in vitro system. The degradation of cyclic AMP is catalyzed by a specific phosphodiesterase which converts it to adenylic (Figure 1). Again, the effect of nucleotide analogues or other potential inhibitors on the degradation of cyclic AMP can be studied in vitro in this enzyme system, and the possible significance of such agents will be discussed below. Cyclic 3',5'-AMF'as a Hormone Mediator
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The phosphorylase system While investigating the site of the glycogenolytic action of epinephrine, Sutherland and Cori4' demonstrated the stimulation of phosphorylase activity by this catecholamine and subsequent studies' '32' 43
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implicated c y c l i c AMP i n t h i s process. Krebs, e t al.32, have r e c e n t l y r e viewed t h e s u b j e c t o f a c t i v a t i o n of s k e l e t a l muscle phosphorylase under t h e influence of epinephrine, mediated by c y c l i c AMP. Figure 2 , adapted from t h e i r paper, summarizes t h e s t e p s which have been shown t o be involved i n t h i s process. The d i r e c t a c t i v a t i o n of phosphorylase b k i n a s e i n muscle e x t r a c t can be demonstrated upon t h e a d d i t i o n of c y c l i c AMP i n v i t r o 4 3 , 4 4 . The a c t i v a t i o n of phosphorylase by epinephrine has a l s o been demonstrated i n t h e perfused h e a r t and i n t a c t h e a r t i n s i t u 3 5 . Thus, t h r e e enzymatic systems a r e a v a i l a b l e which can be s t u d i e d i n i s o l a t e d c e l l p r e p a r a t i o n s f o r e f f e c t s of analogues or chemically u n r e l a t e d compounds on e i t h e r t h e s y n t h e s i s or degradation of c y c l i c AMP or enzymatic a c t i v a t i o n by t h i s nucleotide. The p h o ~ p h o d i e s t e r a s e ~which l cleaves c y c l i c AMP i s i n h i b i t e d by methyl xanthines and it i s e n t i r e l y p o s s i b l e t h a t some or a l l of t h e b i o l o g i c a l a c t i v i t y observed f o r t h e s e i n h i b i t o r s of c y c l i c AMP degradation may be due t o t h i s a c t i o n . For example, t h e o p h y l l i n e has been shown t o i n c r e a s e phosphorylase a a c t i v i t y i n t h e i s o l a t e d h e a r t and p o t e n t i a t e t h e e f f e c t s of epinephrine in t h i s assaya5. E f f e c t s on l i p o l y s i s - I n a r e c e n t a r t i c l e , Butcher37 reviews t h e h i s t o r y o f t h e i r experiments on t h e r o l e of t h e c y c l i c AMF i n t h e l i p o l y t i c process. T h e i r i n v e s t i g a t i o n s showed an i n c r e a s e i n t h e concentration of c y c l i c AMP upon t h e a d d i t i o n of epinephrine t o f a t pads i n v i t r o , followed by a s t i m u l a t i o n of t h e r e l e a s e of f r e e f a t t y a c i d s . Caffeine, a known i n h i b i t o r o f t h e phosphodiesterase which degrades c y c l i c AMP, a c t e d s y n e r g i s t i c a l l y with epinephrine on t h e accumulation of c y c l i c AMP and t h e subsequent r e l e a s e of f r e e f a t t y acid^^^,^^. It should be noted t h a t t h e l i p o l y t i c e f f e c t of epinep h r i n e w a s decreased by d i c h l o r o i s o p r o t e r e n o l , a known i n h i b i t o r of t h e s t i m u l a t i o n of adenyl cyclase by c a t e ~ h o l a r n i n e ~ " ~ ~I n . s p i t e of t h i s e v i dence f o r t h e r o l e of c y c l i c AMP as a mediator i n t h e l i p o l y t i c process, t h e d i r e c t a d d i t i o n of t h i s n u c l e o t i d e t o f a t pads i n v i t r o d i d not s t i m u l a t e f a t t y a c i d r e l e a s e 4 5 , although p e n e t r a t i o n i n t o t i s s u e by t h e n u c l e o t i d e w a s low. When t h e Ne-2'-O,dibutyryl e s t e r of c y c l i c 3',5'-AMP4e w a s added t o f a t pads or f a t pads were perfused with t h i s compound, s t i m u l a t i o n of t i s s u e f r e e f a t t y a c i d s y n t h e s i s and f r e e f a t t y a c i d r e l e a s e w a s noted. The subs t i t u t e d compound w a s a t l e a s t t e n times as a c t i v e i n t h i s system as w a s c y c l i c AMF per se. The d i b u t y r y l d e r i v a t i v e may p e n e t r a t e t i s s u e more e f f e c t i v e l y t h a n does t h e u n s u b s t i t u t e d n u c l e o t i d e , but a l s o it i s degraded by t h e c y c l i c phosphodiesterase more slowly t h a n i s t h e p a r e n t compound 3 7 . T h i s i s indeed a n e x c i t i n g example t o t h e medicinal chemist of a r e l a t i v e l y simple chemical s u b s t i t u t i o n which r e s u l t s i n a marked change i n t h e a b i l i t y of a compound t o p e n e t r a t e t i s s u e and withstand t h e degradation t h a t i t s p r o g e n i t o r undergoes. A t t h e same time, t h e substance obviously can be conv e r t e d , a l b e i t slowly, t o t h e a c t i v e c y c l i c AMP and a c t as a " s l o w f e e d " of the latter. E f f e c t s on s t e r o i d o g e n e s i s - E a r l y investigation^^^'^ showed t h a t incubation of beef a d r e n a l s l i c e s with ACTH r e s u l t e d i n an i n c r e a s e i n t h e c y c l i c AMP l e v e l i n t h i s t i s s u e and t h a t added c y c l i c AMP s t i m u l a t e d c o r t i c o i d sylrthesis i n r a t a d r e n a l s l i c e s . More r e c e n t l y , Karaboyas and K o r i t z & s t u d i e d t h e
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mechanism of s t i m u l a t i o n of c o r t i c o i d s y n t h e s i s i n r a t and beef a d r e n a l c o r t e x s l i c e s and concluded t h a t c y c l i c AMP and ACTH a f f e c t t h e same s i t e i n t h e b i o s y n t h e t i c pathway between c h o l e s t e r o l and pregnenolone. They f u r t h e r suggested t h a t c y c l i c AMP i s a n o b l i g a t o r y intermediate i n t h e a c t i o n ~~ that of ACTH. I n another r e c e n t paper, Roberts and c o - w ~ r k e r sconcluded 5 l - M (as w e l l as c y c l i c AMP) s t i m u l a t e s s t e r o i d hydroxylation i n adrenal mitochondria. The mechanism whereby c y c l i c AMP enhances s t e r o i d hydroxyla t i o n i n t h i s system i s unknown. I n t h e i r hands, hydroxylation of s t e r o i d could not be obtained even a t high l e v e l s of c y c l i c AMP i f t h e a d r e n a l mitoc h o n d r i a l system or homogenate d i d not c o n t a i n reduced NADP, and t h e y suggest as p o s s i b l e explanations f o r t h e a c t i o n of t h i s n u c l e o t i d e 1) increased s t i m u l a t i o n of t h e t r a n s p o r t of s t e r o i d ( o r co-factor ) a c r o s s mitochondria1 membranes o r 2 ) d i r e c t a c t i v a t i o n of t h e s t e r o i d hydroxylase system. Creange and Roberts on t h e o t h e r hand, a l s o studying t h e mechanism of ll-fbhydroxyla t i o n , concluded t h a t c y c l i c AMP s e l e c t i v e l y stimulated t h i s process i n t h e a d r e n a l c o r t e x by a mechanism which i s independent of glycogen phosphoryla t i o n , reduced NADP g e n e r a t i o n and endogenous c o r t i c o i d precursorss1. I n 1964, Marsh and Savards2 r e p o r t e d t h a t l u t e i n i z i n g hormone increased t h e l e v e l of phosphorylase a c t i v i t y i n bovine corpora l u t e a , t h a t t h i s response w a s s p e c i f i c f o r t h e hormone and t h a t a high degree of c o r r e l a t i o n e x i s t e d between t h e e x t e n t o f phosphorylase s t i m u l a t i o n and t h e degree o f progesterone s t i m u l a t i o n by l u t e i n i z i n g hormone. I n t h e i r s t u d i e s , exogenous c y c l i c AMP d i d not s t i m u l a t e l u t e a l phosphorylase a c t i v i t y . More r e c e n t l y , Marsh and c o l l a b o r a t o r s 5 showed t h a t l u t e i n i z i n g hormone s t i m u l a t e d t h e production of c y c l i c AMP i n bovine corpora l u t e a incubated i n v i t r o and t h a t t h i s i n c r e a s e preceded progesterone s y n t h e s i s . Again t h e e f f e c t w a s r e p o r t e d t o be s p e c i f i c f o r l u t e i n i z i n g hormone. These i n v e s t i g a t o r s a l s o demonstrated a n i n c r e a s e i n t h e c o n c e n t r a t i o n o f c y c l i c AMP i n a human corpus luteum s t i m u l a t e d Ln v i t r o by human chorionic gonadotrophin. Other e f f e c t s of c y c l i c AMP i n v i t r o - I n a r e c e n t paper, Appleman and coworkers5 concluded t h a t many s i m i l a r i t i e s e x i s t between t h e ATP-cyclic AMP a c t i v a t i o n of phosphorylase b k i n a s e and t h e conversion of glycogen synthetase from t h e independent t o t h e glucose 6-phosphate-dependent form. C e r t a i n i n t e r e s t i n g e f f e c t s on i o n and water permeability a r e a l s o thought t o be mediated by c y c l i c AMP. Handler, e t a1.=, r e p o r t e d t h a t e i t h e r a r g i n i n e vasopressin or t h e o p h y l l i n e s i g n i f i c a n t l y i n c r e a s e d t h e concentration of c y c l i c AMP i n t h e i s o l a t e d toad bladder and t h a t t h e two t o g e t h e r were synergistic. Cyclic AMP w a s a l s o r e p o r t e d t o have t h e same e f f e c t as vasop r e s s i n on t h e permeability of t h e bladder t o water and sodium i o n and, accordingly, w a s concluded t o a c t as a n i n t r a c e l l u l a r mediator of t h e vasop r e s s i n e f f e c t . It i s i n t e r e s t i n g t h a t t h i s n u c l e o t i d e has a l s o been r e p o r t e d t o i n c r e a s e d i u r e s i s i n vivo, as described below. Although t h y r o i d s t i m u l a t i n g hormone (TSH) has been r e p o r t e d t o i n c r e a s e l e v e l s of c y c l i c AMP i n t h y r o i d homogenates and s l i c e s = ' s 6 , t h e n u c l e o t i d e d i d not i n c r e a s e Ci402 production from glucose nor t h e i n c o r p o r a t i o n of $2phosphate i n t o phospholipids i n v i t r o , as w a s observed with TSH p e r seS6 ' 5 7 . More r e c e n t l y , Pastans8 r e p o r t e d a s i g n i f i c a n t s t i m u l a t i o n of both of t h e s e
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p r o c e s s e s by t h e d i b u t y r y l d e r i v a t i v e o f c y c l i c AMP a t c o n c e n t r a t i o n s as low as 50 pg/mL b u t not by c y c l i c AMP a t f i v e t i m e s t h i s c o n c e n t r a t i o n . TSH, as expected, s t i m u l a t e d b o t h p r o c e s s e s a t low c o n c e n t r a t i o n . Again, as mentioned above under l i p o l y t i c s t u d i e s , t h e enhanced a c t i v i t y of t h e d i b u t y r y l s u b s t i t u t e d n u c l e o t i d e should be noted. Metabolic E f f e c t s of Cyclic AMP i n Whole Animals A s mentioned e a r l i e r , c y c l i c AMP has been p o s t u l a t e d as t h e c e l l u l a r i n t e r mediate i n t h e a n t i - d i u r e t i c a c t i o n of v a s o p r e ~ s i '21y25 n ~ ~ j 5 9 y60 i n i s o l a t e d dog kidney homogenates and i s o l a t e d t o a d b l a d d e r . Based orAt h e s e o b s e r v a tions, Levine s t u d i e d t h e e f f e c t of c y c l i c AMP on d i u r e s i s i n v i v o and r e p o r t e d a prompt a n t i - d i u r e t i c e f f e c t a f t e r i n j e c t i o n of t h e n u c l e o t i d e Levine and VogelG2 a l s o determined t h e e f f e c t of t h e c y c l i c i n man61. n u c l e o t i d e on h e a r t r a t e , c a r d i a c o u t p u t , mean blood p r e s s u r e , t o t a l p e r i p h e r a l r e s i s t a n c e , s t r o k e volume,pulse p r e s s u r e , blood g l u c o s e and plasma f r e e f a t t y a c i d s i n i n t a c t dogs and r e p o r t e d t h a t a d m i n i s t r a t i o n of a s i n g l e dose produced e f f e c t s i n t h e whole animal resembling t h o s e of c a t e cholamines. They r e p o r t e d no s i g n i f i c a n t changes i n c a r d i a c o r m e t a b o l i t e f u n c t i o n s f o l l o w i n g a d m i n i s t r a t i o n of t h e 2 l - , 3 * - o r 5'-AMP, ATP or s a l i n e , a l t h o u g h p r i o r a d m i n i s t r a t i o n of d i c h l o r o i s o p r o t e r e n o l p r e v e n t e d t h e c a r d i o v a s c u l a r (but n o t t h e m e t a b o l i c e f f e c t s ) of c y c l i c AMP. The l a t t e r nucleot i d e has a l s o been i m p l i c a t e d i n t h e mechanism o f myocardial ~ o n t r a c t i l i t ~ ~ - "
General C o n s i d e r a t i o n s Examination o f a v a i l a b l e d a t a c l e a r l y i n d i c a t e s a r o l e f o r c y c l i c AMP as a mediator of t h e catecholamine s t i m u l a t i o n of g l y c o g e n o l y s i s , b y t h e mechanism d e p i c t e d i n F i g u r e 2. Unlike t h e a t t e m p t s t o demonstrate t h e s i t e of a c t i o n of most p r o t e i n and s t e r o i d hormones66, t h e expected i n c r e a s e s i n each of t h e enzyme a c t i v i t i e s presumed t o b e mediated by c y c l i c AMP can be demons t r a t e d with d i r e c t i n v i t r o techniques after treatment of t h e appropriate t i s s u e w i t h s p e c i f i c hormones. A s a n example, adenyl c y c l a s e a c t i v i t y and c y c l i c AMP i n c r e a s e upon t r e a t m e n t w i t h e p i n e p h r i n e and t h i s i n t u r n can b e shown, a t t h e enzymatic l e v e l , t o a c t i v a t e phosphorylase k i n a s e . The l a t t e r enzyme c o n v e r t s phosphorylase b t o phosphorylase a which i s t h e a c t i v e form o f t h e enzyme c a t a l y z i n g t h e s y n t h e s i s of glucose-1-phosphate from glucagon. The o p p o r t u n i t i e s f o r t h e m e d i c i n a l chemist t o s t u d y analogues and d e r i v a t i v e s o f t h e simple n u c l e o t i d e s o r o f compounds r e l a t e d t o t h e catecholamines a r e unusual i n t h a t s e v e r a l enzymatic s t e p s i n a known sequence of r e a c t i o n s can b e measured q u a n t i t a t i v e l y i n v i t r o and e f f e c t s of s t i m u l a t o r s o r i n h i b i t o r s can b e d i r e c t l y a s s e s s e d . The d a t a i m p l i c a t i n g c y c l i c AMP i n t h e l i p o l y t i c p r o c e s s are a l s o q u i t e convincing, and t h e concept of c y c l i c n u c l e o t i d e mediation of ACTH i n s t e r o i d o g e n e s i s and v a s o p r e s s i n a c t i o n on water and i o n t r a n s p o r t a c r o s s nembranes i s a l s o sound. I n v i v o , c y c l i c AMP e x e r t s many of t h e e f f e c t s observed w i t h c a t e cholamines and, i n v a r i o u s systems, t h e methylxanthines (e.g., t h e o p h y l l i n e ) , which a r e known t o i n h i b i t t h e p h o s p h o d i e s t e r a s e t h a t c l e a v e s t h e n u c l e o t i d e , a l s o p o t e n t i a t e i t s a c t i o n . I n a d d i t i o n , t h e c y c l i c n u c l e o t i d e has been i m p l i c a t e d i n v a r i o u s s t e p s i n t h e gluconeogenic pathway, t h e mechanism o f a c t i o n of l u t e i n i z i n g hormone, glycogen s y n t h e t a s e and t h e metabolism of
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thyroid t i ssue. Although it is not uncommon t o a s s o c i a t e s i n g l e co-enzymes with d i f f e r e n t enzymes c a t a l y z i n g s i m i l a r t y p e s of r e a c t i o n s (e.g., ATP w i t h k i n a s e s , NADP w i t h reductases, and t h e l i k e ) , it i s not common t o t h i n k of a s i n g l e small molecule as a mediator of a wide v a r i e t y of hormonal o r metabolic r e a c t i o n s . Since adenyl c y c l a s e i s membrane bound, it i s p o s s i b l e t h a t s t e r e o - s p e c i f i c attachment s i t e s on t h e c e l l s u r f a c e t o which t h e p a r t i c u l a r p r o t e i n o r s t e r o i d hormone binds a c t i v a t e t h e s y n t h e s i s of t h e c y c l i c nucleotide i n a p a r t i c u l a r c e l l u l a r compartment, i n which it t h e n a f f e c t s t h e a c t i v i t y of another enzyme (e.g., phosphorylase kinase, l i p a s e , o r t h e l i k e ) , thereby ~~ completing t h e response i n i t i a t e d a t t h e c e l l surface. S ~ t h e r l a n dand T a l w a r 6 7 e l a b o r a t e d a d d i t i o n a l p o s s i b i l i t i e s whereby adenyl cyclase can mediate d i f f e r e n t hormone e f f e c t s : 1) d i f f e r e n t a f f i n i t i e s of a s i n g l e r e c e p t o r s i t e f o r d i f f e r e n t hormones, 2 ) m u l t i p l e s i t e s on a s i n g l e r e c e p t o r molecule, 3 ) unique responses i n d i f f e r e n t c e l l s t o t h e a c t i v a t i o n of adenyl cyclase , 4 ) d i f f e r e n t c o f a c t o r requirements of t h e v a r i o u s cyclase:;, and 5 ) c e l l u l a r compartmentalization e f f e c t s . Regardless of t h e exact mechanism of a c t i o n , epinephrine can b e shown both i n v i t r o and i n vivo t o i n c r e a s e t h e a c t i v i t y of adenyl cyclase and t h i s e f f e c t can be s t i m u l a t e d or i n h i b i t e d by substances which antagonize t h e s y n t h e s i s o r degradation of c y c l i c AMP. I n t h e case of l i p o l y s i s , t h e r e appears t o be a d i r e c t c o r r e l a t i o n between t h e l e v e l s of c y c l i c AMP achieved and t h e amount of f r e e f a t t y a c i d r e l e a s e d , which a l s o s t r o n g l y i m p l i c a t e s t h e c y c l i c n u c l e o t i d e as a hormone mediator i n t h i s system as well. Coupled with t h i s f a c t , it m u s t be r e c a l l e d t h a t c y c l i c AMP mimics t h e e f f e c t of epinephrine i n l i v e r s l i c e s and causes hyperzlycemia i n v a r i o u s i n t a c t animals including humans"'. The f i n d i n g t h a t N6-21-O-dib~tyryl-3',5'-adenylic acid r e s i s t s the cyclic phosphodiesterase but p e n e t r a t e s c e l l s more r e a d i l y t h a n c y c l i c AMP i t s e l f i s of g r e a t t h e o r e t i c a l and p r a c t i c a l i n t e r e s t t o t h e medicinal chemist. This r e p r e s e n t s a s i t u a t i o n toward which many i n v e s t i g a t o r s s t r i v e i n prep a r i n g s u b s t i t u t e d d e r i v a t i v e s and analogues of pharmacologically a c t i v e compounds. Very seldom, however, a r e such c l e a r - c u t advantages f o r a r e l a It i s s u r p r i s i n g t o t h i s reviewer t h a t t i v e l y simple d e r i v a t i v e r e a l i z e d . considerably more work has not been done t o d a t e , both i n v i t r o and i n whole animals, with t h e d i b u t y r y l compound. It i s not only imperative t h a t s u f f i c i e n t q u a n t i t i e s of t h i s substance be prepared f o r broad e v a l u a t i o n i n experimental animals t o determine t h e o v e r a l l pharmacological p r o p e r t i e s o f t h i s unique substance, b u t a d d i t i o n a l r e l a t e d d e r i v a t i v e s w i t h v a r i e d s i d e chains a l s o seem worth i n v e s t i g a t i n g . It i s p o s s i b l e t h a t t h e p r e p a r a t i o n of s i m i l a r s u b s t i t u t e d nucleosides o r n u c l e o t i d e s o f bases o t h e r t h a n adenine might r e s u l t i n increased p e r m e a b i l i t y i n s p e c i f i c t i s s u e s and decreased r a t e of h y d r o l y s i s t o nucleosides. Analqgues o f c y c l i c AMP p e r s e may a l s o be worthy of i n v e s t i g a t i o n i n view o f t h e f a c t t h a t a s p e c i f i c enzyme s y n t h e s i z e s t h i s compound. The enzyme may be l o c a l i z e d s e l e c t i v e l y i n v a r i o u s t i s s u e s and a s p e c i f i c c y c l i c phosphod i e s t e r a s e i n a c t i v a t e s t h e substance by forming 5l-AVP. Thus, enzyme systems i n which t h e medicinal chemist can e v a l u a t e compounds i n a di-ect manner f o r t h e i r a b i l i t y t o a c t i n p l a c e o f c y c l i c AMP o r i n h i b i t i t s a c t i o n o r degradation
Chap. 27
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a r e a v a i l a b l e . One c l o s e r e l a t i v e of c y c l i c AMP, c y c l i c t u b e r c i d i n phosphate6', has been t e s t e d by Butcher and Sutherland (unpublished o b s e r v a t i o n s ) and shown t o r e p l a c e c y c l i c AMP as an a c t i v a t o r of phosphorylase. C e r t a i n l y a wide v a r i e t y of s u b s t i t u t e d p u r i n e analogues and 2 ' - s u b s t i t u t e d c y c l i c AMP d e r i v a t i v e s or analogues should be prepared and i n v e s t i g a t e d i n t h e abovementioned enzyme systems. The u b i q u i t y of c y c l i c AMP (animal t i s s u e s , u r i n e and E. -~ 0 l i 6 9 )and t h e occurrence of t h e c y c l i c monophosphates of guanosine and p o s s i b l y u r i d i n e i n nature70 '7 f u r t h e r s t r e n g t h e n t h e p o t e n t i a l importance of t h e s e compounds i n o v e r a l l r e g u l a t i o n of metabolism, and extend t h e f i e l d of s y n t h e s i s of p o s s i b l e analogues or d e r i v a t i v e s t o t h i s e n t i r e family o f n u c l e o t i d e s . It should be emphasized t h a t s e l e c t i v e or s p e c i f i c i n h i b i t i o n of t h e a c t i o n of t h e progenitor substance i s not t h e only b a s i s f o r i n t e r e s t i n d e r i v a t i v e s and analogues of compounds r e l a t e d t o c y c l i c AMP. Increased h a l f - l i f e of t h e molecule i n t h e animal body, allowing it t o b e c a r r i e d t o s p e c i f i c organs i n higher concentration t h a n t h e p a r e n t compound, can be a decided advantage. Likewise, s e l e c t i v e organ concentration of d e r i v a t i v e s o r analoguesorchanges i n r a t e of c e l l u l a r k i n e t i c s can a l t e r t h e o v e r a l l metabolism and d i s t r i b u t i o n p i c t u r e as w e l l as t h e b i o l o g i c a l e f f e c t s observed w i t h s u b s t i t u t e d nucleotides68 '72. Marked changes i n p o l a r i t y of t h e compound may s i g n i f i c a n t l y i n f l u e n c e passage a c r o s s t h e blood-brain b a r r i e r r e s u l t i n g i n pharmacologically a c t i v e doses of a compound i n t h e nervous system where t h e p a r e n t accumulates i n only v e r y low concentrations. Obviously, it cannot be p r e d i c t e d whether such changes w i l l be f a v o r a b l e of unfavora5le t o t h e animal i n which t h e y are induced. When t h e c y c l i c AMP and c y c l i c n u c l e o t i d e p i c t u r e i s considered i n t o t o , it emerges as a f a s c i n a t i n g a r e a of biochemistry and biology i n which t h e medicinal chemist ought t o p l a y a s i g n i f i c a n t r o l e i n t a i l o r i n g molecules f o r c e r t a i n enzymatic a c t i v i t i e s which can be d i r e c t l y measured i n v i t r o and evaluated i n v a r i o u s s p e c i e s of whole animals, including man, where i n d i c a t e d .
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Topics i n Biology
Figure 1
0
I
0
II H OI - P 1- O - P - O - ~ ~ ~ II
0 I
0
-7OH
0- -OH
H
OH
OH
Adenyl Cyclase
9
Activated by hormones
I
ATP
Cyclic-AMP
Phosphod i esterase
T
F2
I n h i b i t e d by methyl x a n t h i n e s
0
II
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OH
5'
B i o s y n t h e s i s and Degradation of Cyclic AMP
Smith
Cell Metabolism
Chap. 27
Figure 2 The Mechanism of Action of Epinephrine oh Glycogenolysis in Muscle Epinephrine
J
Adenyl cyclase
n
ATP
Cyclic 3' ,5'-AMP Enzyme ( ? )
Nonactivated
Activated
phosphorylase
phosphorylase kinase
kinase
2
+ ATP Phosphorylase
Glycogen
+ Pi
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Glucose-1-P
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Topics i n Biology
Tabachnick, Ed.
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The phosphorylase system While investigating the site of the glycogenolytic action of epinephrine, Sutherland and Cori4' demonstrated the stimulation of phosphorylase activity by this catecholamine and subsequent studies' '32' 43
'la,
Chap. 27
Cell Metabolism
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implicated c y c l i c AMP i n t h i s process. Krebs, e t al.32, have r e c e n t l y r e viewed t h e s u b j e c t o f a c t i v a t i o n of s k e l e t a l muscle phosphorylase under t h e influence of epinephrine, mediated by c y c l i c AMP. Figure 2 , adapted from t h e i r paper, summarizes t h e s t e p s which have been shown t o be involved i n t h i s process. The d i r e c t a c t i v a t i o n of phosphorylase b k i n a s e i n muscle e x t r a c t can be demonstrated upon t h e a d d i t i o n of c y c l i c AMP i n v i t r o 4 3 , 4 4 . The a c t i v a t i o n of phosphorylase by epinephrine has a l s o been demonstrated i n t h e perfused h e a r t and i n t a c t h e a r t i n s i t u 3 5 . Thus, t h r e e enzymatic systems a r e a v a i l a b l e which can be s t u d i e d i n i s o l a t e d c e l l p r e p a r a t i o n s f o r e f f e c t s of analogues or chemically u n r e l a t e d compounds on e i t h e r t h e s y n t h e s i s or degradation of c y c l i c AMP or enzymatic a c t i v a t i o n by t h i s nucleotide. The p h o ~ p h o d i e s t e r a s e ~which l cleaves c y c l i c AMP i s i n h i b i t e d by methyl xanthines and it i s e n t i r e l y p o s s i b l e t h a t some or a l l of t h e b i o l o g i c a l a c t i v i t y observed f o r t h e s e i n h i b i t o r s of c y c l i c AMP degradation may be due t o t h i s a c t i o n . For example, t h e o p h y l l i n e has been shown t o i n c r e a s e phosphorylase a a c t i v i t y i n t h e i s o l a t e d h e a r t and p o t e n t i a t e t h e e f f e c t s of epinephrine in t h i s assaya5. E f f e c t s on l i p o l y s i s - I n a r e c e n t a r t i c l e , Butcher37 reviews t h e h i s t o r y o f t h e i r experiments on t h e r o l e of t h e c y c l i c AMF i n t h e l i p o l y t i c process. T h e i r i n v e s t i g a t i o n s showed an i n c r e a s e i n t h e concentration of c y c l i c AMP upon t h e a d d i t i o n of epinephrine t o f a t pads i n v i t r o , followed by a s t i m u l a t i o n of t h e r e l e a s e of f r e e f a t t y a c i d s . Caffeine, a known i n h i b i t o r o f t h e phosphodiesterase which degrades c y c l i c AMP, a c t e d s y n e r g i s t i c a l l y with epinephrine on t h e accumulation of c y c l i c AMP and t h e subsequent r e l e a s e of f r e e f a t t y acid^^^,^^. It should be noted t h a t t h e l i p o l y t i c e f f e c t of epinep h r i n e w a s decreased by d i c h l o r o i s o p r o t e r e n o l , a known i n h i b i t o r of t h e s t i m u l a t i o n of adenyl cyclase by c a t e ~ h o l a r n i n e ~ " ~ ~I n . s p i t e of t h i s e v i dence f o r t h e r o l e of c y c l i c AMP as a mediator i n t h e l i p o l y t i c process, t h e d i r e c t a d d i t i o n of t h i s n u c l e o t i d e t o f a t pads i n v i t r o d i d not s t i m u l a t e f a t t y a c i d r e l e a s e 4 5 , although p e n e t r a t i o n i n t o t i s s u e by t h e n u c l e o t i d e w a s low. When t h e Ne-2'-O,dibutyryl e s t e r of c y c l i c 3',5'-AMP4e w a s added t o f a t pads or f a t pads were perfused with t h i s compound, s t i m u l a t i o n of t i s s u e f r e e f a t t y a c i d s y n t h e s i s and f r e e f a t t y a c i d r e l e a s e w a s noted. The subs t i t u t e d compound w a s a t l e a s t t e n times as a c t i v e i n t h i s system as w a s c y c l i c AMF per se. The d i b u t y r y l d e r i v a t i v e may p e n e t r a t e t i s s u e more e f f e c t i v e l y t h a n does t h e u n s u b s t i t u t e d n u c l e o t i d e , but a l s o it i s degraded by t h e c y c l i c phosphodiesterase more slowly t h a n i s t h e p a r e n t compound 3 7 . T h i s i s indeed a n e x c i t i n g example t o t h e medicinal chemist of a r e l a t i v e l y simple chemical s u b s t i t u t i o n which r e s u l t s i n a marked change i n t h e a b i l i t y of a compound t o p e n e t r a t e t i s s u e and withstand t h e degradation t h a t i t s p r o g e n i t o r undergoes. A t t h e same time, t h e substance obviously can be conv e r t e d , a l b e i t slowly, t o t h e a c t i v e c y c l i c AMP and a c t as a " s l o w f e e d " of the latter. E f f e c t s on s t e r o i d o g e n e s i s - E a r l y investigation^^^'^ showed t h a t incubation of beef a d r e n a l s l i c e s with ACTH r e s u l t e d i n an i n c r e a s e i n t h e c y c l i c AMP l e v e l i n t h i s t i s s u e and t h a t added c y c l i c AMP s t i m u l a t e d c o r t i c o i d sylrthesis i n r a t a d r e n a l s l i c e s . More r e c e n t l y , Karaboyas and K o r i t z & s t u d i e d t h e
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mechanism of s t i m u l a t i o n of c o r t i c o i d s y n t h e s i s i n r a t and beef a d r e n a l c o r t e x s l i c e s and concluded t h a t c y c l i c AMP and ACTH a f f e c t t h e same s i t e i n t h e b i o s y n t h e t i c pathway between c h o l e s t e r o l and pregnenolone. They f u r t h e r suggested t h a t c y c l i c AMP i s a n o b l i g a t o r y intermediate i n t h e a c t i o n ~~ that of ACTH. I n another r e c e n t paper, Roberts and c o - w ~ r k e r sconcluded 5 l - M (as w e l l as c y c l i c AMP) s t i m u l a t e s s t e r o i d hydroxylation i n adrenal mitochondria. The mechanism whereby c y c l i c AMP enhances s t e r o i d hydroxyla t i o n i n t h i s system i s unknown. I n t h e i r hands, hydroxylation of s t e r o i d could not be obtained even a t high l e v e l s of c y c l i c AMP i f t h e a d r e n a l mitoc h o n d r i a l system or homogenate d i d not c o n t a i n reduced NADP, and t h e y suggest as p o s s i b l e explanations f o r t h e a c t i o n of t h i s n u c l e o t i d e 1) increased s t i m u l a t i o n of t h e t r a n s p o r t of s t e r o i d ( o r co-factor ) a c r o s s mitochondria1 membranes o r 2 ) d i r e c t a c t i v a t i o n of t h e s t e r o i d hydroxylase system. Creange and Roberts on t h e o t h e r hand, a l s o studying t h e mechanism of ll-fbhydroxyla t i o n , concluded t h a t c y c l i c AMP s e l e c t i v e l y stimulated t h i s process i n t h e a d r e n a l c o r t e x by a mechanism which i s independent of glycogen phosphoryla t i o n , reduced NADP g e n e r a t i o n and endogenous c o r t i c o i d precursorss1. I n 1964, Marsh and Savards2 r e p o r t e d t h a t l u t e i n i z i n g hormone increased t h e l e v e l of phosphorylase a c t i v i t y i n bovine corpora l u t e a , t h a t t h i s response w a s s p e c i f i c f o r t h e hormone and t h a t a high degree of c o r r e l a t i o n e x i s t e d between t h e e x t e n t o f phosphorylase s t i m u l a t i o n and t h e degree o f progesterone s t i m u l a t i o n by l u t e i n i z i n g hormone. I n t h e i r s t u d i e s , exogenous c y c l i c AMP d i d not s t i m u l a t e l u t e a l phosphorylase a c t i v i t y . More r e c e n t l y , Marsh and c o l l a b o r a t o r s 5 showed t h a t l u t e i n i z i n g hormone s t i m u l a t e d t h e production of c y c l i c AMP i n bovine corpora l u t e a incubated i n v i t r o and t h a t t h i s i n c r e a s e preceded progesterone s y n t h e s i s . Again t h e e f f e c t w a s r e p o r t e d t o be s p e c i f i c f o r l u t e i n i z i n g hormone. These i n v e s t i g a t o r s a l s o demonstrated a n i n c r e a s e i n t h e c o n c e n t r a t i o n o f c y c l i c AMP i n a human corpus luteum s t i m u l a t e d Ln v i t r o by human chorionic gonadotrophin. Other e f f e c t s of c y c l i c AMP i n v i t r o - I n a r e c e n t paper, Appleman and coworkers5 concluded t h a t many s i m i l a r i t i e s e x i s t between t h e ATP-cyclic AMP a c t i v a t i o n of phosphorylase b k i n a s e and t h e conversion of glycogen synthetase from t h e independent t o t h e glucose 6-phosphate-dependent form. C e r t a i n i n t e r e s t i n g e f f e c t s on i o n and water permeability a r e a l s o thought t o be mediated by c y c l i c AMP. Handler, e t a1.=, r e p o r t e d t h a t e i t h e r a r g i n i n e vasopressin or t h e o p h y l l i n e s i g n i f i c a n t l y i n c r e a s e d t h e concentration of c y c l i c AMP i n t h e i s o l a t e d toad bladder and t h a t t h e two t o g e t h e r were synergistic. Cyclic AMP w a s a l s o r e p o r t e d t o have t h e same e f f e c t as vasop r e s s i n on t h e permeability of t h e bladder t o water and sodium i o n and, accordingly, w a s concluded t o a c t as a n i n t r a c e l l u l a r mediator of t h e vasop r e s s i n e f f e c t . It i s i n t e r e s t i n g t h a t t h i s n u c l e o t i d e has a l s o been r e p o r t e d t o i n c r e a s e d i u r e s i s i n vivo, as described below. Although t h y r o i d s t i m u l a t i n g hormone (TSH) has been r e p o r t e d t o i n c r e a s e l e v e l s of c y c l i c AMP i n t h y r o i d homogenates and s l i c e s = ' s 6 , t h e n u c l e o t i d e d i d not i n c r e a s e Ci402 production from glucose nor t h e i n c o r p o r a t i o n of $2phosphate i n t o phospholipids i n v i t r o , as w a s observed with TSH p e r seS6 ' 5 7 . More r e c e n t l y , Pastans8 r e p o r t e d a s i g n i f i c a n t s t i m u l a t i o n of both of t h e s e
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p r o c e s s e s by t h e d i b u t y r y l d e r i v a t i v e o f c y c l i c AMP a t c o n c e n t r a t i o n s as low as 50 pg/mL b u t not by c y c l i c AMP a t f i v e t i m e s t h i s c o n c e n t r a t i o n . TSH, as expected, s t i m u l a t e d b o t h p r o c e s s e s a t low c o n c e n t r a t i o n . Again, as mentioned above under l i p o l y t i c s t u d i e s , t h e enhanced a c t i v i t y of t h e d i b u t y r y l s u b s t i t u t e d n u c l e o t i d e should be noted. Metabolic E f f e c t s of Cyclic AMP i n Whole Animals A s mentioned e a r l i e r , c y c l i c AMP has been p o s t u l a t e d as t h e c e l l u l a r i n t e r mediate i n t h e a n t i - d i u r e t i c a c t i o n of v a s o p r e ~ s i '21y25 n ~ ~ j 5 9 y60 i n i s o l a t e d dog kidney homogenates and i s o l a t e d t o a d b l a d d e r . Based orAt h e s e o b s e r v a tions, Levine s t u d i e d t h e e f f e c t of c y c l i c AMP on d i u r e s i s i n v i v o and r e p o r t e d a prompt a n t i - d i u r e t i c e f f e c t a f t e r i n j e c t i o n of t h e n u c l e o t i d e Levine and VogelG2 a l s o determined t h e e f f e c t of t h e c y c l i c i n man61. n u c l e o t i d e on h e a r t r a t e , c a r d i a c o u t p u t , mean blood p r e s s u r e , t o t a l p e r i p h e r a l r e s i s t a n c e , s t r o k e volume,pulse p r e s s u r e , blood g l u c o s e and plasma f r e e f a t t y a c i d s i n i n t a c t dogs and r e p o r t e d t h a t a d m i n i s t r a t i o n of a s i n g l e dose produced e f f e c t s i n t h e whole animal resembling t h o s e of c a t e cholamines. They r e p o r t e d no s i g n i f i c a n t changes i n c a r d i a c o r m e t a b o l i t e f u n c t i o n s f o l l o w i n g a d m i n i s t r a t i o n of t h e 2 l - , 3 * - o r 5'-AMP, ATP or s a l i n e , a l t h o u g h p r i o r a d m i n i s t r a t i o n of d i c h l o r o i s o p r o t e r e n o l p r e v e n t e d t h e c a r d i o v a s c u l a r (but n o t t h e m e t a b o l i c e f f e c t s ) of c y c l i c AMP. The l a t t e r nucleot i d e has a l s o been i m p l i c a t e d i n t h e mechanism o f myocardial ~ o n t r a c t i l i t ~ ~ - "
General C o n s i d e r a t i o n s Examination o f a v a i l a b l e d a t a c l e a r l y i n d i c a t e s a r o l e f o r c y c l i c AMP as a mediator of t h e catecholamine s t i m u l a t i o n of g l y c o g e n o l y s i s , b y t h e mechanism d e p i c t e d i n F i g u r e 2. Unlike t h e a t t e m p t s t o demonstrate t h e s i t e of a c t i o n of most p r o t e i n and s t e r o i d hormones66, t h e expected i n c r e a s e s i n each of t h e enzyme a c t i v i t i e s presumed t o b e mediated by c y c l i c AMP can be demons t r a t e d with d i r e c t i n v i t r o techniques after treatment of t h e appropriate t i s s u e w i t h s p e c i f i c hormones. A s a n example, adenyl c y c l a s e a c t i v i t y and c y c l i c AMP i n c r e a s e upon t r e a t m e n t w i t h e p i n e p h r i n e and t h i s i n t u r n can b e shown, a t t h e enzymatic l e v e l , t o a c t i v a t e phosphorylase k i n a s e . The l a t t e r enzyme c o n v e r t s phosphorylase b t o phosphorylase a which i s t h e a c t i v e form o f t h e enzyme c a t a l y z i n g t h e s y n t h e s i s of glucose-1-phosphate from glucagon. The o p p o r t u n i t i e s f o r t h e m e d i c i n a l chemist t o s t u d y analogues and d e r i v a t i v e s o f t h e simple n u c l e o t i d e s o r o f compounds r e l a t e d t o t h e catecholamines a r e unusual i n t h a t s e v e r a l enzymatic s t e p s i n a known sequence of r e a c t i o n s can b e measured q u a n t i t a t i v e l y i n v i t r o and e f f e c t s of s t i m u l a t o r s o r i n h i b i t o r s can b e d i r e c t l y a s s e s s e d . The d a t a i m p l i c a t i n g c y c l i c AMP i n t h e l i p o l y t i c p r o c e s s are a l s o q u i t e convincing, and t h e concept of c y c l i c n u c l e o t i d e mediation of ACTH i n s t e r o i d o g e n e s i s and v a s o p r e s s i n a c t i o n on water and i o n t r a n s p o r t a c r o s s nembranes i s a l s o sound. I n v i v o , c y c l i c AMP e x e r t s many of t h e e f f e c t s observed w i t h c a t e cholamines and, i n v a r i o u s systems, t h e methylxanthines (e.g., t h e o p h y l l i n e ) , which a r e known t o i n h i b i t t h e p h o s p h o d i e s t e r a s e t h a t c l e a v e s t h e n u c l e o t i d e , a l s o p o t e n t i a t e i t s a c t i o n . I n a d d i t i o n , t h e c y c l i c n u c l e o t i d e has been i m p l i c a t e d i n v a r i o u s s t e p s i n t h e gluconeogenic pathway, t h e mechanism o f a c t i o n of l u t e i n i z i n g hormone, glycogen s y n t h e t a s e and t h e metabolism of
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thyroid t i ssue. Although it is not uncommon t o a s s o c i a t e s i n g l e co-enzymes with d i f f e r e n t enzymes c a t a l y z i n g s i m i l a r t y p e s of r e a c t i o n s (e.g., ATP w i t h k i n a s e s , NADP w i t h reductases, and t h e l i k e ) , it i s not common t o t h i n k of a s i n g l e small molecule as a mediator of a wide v a r i e t y of hormonal o r metabolic r e a c t i o n s . Since adenyl c y c l a s e i s membrane bound, it i s p o s s i b l e t h a t s t e r e o - s p e c i f i c attachment s i t e s on t h e c e l l s u r f a c e t o which t h e p a r t i c u l a r p r o t e i n o r s t e r o i d hormone binds a c t i v a t e t h e s y n t h e s i s of t h e c y c l i c nucleotide i n a p a r t i c u l a r c e l l u l a r compartment, i n which it t h e n a f f e c t s t h e a c t i v i t y of another enzyme (e.g., phosphorylase kinase, l i p a s e , o r t h e l i k e ) , thereby ~~ completing t h e response i n i t i a t e d a t t h e c e l l surface. S ~ t h e r l a n dand T a l w a r 6 7 e l a b o r a t e d a d d i t i o n a l p o s s i b i l i t i e s whereby adenyl cyclase can mediate d i f f e r e n t hormone e f f e c t s : 1) d i f f e r e n t a f f i n i t i e s of a s i n g l e r e c e p t o r s i t e f o r d i f f e r e n t hormones, 2 ) m u l t i p l e s i t e s on a s i n g l e r e c e p t o r molecule, 3 ) unique responses i n d i f f e r e n t c e l l s t o t h e a c t i v a t i o n of adenyl cyclase , 4 ) d i f f e r e n t c o f a c t o r requirements of t h e v a r i o u s cyclase:;, and 5 ) c e l l u l a r compartmentalization e f f e c t s . Regardless of t h e exact mechanism of a c t i o n , epinephrine can b e shown both i n v i t r o and i n vivo t o i n c r e a s e t h e a c t i v i t y of adenyl cyclase and t h i s e f f e c t can be s t i m u l a t e d or i n h i b i t e d by substances which antagonize t h e s y n t h e s i s o r degradation of c y c l i c AMP. I n t h e case of l i p o l y s i s , t h e r e appears t o be a d i r e c t c o r r e l a t i o n between t h e l e v e l s of c y c l i c AMP achieved and t h e amount of f r e e f a t t y a c i d r e l e a s e d , which a l s o s t r o n g l y i m p l i c a t e s t h e c y c l i c n u c l e o t i d e as a hormone mediator i n t h i s system as well. Coupled with t h i s f a c t , it m u s t be r e c a l l e d t h a t c y c l i c AMP mimics t h e e f f e c t of epinephrine i n l i v e r s l i c e s and causes hyperzlycemia i n v a r i o u s i n t a c t animals including humans"'. The f i n d i n g t h a t N6-21-O-dib~tyryl-3',5'-adenylic acid r e s i s t s the cyclic phosphodiesterase but p e n e t r a t e s c e l l s more r e a d i l y t h a n c y c l i c AMP i t s e l f i s of g r e a t t h e o r e t i c a l and p r a c t i c a l i n t e r e s t t o t h e medicinal chemist. This r e p r e s e n t s a s i t u a t i o n toward which many i n v e s t i g a t o r s s t r i v e i n prep a r i n g s u b s t i t u t e d d e r i v a t i v e s and analogues of pharmacologically a c t i v e compounds. Very seldom, however, a r e such c l e a r - c u t advantages f o r a r e l a It i s s u r p r i s i n g t o t h i s reviewer t h a t t i v e l y simple d e r i v a t i v e r e a l i z e d . considerably more work has not been done t o d a t e , both i n v i t r o and i n whole animals, with t h e d i b u t y r y l compound. It i s not only imperative t h a t s u f f i c i e n t q u a n t i t i e s of t h i s substance be prepared f o r broad e v a l u a t i o n i n experimental animals t o determine t h e o v e r a l l pharmacological p r o p e r t i e s o f t h i s unique substance, b u t a d d i t i o n a l r e l a t e d d e r i v a t i v e s w i t h v a r i e d s i d e chains a l s o seem worth i n v e s t i g a t i n g . It i s p o s s i b l e t h a t t h e p r e p a r a t i o n of s i m i l a r s u b s t i t u t e d nucleosides o r n u c l e o t i d e s o f bases o t h e r t h a n adenine might r e s u l t i n increased p e r m e a b i l i t y i n s p e c i f i c t i s s u e s and decreased r a t e of h y d r o l y s i s t o nucleosides. Analqgues o f c y c l i c AMP p e r s e may a l s o be worthy of i n v e s t i g a t i o n i n view o f t h e f a c t t h a t a s p e c i f i c enzyme s y n t h e s i z e s t h i s compound. The enzyme may be l o c a l i z e d s e l e c t i v e l y i n v a r i o u s t i s s u e s and a s p e c i f i c c y c l i c phosphod i e s t e r a s e i n a c t i v a t e s t h e substance by forming 5l-AVP. Thus, enzyme systems i n which t h e medicinal chemist can e v a l u a t e compounds i n a di-ect manner f o r t h e i r a b i l i t y t o a c t i n p l a c e o f c y c l i c AMP o r i n h i b i t i t s a c t i o n o r degradation
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a r e a v a i l a b l e . One c l o s e r e l a t i v e of c y c l i c AMP, c y c l i c t u b e r c i d i n phosphate6', has been t e s t e d by Butcher and Sutherland (unpublished o b s e r v a t i o n s ) and shown t o r e p l a c e c y c l i c AMP as an a c t i v a t o r of phosphorylase. C e r t a i n l y a wide v a r i e t y of s u b s t i t u t e d p u r i n e analogues and 2 ' - s u b s t i t u t e d c y c l i c AMP d e r i v a t i v e s or analogues should be prepared and i n v e s t i g a t e d i n t h e abovementioned enzyme systems. The u b i q u i t y of c y c l i c AMP (animal t i s s u e s , u r i n e and E. -~ 0 l i 6 9 )and t h e occurrence of t h e c y c l i c monophosphates of guanosine and p o s s i b l y u r i d i n e i n nature70 '7 f u r t h e r s t r e n g t h e n t h e p o t e n t i a l importance of t h e s e compounds i n o v e r a l l r e g u l a t i o n of metabolism, and extend t h e f i e l d of s y n t h e s i s of p o s s i b l e analogues or d e r i v a t i v e s t o t h i s e n t i r e family o f n u c l e o t i d e s . It should be emphasized t h a t s e l e c t i v e or s p e c i f i c i n h i b i t i o n of t h e a c t i o n of t h e progenitor substance i s not t h e only b a s i s f o r i n t e r e s t i n d e r i v a t i v e s and analogues of compounds r e l a t e d t o c y c l i c AMP. Increased h a l f - l i f e of t h e molecule i n t h e animal body, allowing it t o b e c a r r i e d t o s p e c i f i c organs i n higher concentration t h a n t h e p a r e n t compound, can be a decided advantage. Likewise, s e l e c t i v e organ concentration of d e r i v a t i v e s o r analoguesorchanges i n r a t e of c e l l u l a r k i n e t i c s can a l t e r t h e o v e r a l l metabolism and d i s t r i b u t i o n p i c t u r e as w e l l as t h e b i o l o g i c a l e f f e c t s observed w i t h s u b s t i t u t e d nucleotides68 '72. Marked changes i n p o l a r i t y of t h e compound may s i g n i f i c a n t l y i n f l u e n c e passage a c r o s s t h e blood-brain b a r r i e r r e s u l t i n g i n pharmacologically a c t i v e doses of a compound i n t h e nervous system where t h e p a r e n t accumulates i n only v e r y low concentrations. Obviously, it cannot be p r e d i c t e d whether such changes w i l l be f a v o r a b l e of unfavora5le t o t h e animal i n which t h e y are induced. When t h e c y c l i c AMP and c y c l i c n u c l e o t i d e p i c t u r e i s considered i n t o t o , it emerges as a f a s c i n a t i n g a r e a of biochemistry and biology i n which t h e medicinal chemist ought t o p l a y a s i g n i f i c a n t r o l e i n t a i l o r i n g molecules f o r c e r t a i n enzymatic a c t i v i t i e s which can be d i r e c t l y measured i n v i t r o and evaluated i n v a r i o u s s p e c i e s of whole animals, including man, where i n d i c a t e d .
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Sect. V
Tabachnick, Ed.
Topics i n Biology
Figure 1
0
I
0
II H OI - P 1- O - P - O - ~ ~ ~ II
0 I
0
-7OH
0- -OH
H
OH
OH
Adenyl Cyclase
9
Activated by hormones
I
ATP
Cyclic-AMP
Phosphod i esterase
T
F2
I n h i b i t e d by methyl x a n t h i n e s
0
II
OH
OH
5'
B i o s y n t h e s i s and Degradation of Cyclic AMP
Smith
Cell Metabolism
Chap. 27
Figure 2 The Mechanism of Action of Epinephrine oh Glycogenolysis in Muscle Epinephrine
J
Adenyl cyclase
n
ATP
Cyclic 3' ,5'-AMP Enzyme ( ? )
Nonactivated
Activated
phosphorylase
phosphorylase kinase
kinase
2
+ ATP Phosphorylase
Glycogen
+ Pi
Phosphorylase 5
Glucose-1-P
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Sect. V
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Topics i n Biology
Tabachnick, Ed.
References
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